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Blockade of ataxia telangiectasia mutated sensitizes hepatoma cell lines to sorafenib by interfering with Akt signaling
Abstract
Sorafenib is a multi-kinase inhibitor applicable to hepatocellular carcinoma (HCC), but its limited therapeutic effects are a major problem to be solved. Here, we show that blockade of ataxia telangiectasia mutated (ATM) improves the antitumor effects of sorafenib. When hepatoma cell lines HepG2 and PLC/PRF/5 were treated with sorafenib plus ATM small inhibitory RNAs, ATM inhibitor KU55933 or caffeine, Akt signaling was suppressed and the cytotoxic effects were significantly potentiated. Moreover, ATM inhibition effectively suppressed the sorafenib-induced cell migration. Taken together, manipulation of ATM activity might be a useful strategy for improving sorafenib treatment of HCC.
... Most HCC patients have severe liver dysfunction related to conditions such as chronic hepatitis B virus (HBV) or hepatitis C virus (HCV) viral infections, alcohol abuse, and metabolic disease [2]. Such conditions lead to 80 % of cases being ineligible for curative surgical treatment [3]. Patients with unresectable HCC cannot generally be cured by systemic chemotherapy or radiotherapy [3], because hepatoma cells respond poorly to conventional approaches [4][5][6]. ...
... Such conditions lead to 80 % of cases being ineligible for curative surgical treatment [3]. Patients with unresectable HCC cannot generally be cured by systemic chemotherapy or radiotherapy [3], because hepatoma cells respond poorly to conventional approaches [4][5][6]. Given the limited efficacy of existing treatments for hepatoma cells, the molecular mechanisms underlying the pathogenesis of HCC need to be understood so that novel and efficient targeted therapies may be developed for these patients. ...
... There are comparatively few studies of the relationship between HBX and ATM. ATM regulates the DDR and stimulates redox signaling [3,51]. In addition, we have recently reported that ATM can be activated by HBX both in vitro and in vivo [52]. ...
Patients with unresectable hepatocellular carcinoma (HCC) cannot generally be cured by systemic chemotherapy or radiotherapy due to their poor response to conventional therapeutic agents. The development of novel and efficient targeted therapies to increase their treatment options depends on the elucidation of the molecular mechanisms that underlie the pathogenesis of HCC. The DNA damage response (DDR) is a network of cell-signaling events that are triggered by DNA damage. Its dysregulation is thought to be one of the key mechanisms underlying the generation of HCC. Sphingosine-1-phosphate (S1P), a lipid mediator, has emerged as an important signaling molecule that has been found to be involved in many cellular functions. In the liver, the alteration of S1P signaling potentially affects the DDR pathways. In this review, we explore the role of the DDR in hepatocarcinogenesis of various etiologies, including hepatitis B and C infection and non-alcoholic steatohepatitis. Furthermore, we discuss the metabolism and functions of S1P that may affect the hepatic DDR. The elucidation of the pathogenic role of S1P may create new avenues of research into therapeutic strategies for patients with HCC.
... A small molecular inhibitor, CGK733, which targets kinase activity of ATM, can obviously reverse the resistance of HepG2 to taxol treatment (Tables 1 and 3) [147]. Besides, a synthetic cytotoxic effect was obtained in HepG2 cells when cotreated with sorafenib (multikinase inhibitor) and KU55933 (ATM inhibitor) (Tables 2 and 3) [148]. These results suggest that the ATM signaling pathway may serve as a novel target for the clinical treatment of HCC patients with chronic hepatitis B. ...
... [ [146][147][148] XPC Increased XPC is associated with liver fibrogenesis and cancer and could be related to the well-recognized resistance of HCC to chemotherapeutics. ...
... ATM HR or NHEJ Small molecular inhibitor, CGK733, which targets kinase activity of ATM can obviously reverse HBV-positive HCC cells, [147] KU55933, ATM inhibitor cotreat with sorafenib, multikinase inhibitor, shows synthetic cytotoxicity to HepG2 cells. [148] 3.1. HBV. ...
Hepatocellular carcinoma (HCC) has been known as one of the most lethal human malignancies, due to the difficulty of early detection, chemoresistance, and radioresistance, and is characterized by active angiogenesis and metastasis, which account for rapid recurrence and poor survival. Its development has been closely associated with multiple risk factors, including hepatitis B and C virus infection, alcohol consumption, obesity, and diet contamination. Genetic alterations and genomic instability, probably resulted from unrepaired DNA lesions, are increasingly recognized as a common feature of human HCC. Dysregulation of DNA damage repair and signaling to cell cycle checkpoints, known as the DNA damage response (DDR), is associated with a predisposition to cancer and affects responses to DNA-damaging anticancer therapy. It has been demonstrated that various HCC-associated risk factors are able to promote DNA damages, formation of DNA adducts, and chromosomal aberrations. Hence, alterations in the DDR pathways may accumulate these lesions to trigger hepatocarcinogenesis and also to facilitate advanced HCC progression. This review collects some of the most known information about the link between HCC-associated risk factors and DDR pathways in HCC. Hopefully, the review will remind the researchers and clinicians of further characterizing and validating the roles of these DDR pathways in HCC.
... agents in cancer cells including hepatoma cell lines. [34][35][36] Caffeine has been reported to affect cell cycle and induce apoptosis 23,24,[37][38][39] but the molecular mechanism of its anti-carcinogenic effect are still not completely elucidated. Interestingly, caffeine has been shown to inhibit various isoforms of phosphoinositide 3-kinases (PI3Ks) in vitro. ...
... Interestingly, although it was previously reported that caffeine slightly induces apoptosis in pancreatic cancer cells, 66 in a neuroblastoma cell line 67 and in a mouse epithelial cell line JB6 24 we observed that caffeine was not able to induce apoptosis in hepatic cancer cells at the concentrations used in our study. This was consistent with previous studies reporting an effect on cell proliferation with no effect on apoptosis in hepatic cancer cells 23,[34][35][36] Our data support the conclusion that caffeine could have anticarcinogenic effects in HCC in addition to chemopreventive properties. However the high concentrations of caffeine (1-2 mM) necessary to exert its inhibitory effect clearly indicate that caffeine itself is not likely to represent a suitable chemotherapeutic agent. ...
... Inhibition of Akt phosphorylation was previously reported in HepG2 and PLC/PRF/5 cells treated with caffeine at concentrations as high as 2.5 mM. 34 Here we further extended this observation by showing that CGS 15943 was also able to affect Akt phosphorylation in HCC cell lines by acting on the PI3K/Akt pathway. ...
Caffeine is a naturally occurring methylxanthine that acts as a non-selective adenosine receptor antagonist. Epidemiological studies demonstrated habitual coffee drinking to be significantly associated with liver cancer survival. We aimed to investigate the effects of caffeine and its analog CGS 15943 on hepatocellular carcinoma (HCC) and pancreatic cancer adenocarcinoma (PDAC). We demonstrate that caffeine and CGS 15943 block proliferation in HCC and PDAC cell lines by inhibiting the PI3K/Akt pathway. Importantly a kinase profiling assay reveals that CGS 15943 targets specifically the catalytic subunit of the class IB PI3K isoform (p110γ). These data give mechanistic insight into the action of caffeine and its analogs and they identify these compounds as promising lead compounds to develop drugs that can specifically target this PI3K isoform whose key role in cancer progression is emerging.
... For instance, sorafenib activates DNA repair signaling, and inhibition of DNA repair increases sorafenib cytotoxicity in cancers. 36 Inhibition of another DNA repair protein, ataxia telangiectasia mutated, enhanced the effects of sorafenib on hepatoma cells. 36 These findings are in line with our data on cytotoxicity of RAD51 inhibitor plus sorafenib in four different human HCC cell lines (Figure 2A-D, middle panel; Figure S2A-D, upper panel). ...
... 36 Inhibition of another DNA repair protein, ataxia telangiectasia mutated, enhanced the effects of sorafenib on hepatoma cells. 36 These findings are in line with our data on cytotoxicity of RAD51 inhibitor plus sorafenib in four different human HCC cell lines (Figure 2A-D, middle panel; Figure S2A-D, upper panel). ...
The multityrosine kinase inhibitor sorafenib remains an important systemic treatment option for hepatocellular carcinoma (HCC). Signaling pathways, which are targeted by sorafenib, are involved in checkpoint and DNA repair response, RAD51 being a candidate protein. Here, we aim to evaluate the effect of the human RAD51 inhibitor B02 in combination with sorafenib in human HCC cells. Impact of RAD51 expression on HCC patient survival was evaluated by an in silico approach using Human Protein Atlas dataset. Cell viability of HUH7, AKH12, AKH13, and 3P was assessed by neutral red assay. To measure the cytotoxicity, we quantified loss of membrane integrity by lactate dehydrogenase release. We also employed colony formation assay and hanging drop method to assess clonogenic and invasive ability of HCC cell lines upon sorafenib and B02 treatment. Cell cycle distribution and characterization of apoptosis was evaluated by flow cytometry. In silico approach revealed that HCC patients with higher expression of RAD51 messenger RNA had a significantly shorter overall survival. The RAD51 inhibitor B02 alone and in combination with sorafenib significantly reduced viability, colony formation ability, and invasion capacity of HCC cells. Cell cycle analysis revealed that the combination of both agents reduces the proportion of cells in the G2/M phase while leading to an accumulating in the subG1 phase. The RAD51 inhibitor B02 seems to be a promising agent for HCC treatment and enhances the antitumor effects of sorafenib in vitro.
... Indeed, ATM inhibitors have been in clinical development for the past two decades [12,13]. Multiple pre-clinical studies have indicated that ATM inhibitor monotherapy is not likely to be effective [13,14,15,16,17]. However, combined inhibition of ATM and DNA damaging agents such as PARP inhibitors and irradiation is synergistic [17], and recently a Phase I clinical trial using the ATM inhibitor AZD0156 in combination with a variety of DNA damaging agents has commenced (clinicaltrials.gov). ...
... ATM may be an actionable target in HGSOC. However, many preclinical studies have demonstrated that inhibition of ATM as a single agent is not likely to be effective [13,14,15,16,17]. Combined inhibition of ATM and other therapeutic agents has shown potential to inhibit cancer cell survival both in vitro and in vivo [12,13,17,31]. ...
While therapies targeting deficiencies in the homologous recombination (HR) pathway are emerging as the standard treatment for high grade serous ovarian cancer (HGSOC) patients, this strategy is limited to the ~50% of patients with a deficiency in this pathway. Therefore, patients with HR-proficient tumors are likely to be resistant to these therapies and require alternative strategies. We found that the HR gene Ataxia Telangiectasia Mutated (ATM) is wildtype and its activity is upregulated in HGSOC compared to normal fallopian tube tissue. Interestingly, multiple pathways related to metabolism are inversely correlated with ATM expression in HGSOC specimens, suggesting that combining ATM inhibition with metabolic drugs would be effective. Analysis of FDA-approved drugs from the Dependency Map demonstrated that ATM-low cells are more sensitive to fenofibrate, a PPARα agonist that affects multiple cellular metabolic pathways. Consistently, PPARα signaling is associated with ATM expression. We validated that combined inhibition of ATM and treatment with fenofibrate is synergistic in multiple HGSOC cell lines by inducing senescence. Together, our results suggest that metabolic changes induced by ATM inhibitors are a potential target for the treatment of HGSOC.
... Indeed, ATM inhibitors have been in clinical development for the past two decades (13,14). Multiple pre-clinical studies have indicated that ATM inhibitor monotherapy is not likely to be effective (14)(15)(16)(17)(18). ...
... ATM may be an actionable target in HGSOC. However, many preclinical studies have demonstrated that inhibition of ATM as a single agent is not likely to be effective (14)(15)(16)(17)(18). ...
Background
Epithelial ovarian cancer (EOC) is the deadliest gynecological malignancy in the United States with high grade serous ovarian cancer (HGSOC) as the most commonly diagnosed subtype. While therapies targeting deficiencies in the homologous recombination (HR) pathway are emerging as the standard treatment for HGSOC patients, this strategy is limited to the 50% of patients with a deficiency in this pathway. Therefore, patients with HR-proficient tumors are likely to be resistant to these therapies and require alternative strategies.
Methods
Data from HGSOC patients in The Cancer Genome Atlas (TCGA) were analyzed for ATM status, ATM and PPARα expression, and used to perform Gene Set Enrichment Analysis (GSEA). Screening data from the Dependency Map were analyzed to identify FDA-approved drugs that preferentially inhibit ATM-low cancer cells. In vitro studies were performed to determine whether ATM inhibitors synergize with the PPARα agonist fenofibrate in HGSOC cell lines.
Results
The HR gene Ataxia Telangiectasia Mutated (ATM) is wildtype in the majority of HGSOC patients and its kinase activity is upregulated compared to normal fallopian tube tissue. As high ATM has been associated with poor overall and progression-free survival, targeting ATM may be beneficial for a subset of HGSOC patients. Clinical trials of ATM inhibitors are commencing; however, ATM inhibitors are not effective as single agents. We aimed to explore novel therapeutic vulnerabilities of ATM deficient cells to develop a combinatorial therapy. Using data from TCGA, we found that multiple pathways related to metabolism are inversely correlated with ATM expression, suggesting that combining ATM inhibition and metabolic inhibition would be effective. Indeed, analysis of FDA-approved drugs from the Dependency Map demonstrated that ATM low cell lines are more sensitive to fenofibrate, a PPARα agonist that has been previously shown to affect multiple cellular metabolic pathways. Consistently, PPARα signaling is associated with ATM expression. We validated that combined inhibition of ATM and treatment with fenofibrate is synergistic in multiple HGSOC cell lines by inducing senescence.
Conclusions
Our results suggest that metabolic changes induced by ATM inhibitors are a potential target for the treatment for HGSOC.
... The p-Akt (Thr308) levels were also considerably inhibited by the sorafenib-plus-CH12 combination treatment compared with the singleagent treatment. Recent studies indicated that sorafenib, as a single agent, could increase the Akt (Ser473) phosphorylation, and the Akt (Ser473) may be one of the main mediators of sorafenib resistance [23] [24]. Here we also detected the levels of p-Akt (Ser473). ...
... Recently, it has been proposed that Akt may be one of the main mediators of sorafenib resistance, although several studies have produced controversial results. Fujimaki et al. [24] revealed that sorafenib significantly decreased the levels of p-Akt (Thr308) and increased the levels of p-Akt (Ser473) in both HepG2 and PLC/PRF/5 cells. In the present study, our data illustrate that sorafenib alone or in combination with CH12 can downregulate the levels of p-Akt (Thr308) both in Huh-7–EGFRvIII and in SMMC- 7721 cells. ...
The multikinase inhibitor sorafenib is the first oral agent to show activity against human hepatocellular carcinoma (HCC). Although the clinical application of sorafenib has shown good tolerability in the studied populations, it also causes multiple human dose-limiting toxicities. Thus, there is a strong need to reduce the overall dose of sorafenib. We have reported that the epidermal growth factor receptor variant III (EGFRvIII) expression can decrease the sensitivity of HCC cells to chemotherapeutic drugs. Therefore, we sought to explore whether EGFRvIII can affect the sensitivity of HCC cells to sorafenib. In this study, we observed that EGFRvIII expression significantly decreased the sensitivity of HCC cells to sorafenib. To enhance the antitumor effect and reduce the overall dose of sorafenib, we evaluated the combined effects of CH12, a monoclonal antibody against EGFRvIII, and sorafenib on the growth of HCC cells expressing EGFRvIII in vitro and in vivo. The results showed that, when CH12 was combined with sorafenib, the tumor growth suppression effect was significantly increased, and the concentration of sorafenib required for growth inhibition was substantially reduced. Mechanistically, the combination could more noticeably downregulate the phosphorylation of constitutively active extracellular signal-regulated kinase (ERK), Akt (Thr308), and signal transducer and activator of transcription 3 (STAT3) than sorafenib alone. Collectively, these findings demonstrate that CH12 interacts additively with sorafenib to strongly inhibit the tumor growth of HCC xenografts expressing EGFRvIII by enhancing the sorafenib-mediated inhibition of the MEK/ERK, phosphoinositide 3-kinase/AKT, and STAT3 pathways.
... Aberrant AKT activation contributes to CSC features in HCC; however, the effect of siRNA against ATM in CSC has not been explored. 91 ...
Hepatocellular carcinoma (HCC) is the most common liver cancer. It is highly lethal and has high recurrence. Death among HCC patients occur mainly due to tumor progression, recurrence, metastasis, and chemoresistance. Cancer stem cells (CSCs) are cell subpopulations within the tumor that promote invasion, recurrence, metastasis, and drug resistance. Hepatic stellate cells (HSCs) are important components of the tumor microenvironment (TME) responsible for primary secretory ECM proteins during liver injury and inflammation. These cells promote fibrogenesis, infiltrate the tumor stroma, and contribute to HCC development. Interactions between HSC and CSC and their microenvironment help promote carcinogenesis through different mechanisms. This review summarizes the roles of CSCs and HSCs in establishing the TME in primary liver tumors and describes their involvement in HCC chemoresistance.
... Whether ATM similarly alters nutrient uptake and cellular metabolism in the context of cancers with mutated p53 and high c-MYC expression is unclear. Moreover, ATM inhibitors are currently undergoing clinical trials (Jin and Oh, 2019), although they are not generally effective as a monotherapy (Batey et al., 2013;Chen et al., 2020;Fujimaki et al., 2012;Golding et al., 2012;Jin and Oh, 2019;Riches et al., 2020). Thus, understanding how the inhibition of ATM drives metabolic reprogramming may be important toward identifying potential resistance mechanisms or other targets that could be used in combination with these inhibitors in ATM-WT tumors. ...
Macropinocytosis is a nonspecific endocytic process that may enhance cancer cell survival under nutrient-poor conditions. Ataxia-Telangiectasia mutated (ATM) is a tumor suppressor that has been previously shown to play a role in cellular metabolic reprogramming. We report that the suppression of ATM increases macropinocytosis to promote cancer cell survival in nutrient-poor conditions. Combined inhibition of ATM and macropinocytosis suppressed proliferation and induced cell death both in vitro and in vivo. Supplementation of ATM-inhibited cells with amino acids, branched-chain amino acids (BCAAs) in particular, abrogated macropinocytosis. Analysis of ATM-inhibited cells in vitro demonstrated increased BCAA uptake, and metabolomics of ascites and interstitial fluid from tumors indicated decreased BCAAs in the microenvironment of ATM-inhibited tumors. These data reveal a novel basis of ATM-mediated tumor suppression whereby loss of ATM stimulates protumorigenic uptake of nutrients in part via macropinocytosis to promote cancer cell survival and reveal a potential metabolic vulnerability of ATM-inhibited cells.
... According to recent reports, 60% of the anticancer medications in current use have been obtained from natural sources (Rawat et al., 2018). Dietary phytochemical caffeine has been found to be useful for treating HCC and other diseases (Fujimaki et al., 2012;Estari et al., 2021). As early as 1989, studies reported on the use of caffeine-potentiated chemotherapy (Kawano et al., 2012). ...
We have previously shown that adenosine A1AR antagonists, adenosine A2aAR antagonists, and caffeine have significant inhibitory effects on the activation and proliferation of hepatic stellate cells in alcoholic liver fibrosis. Many recent studies have found that moderate coffee consumption is beneficial for various liver diseases. The main active ingredient of coffee is caffeine, which is a natural non-selective adenosine receptor antagonist. Moreover, numerous preclinical epidemiological studies and clinical trials have examined the association between frequent coffee consumption and the risk of developing different liver diseases. In this review, we summarize and analyze the prophylactic and therapeutic effects of caffeine on various liver diseases, with an emphasis on cellular assays, animal experiments, and clinical trials. To review the prevention and treatment effects of caffeine on different liver diseases, we searched all literature before 19 July 2022, using “caffeine” and “liver disease” as keywords from the PubMed and ScienceDirect databases. We found that moderate coffee consumption has beneficial effects on various liver diseases, possibly by inhibiting adenosine binding to its receptors. Caffeine is a potential drug for the prevention and treatment of various liver diseases.
... Somatic mutations in ATM occur in many tumor types, particularly hematologic malignancies, and generally have been associated with inferior prognosis [71][72][73]. On the other hand, it has been reported that blockade of ATM improves the antitumor effects of sorafenib in HCC cells, with suppression of Akt signaling and significant potentiation of the cytotoxic effects [74,75]. Involvement of this gene in LEN treatment resistance is unknown, but it may play a role. ...
Background
There has been a recent surge in interest in predicting biological effects associated with genomic alterations in order to implement personalized cancer treatment strategies. However, no reports have yet evaluated the utility of profiling blood-based circulating tumor DNA (ctDNA) in hepatocellular carcinoma (HCC) patients treated with lenvatinib (LEN).
Method
We retrospectively performed ctDNA next-generation sequencing (NGS) analysis in 24 patients with advanced HCC at baseline and 4 weeks after initiation of LEN. Association of the changes in variant allele frequencies (VAFs) during treatment and clinical outcome were evaluated.
Results
In total, 131 single nucleotide variants, 17 indels, and 23 copy number variations were detected as somatic alterations in 28, 6, and 12 genes, respectively in 23 of 24 patients. The most frequently altered genes were TP53 (54%), CTNNB1 (42%), TERT (42%), ATM (25%), and ARID1A (13%). The reduction in the mean frequency of variants (VAF mean ) following 4 weeks of LEN treatment was associated with longer progression-free survival. The specificity and sensitivity of the reduction of VAF mean for predicting partial response were 0.67 and 1.0, respectively, which were higher than those of serum α-fetoprotein level (0.10 and 0.93, respectively). No association between the mutation status at baseline and the effectiveness of LEN was observed.
Conclusion
Our study demonstrated that somatic alterations could be detected in the majority of advanced HCC patients by ctDNA profiling and that ctDNA-kinetics during LEN treatment was a useful marker of disease progression. These results suggest that ctDNA profiling is a promising method that provides valuable information in clinical practice.
... In this regard, the present study demonstrates that direct targeting of ATM kinase by KU55933 induces apoptosis in cancer cells (Figures 5 and 6, Supplementary Figures S3-S6). Several studies also show that KU55933 can increase apoptosis in combination with radiation or chemotherapeutic drugs [29,[52][53][54]. Furthermore, our transcriptomic analysis suggests that KU55933 can modulate cellular responses to autophagy, cisplatin, glutathione and lipid metabolism, interferon, and many others. ...
ATM and BRCA1 are DNA repair genes that play a central role in homologous recombination repair. Alterations of ATM and BRCA1 gene expression are found in cancers, some of which are correlated with treatment response and patient outcome. However, the role of ATM and BRCA1 gene expression in head and neck cancer (HNC) is not well characterized. Here, we examined the prognostic role of ATM and BRCA1 expression in two HNC cohorts with and without betel quid (BQ) exposure. The results showed that the expression of ATM and BRCA1 was downregulated in BQ-associated HNC, as the BQ ingredient arecoline could suppress the expression of both genes. Low expression of either ATM or BRCA1 was correlated with poor overall survival (OS) and was an independent prognostic factor in multivariate analysis (ATM HR: 1.895, p = 0.041; BRCA1 HR: 2.163, p = 0.040). The combination of ATM and BRCA1 expression states further improved on the prediction of OS (HR: 4.195, p = 0.001, both low vs. both high expression). Transcriptomic analysis showed that inhibition of ATM kinase by KU55933 induced apoptosis signaling and potentiated cisplatin-induced cytotoxicity. These data unveil poor prognosis in the HNC patient subgroup with low expression of ATM and BRCA1 and support the notion of ATM-targeted therapy.
... Unfortunately, those unresectable patients are generally treated by systemic chemotherapy or radiotherapy, which is not enough to cure them. [3][4][5][6] The limited effectiveness of the current therapies for HCC is driven by a limited understanding of the underlying biology. An improved understanding will promote the discovery of novel and more effective targeted therapies for patients with HCC. ...
Aim:
Sphingosine-1-phosphate (S1P) and ceramide are bioactive sphingolipids known to be important in regulating numerous processes involved in cancer progression. The aim of this study was to determine the absolute levels of sphingolipids in hepatocellular carcinoma (HCC) utilizing data obtained from surgical specimens. In addition, we explored the clinical significance of S1P in HCC patients and the biological role of S1P in HCC cells.
Methods:
Tumors and normal liver tissues were collected from 20 HCC patients, and sphingolipids were measured by mass spectrometry. The Cancer Genome Atlas (TCGA) cohort was utilized to evaluate gene expression of enzymes related to sphingolipid metabolism. Immunohistochemistry of phospholyrated-SphK1, an S1P-producing enzyme, was performed for 61 surgical specimens. CRISPR/Cas9-mediated SphK1 knockout cells were used to examine HCC cell biology.
Results:
S1P levels were substantially higher in HCC tissue compared with normal liver tissue. Levels of other sphingolipids upstream of S1P in the metabolic cascade, such as sphingomyelin, monohexosylceramide and ceramide, were also considerably higher in HCC tissue. Enzymes involved in generating S1P and its precursor, ceramide, were found in higher levels in HCC compared with normal liver tissue. Immunohistochemical analysis found that phospholyrated-SphK1 expression was associated with tumor size. Finally, in vitro assays indicated that S1P is involved in the aggressiveness of HCC cells.
Conclusions:
Sphingolipid levels, including S1P and ceramide, were elevated in HCC compared with surrounding normal liver tissue. Our findings suggest S1P plays an important role in HCC tumor progression, and further examination is warranted. This article is protected by copyright. All rights reserved.
... The expression of ataxia telangiectasia mutant (ATM) protein kinase, which participates in the mechanisms of DNA repair through the HR pathway of double strand break repair, is also altered in HCC. Blocking ATM activity in HCC cell lines results in an enhanced antitumor effect of sorafenib, through the inhibition of the AKT pathway (MOC-5) [89]. ...
The poor outcome of patients with non-surgically removable advanced hepatocellular carcinoma (HCC), the most frequent type of primary liver cancer, is mainly due to the high refractoriness of this aggressive tumor to classical chemotherapy. Novel pharmacological approaches based on the use of inhibitors of tyrosine kinases (TKIs), mainly sorafenib and regorafenib, have provided only a modest prolongation of the overall survival in these HCC patients. The present review is an update of the available information regarding our understanding of the molecular bases of mechanisms of chemoresistance (MOC) with a significant impact on the response of HCC to existing pharmacological tools, which include classical chemotherapeutic agents, TKIs and novel immune-sensitizing strategies. Many of the more than one hundred genes involved in seven MOC have been identified as potential biomarkers to predict the failure of treatment, as well as druggable targets to develop novel strategies aimed at increasing the sensitivity of HCC to pharmacological treatments.
... The cells [42]. Moreover, in malignant glioma cells, the ATM-Akt signaling pathway could mediate cell migration, as discovered by Golding et al. [43].There was also evidence that a specific inhibitor of ATM (KU55933) could effectively inhibit cell migration induced by sorafenib, a chemotherapeutic agent, in HCC cells [44]. On the other hand, GSK-3β, which is a downstream gene in the AKT signaling pathway, can lead to the phosphorylation of the Snail transcription factor, thus regulating EMT and participating in tumorinvasion and metastasis [45][46][47].Additionally, Mianen and colleagues also demonstrated that ATM was highly activated in breast cancer tissues with advanced lymph node metastasis, and ATM was associated with the positive expression of Snail, which is an EMT-related molecule [48]. ...
Objective:
To investigate whether miR-203a-3p can regulate the biological behaviors of ovarian cancer cells by targeting ATM to affect the Akt/GSK-3β/Snail signaling pathway.
Methods:
The expression levels of miR-203a-3p and ATM were detected by qRT-PCR, immunohistochemical staining and Western blotting in ovarian cancer tissues and adjacent normal tissues obtained from 152 subjects. A dual-luciferase reporter gene assay was performed to verify the relationship between miR-203a-3p and ATM. Human ovarian cancer cell lines (A2780 and SKOV3) were used to generate the Blank, miR-NC, miR-203a-3p mimic, Control siRNA, ATM siRNA, and miR-203a-3p inhibitor + ATM siRNA groups. The biological behaviors of ovarian cancer cells were evaluated by CCK-8, wound healing, and Transwell invasion assays, annexin V-FITC/PI staining and flow cytometry. The levels of Akt/GSK-3β/Snail pathway-related proteins were assessed by Western blotting.
Results:
Ovarian cancer tissues showed lower miR-203a-3p levels and higher ATM levels than adjacent normal tissues, both of which were associated with the FIGO stage, grade and prognosis of ovarian cancer. As confirmed by a dual-luciferase reporter gene assay, miR-203a-3p could target ATM. Furthermore, the miR-203a-3p mimic had multiple effects, including the inhibition of the proliferation, invasion and migration of A2780 and SKOV3 cells, the promotion of cell apoptosis, the arrest of the cell cycle at the G1 phase, and the blockage of the Akt/GSK-3β/Snail signaling pathway. ATM siRNA had similar effects on the biological behaviors of ovarian cancer cells, and these effects could be reversed by a miR-203a-3p inhibitor.
Conclusion:
miR-203a-3p was capable of hindering proliferation, migration, and invasion and facilitating the apoptosis of ovarian cancer cells through its modulation of the Akt/GSK-3β/Snail signaling pathway by targeting ATM, and therefore it could serve as a potential therapeutic option for ovarian cancer.
... Activated AKT then promotes DNA repair (79) and inhibition of AKT decreases DNA repair (80,81). Consistently, pharmacological inhibition of ATM inhibits AKT phosphorylation and survival in multiple cancer types (82)(83)(84). These findings suggest a vital role for AKT in the maintenance of genome integrity, and inhibition of this DNA repair function may result in accumulation of DNA damage and cell death. ...
The ataxia-telangiectasia mutated (ATM) protein kinase has been extensively studied for its role in the DNA damage response and its association with the disease ataxia telangiectasia. There is increasing evidence that ATM also plays an important role in other cellular processes, including carbon metabolism. Carbon metabolism is highly dysregulated in cancer due to the increased need for cellular biomass. A number of recent studies report a non-canonical role for ATM in the regulation of carbon metabolism. This review highlights what is currently known about ATM’s regulation of carbon metabolism, the implication of these pathways in cancer, and the development of ATM inhibitors as therapeutic strategies for cancer.
... Another interesting but secondary finding of our study is the observation that 24 h of treatment with KU-55933 alone induced cell damage in cell lines of tumor origin (UN-SH-SY5Y and C6 glioma cells) when at concentrations above 10 μM (Tables 1 and 6) and increased the cell damage induced by various detrimental factors (St, Dox or H 2 O 2 ) in some of these cell types ( Fig. 1A and D; Fig. 6D). These results are in line with other reports demonstrating cytotoxic properties of the ATM inhibitor (10 μM) when given alone or in combination with chemotherapeutic agents in various tumor cell lines (HeLa cells, head and neck cancer cells, and melanoma, hepatoma, breast and prostate cancer cell lines) (Golding et al., 2007(Golding et al., , 2009Fujimaki et al., 2012;Hickson et al., 2004;Ivanov et al., 2009;Li and Yang, 2010;Lin et al., 2012) that could be used for future improvements in therapies for various types of tumors. ...
The role of the kinase ataxia-telangiectasia mutated (ATM), a well-known protein engaged in DNA damage repair, in the regulation of neuronal responses to oxidative stress remains unexplored. Thus, the neuroprotective efficacy of KU-55933, a potent inhibitor of ATM, against cell damage evoked by oxidative stress (hydrogen peroxide, H2O2) has been studied in human neuroblastoma SH-SY5Y cells and compared with the efficacy of this agent in models of doxorubicin (Dox)- and staurosporine (St)-evoked cell death. KU-55933 inhibited the cell death induced by H2O2 or Dox but not by St in undifferentiated (UN-) and retinoic acid-differentiated (RA)-SH-SY5Y cells, with a more pronounced effect in the latter cell phenotype. Furthermore, this ATM inhibitor attenuated the Dox- but not H2O2-induced caspase-3 activity in both UN- and RA-SH-SY5Y cells. Although KU-55933 inhibited the H2O2- and Dox-induced activation of ATM, it attenuated the toxin-induced phosphorylation of the proteins H2AX and p53 only in the latter model of cell damage. Moreover, the ATM inhibitor prevented the H2O2-evoked increases in calpain and cathepsin D activity and attenuated cell damage to a similar degree as inhibitors of calpain (MDL28170) and cathepsin D (pepstatin A). Finally, we confirmed the neuroprotective potential of KU-55933 against the H2O2- and Dox-evoked cell damage in primary mouse cerebellar granule cells and in the mouse hippocampal cell line HT-22. Altogether, our results extend the neuroprotective portfolio of KU-55933 to a model of oxidative stress, with this effect not involving inhibition of the γH2AX/p-p53/caspase-3 pathway and instead associated with the attenuation of calpain and cathepsin D activity.
... 8 Small molecule inhibitors of ATM kinase activity were reported to significantly enhance the cytotoxicity of Taxol and sorafenib. 9,10 Recently, we published genome-wide gene expression profiles of cirrhosis and HCC tissues, in comparison with senescence-and immortality-associated genes. 11 Among gene sets that are enriched in HCC, 13 were related to DNA repair, suggesting that a large set of genes involved in DNA repair are overexpressed in HCC. ...
Aim:
To investigate the expression of DNA repair genes and the impact of BRCA1 on chemoresistance of hepatocellular carcinoma (HCC).
Methods:
Microarray gene expression datasets were analyzed using the gene set enrichment analysis method. BRCA1 protein was tested by western blotting. Response of HCC cells to interstrand cross-links (ICL) was investigated by cell viability assay following exposure to Mitomycin-C (MMC), Cisplatin and Melphalan. Effects of BRCA1 ectopic expression were studied in HepG2 cells with BRCA1-expression plasmids. Effects of BRCA1 downregulation were studied in SNU449 cells with BRCA1-specific siRNAs. Response of transfected SNU449 cells to MMC was analyzed by cell viability tests and cell cycle analysis using flow cytometry.
Results:
Expression of Fanconi Anemia and double-strand DNA break repair genes was significantly upregulated in HCC tumors. This upregulation displayed a gradual amplification during tumor progression. BRCA1 and BRCA2 genes were among consistently upregulated genes. Epithelial-like HCC cells had low BRCA1 expression and low chemoresistance, whereas mesenchymal-like HCC cells had high BRCA1 expression and increased chemoresistance. Ectopic expression of BRCA1 increased the chemoresistance of epithelial-like HepG2 cells. Conversely, BRCA1 knock-down chemosensitized mesenchymal-like SNU449 cells. Chemosensitization of SNU449 cells was due to a cell cycle arrest at 4N stage.
Conclusions:
Increased expression of Fanconi Anemia and double-strand DNA repair genes such as BRCA1 is a novel mechanism of HCC chemoresistance. However, functional inactivation of BRCA1 expression is sufficient to reverse such chemoresistance. This article is protected by copyright. All rights reserved.
... Treatment of breast cancer cell lines with KU55933 resulted in an increase of sensitivity to IR and cis- platin, suggesting that ATM inhibition may suppress tumor resistance linked to overexpression of Aurora kinase A [69]. ATM inhibition by KU55933 might be a strategy for im- proving sorafenib treatment of hepatocellular carcinoma (HCC) [70]. Sorafenib is a multi-kinase inhibitor utilized in treatment of HCC but so far its therapeutic effects have been limited. ...
Genotoxic anticancer drugs explicate their effects damaging DNA, thus triggering a coordinated signal-transduction network called DNA Damage Response (DDR). Ataxia Telangiectasia Mutated (ATM) protein plays a central role in this response: activated by DNA damage, ATM phosphorylates itself and downstream effectors that arrest cell cycle allowing for DNA repair or, should DNA damage be too severe and not retrievable, inducing apoptosis. ATM is a worth-investigating target for tumor radio- and chemosensitization. During last years, pharmaceutical industries and research laboratories have developed a series of small molecules, capable to inhibit ATM with increasing specificity. Several preclinical studies have demonstrated that these inhibitors alone or in association with other treatments may improve therapeutic outcomes. In this review we discuss ATM inhibitors so far developed, focussing on recent acquisitions on their potential antineoplastic usefulness.
... ATM appears to have an additional role in ROS regulation, independent of its originally pro-posed role as a DNA damage sensor [26,27]. In the present study, H 2 O 2 treatment increased the phosphorylation of ATM in both HBXnegative and positive cells, and this increase was strongly suppressed by NAC. ...
Hepatitis B virus X (HBX) protein plays a crucial role in carcinogenesis, but its mechanism is unclear. The involvement of ataxia telangiectasia mutated (ATM) kinase in the enhanced redox system was investigated by examining the phosphorylation level of ATM in HBX gene-transfected cells and in transgenic mice following redox system manipulation by treatment with hydrogen peroxide (H2O2) or antioxidant. Western blotting and immunostaining showed that phospho-ATM was significantly increased by HBX both in vitro (3.2-fold; p<0.05) and in vivo (4-fold; p<0.05), and this effect was abrogated by antioxidant treatment. The level of PKC-δ in HBX-expressing cells was increased 3.5-fold compared to controls. Nuclear localized NF-E2-related factor 2 (Nrf2) was increased in HBX-expressing cells exposed to H2O2, but remained at lower levels after the treatment with rottlerin, KU55933, or caffeine. The levels of anti-oxidant molecules were increased in HBX expressing cells and in transgenic mice, indicating that HBX stimulates the Nrf2-mediated redox system. The levels of intracellular reactive oxygen species (ROS) were significantly increased in HBX-expressing cells treated with hydrogen peroxide in the presence of ATM inhibitor KU55933 or caffeine. Treatment of HBX-expressing cells with KU55933 or caffeine before the exposure to H2O2 increased the ratio of cell apoptosis to 33 ± 4% (p<0.05) and 22 ± 4% (p<0.05), respectively. Collectively, HBX stimulates the ATM-mediated PKC-δ/Nrf2 pathway, and maintains the enhanced activity of the redox system. Therefore, manipulating ATM kinase activity might be a useful strategy for treating HBX-induced carcinogenesis.
... Recently, it has been revealed that low-dose of sorafenib (<1 μM; non-cytotoxic concentration) stimulates cell migration through AKT signaling [18,19]. To examine whether VPA can ameliorate sorafenib-induced cell migration, cells were treated with low dose sorafenib (0.5 μM) with or without VPA or LY294002. ...
Sorafenib is a multi-kinase inhibitor approved for hepatocellular carcinoma, but rarely causes tumor regression in patients with chronic liver diseases. To investigate whether growth factor-mediated signaling is involved in sorafenib resistance, HepG2 and PLC/PRF/5 hepatoma cells were exposed to epidermal growth factor (EGF), hepatocyte growth factor (HGF) or transforming growth factor-β (TGF-β) prior to treatment with sorafenib. Furthermore, to identify an effective combination treatment with sorafenib, growth factor-sensitized cells were treated with sorafenib alone or in combination with celecoxib, lovastatin or valproic acid (VPA). Trypan blue staining and Annexin V assays showed that the cytotoxic effect of sorafenib was inhibited by 15-54% in cells sensitized to TGF-β (P<0.05). Western blotting analysis showed that TGF-β significantly activated extracellular signal-regulated kinase (ERK)-mediated AKT signaling, and sorafenib failed to suppress both ERK and AKT in TGF-β-sensitized cells. The decreased anti-tumor effect of sorafenib was rescued by chemical inhibition of ERK and AKT. When TGF-β-sensitized cells were treated with sorafenib plus VPA, the levels of phosphorylated ERK and AKT were considerably suppressed and the numbers of dead cells were increased by 3.7-5.7-fold compared with those exposed to sorafenib alone (P<0.05). Moreover, low dose sorafenib-induced cell migration was effectively suppressed by combination treatment with sorafenib and VPA. Collectively, TGF-β/ERK/AKT signaling might play a critical role in sorafenib resistance in hepatoma cells, and combination treatment with VPA may be effective against this drug resistance.
... Interestingly, it was reported that Mst-1 could suppress HCC growth by interacting with Yap1 [33]. The inhibition of Akt was reported to increase sorafenib sensitivity [34,35]. To the best of our knowledge, this is the first study to reveal the relationship between NORE1A expression and chemotherapeutic resistance in human cancer. ...
NORE1A, identified as a Ras effector, is frequently silenced in human cancers and has been implicated in tumour progression. Reports showing that NORE1A may function as a tumour suppressor have been emerging. However, to date, its expression and relevant significance in hepatocellular carcinoma (HCC) remain elusive. In this study, we examined the expression of NORE1A in HCC cell lines and a cohort of 250 HCC samples. We found that both the mRNA and the protein levels of NORE1A were noticeably downregulated in 14 fresh HCC tissues, compared to corresponding paracarcinoma tissues. Furthermore, NORE1A in tumours was decreased in 72.4 % (181/250) of HCC patients. Low NORE1A expression was significantly associated with poor differentiation (P = 0.003), advanced stage (P = 0.002), high level of serum AFP (P < 0.001), vascular invasion (P = 0.034) and incomplete involucrum (P = 0.018). Multivariate analysis revealed that NORE1A was an independent poor prognostic factor for both overall survival (hazard ratio (HR) 0.622, 95 % confidence interval (95 % CI) 0.405-0.956, P = 0.030) and recurrence-free survival (HR 0.613, 95 % CI 0.390-0.964, P = 0.034). Moreover, low NORE1A expression in advanced-stage HCC predicted disease relapse. In addition, NORE1A overexpression reduced cell viability, inhibited colony formation, and attenuated cell invasion in vitro. Further study demonstrated that NORE1A was capable of sensitising cancer cells to sorafenib-induced apoptosis via the activation of the Mst-1/Akt pathway. Collectively, our data suggest that NORE1A may be a promising prognostic biomarker and therapeutic target in HCC.
Drug resistance is one of the major challenges for treatment of hepatocellular carcinoma (HCC) with sorafenib. Our present study found that sorafenib resistant (SR) HCC cells showed epithelial-mesenchymal transition (EMT) characteristics with the downregulation of epithelial marker and upregulation of mesenchymal makers. The expression of Snail, a core factor of EMT, was increased in HCC/SR cells, while knockdown of Snail can restore sorafenib sensitivity and EMT potential of HCC/SR cells. Further, the upregulation of protein stability was responsible for the upregulation of Snail in HCC/SR cells. ATM and CSN2, which can stabilize Snail protein, were increased in HCC/SR cells. Knockdown of ATM and CSN2 can suppress the expression of Snail and increase sorafenib sensitivity of HCC/SR cells. It indicated that targeted inhibition of Snail might be helpful to overcome sorafenib resistance of HCC patients.
Background
Activin A receptor type 2A (ACVR2A) is one of the most frequently mutated genes in microsatellite instability-high (MSI-H) gastric cancer. However, the clinical relevance of the ACVR2A mutation in MSI-H gastric cancer patients remains unclear. The aims of this study were to explore the effect of ACVR2A mutation on the tumor behavior and to identify the clinicopathological characteristics of gastric cancer patients with ACVR2A mutations.Methods
An in vitro study was performed to investigate the biological role of ACVR2A via CRISPR/Cas9-mediated ACVR2A knockout MKN74 human gastric cancer cells. One hundred twenty-four patients with gastric cancer were retrospectively analyzed, and relations between MSI status, ACVR2A mutations, and clinicopathological factors were evaluated.ResultsACVR2A knockout cells showed less aggressive tumor biology than mock-transfected cells, displaying reduced proliferation, migration, and invasion (P < 0.05). MSI mutations were found in 10% (13/124) of gastric cancer patients, and ACVR2A mutations were found in 8.1% (10/124) of patients. All ACVR2A mutations were accompanied by MSI. The 5-year overall survival rates of ACVR2A wild-type patients and ACVR2A-mutated patients were 57% and 90%, respectively (P = 0.048). Multivariate analysis revealed that older age (P = 0.015), distant metastasis (P < 0.001), and ACVR2A wild-type status (P = 0.040) were independent prognostic factors for overall survival.Conclusions
Our study demonstrated that gastric cancer patients with ACVR2A mutation have a significantly better prognosis than those without. Dysfunction of ACVR2A in MKN74 human gastric cancer cells caused less aggressive tumor biology, indicating the importance of ACVR2A in the progression of MSI-H tumors.
Hepatocellular carcinoma is one of the most common malignant tumors in the world. Chronic hepatitis B and C infections are the most common etiologies of hepatocellular carcinoma worldwide. In this study, we explore the potential DNA damaging effect of some FDA-approved antiviral drugs which may be able to serve as anticancer agents for hepatocellular carcinoma, in order to better elucidate their mode of action. Five antiviral drugs were selected; ribavirin, sofosbuvir, tenofovir disoproxil fumerate, daclatasvir and ledipasvir. Several methods, including absorption spectroscopy, MALDI-TOF mass spectrometry and fluorimetric analysis using the EvaGreen (EG) intercalating dye, were used to probe the drug-induced DNA damage. Results show that only daclatasvir and ledipasvir induced DNA damage. Absorption spectroscopy showed hyperchromicity in the 260-nm DNA absorption band of DNA samples incubated with each drug, indicating disruption of the double-strand structure. Mass spectra for DNA samples incubated with each of the two drugs showed a disappearance of the DNA molecular ion peak with a concomitant appearance of peaks with smaller m/z, indicating DNA strand breaks. EG fluorescence was observed to decrease with increasing incubation time of daclatasvir and ledipasvir with DNA, indicating that the EG detaches from the DNA, likely due to DNA damage. All of these results are consistent with DNA damage, proposed as oxidative damage to both nucleobase and deoxyribose moieties of DNA as the mode of action for these two drugs. Moreover, these results are dependent on the antiviral drug concentration and show that DNA regions rich in guanine are affected more than other regions by these two drugs. Therefore, such antiviral drugs may present a promising therapeutic alternative to the currently used anticancer agents, especially for hepatitis B and C patients with hepatocellular carcinoma resistant to conventional treatment approaches.
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer mortality, accounting for almost 90% of total liver cancer burden. Surgical resection followed by adjuvant and systemic chemotherapy are the most meticulously followed treatment procedures but the complex etiology and high metastatic potential of the disease renders surgical treatment futile in majority of the cases. Another hindrance to the scenario is the acquired resistance to drugs resulting in relapse of the disease. Hence, to provide insights into development of novel therapeutic targets and diagnostic biomarkers, this review focuses on the various molecular mechanisms underlying chemoresistance in HCC. We have provided a comprehensive summary of the various strategies adopted by HCC cells, extending from apoptosis evasion, autophagy activation, drug expulsion to epigenetic transformation as modes of therapy resistance. The role of stem cells in imparting chemoresistance is also discussed. Furthermore, the review also focuses on how this knowledge might be exploited for the development of an effective, prospective therapy against HCC.
Background
To provide support for combined usage of phosphoinositide 3-kinase (PI3K) inhibitors or mitogen-activated protein kinase pathway inhibitors together with sorafenib in treatment of sorafenib-resistant hepatocellular carcinoma.
Materials and methods
The sorafenib-resistant cell lines were established to evaluate the effects of MK-2206 2HCL, a dual PI3K/mammalian target of rapamycin (mTOR) inhibitor, and PD0325901, an rat sarcoma (RAS) and/or extracellular signal-regulated kinase (ERK) inhibitor, on cell proliferation and apoptosis, as both single and combined treatments with sorafenib. In addition, multidrug resistance 1 gene expression, mutation status of key members in PI3K/mTOR, and RAS/ERK pathways and pathway activation were analyzed to identify predictors of drug response.
Results
Molecular studies reveal that combining MK-2206 2HCL or PD0325901 with sorafenib not only has a synergistic effect, in suppressing PI3K/protein kinase B/mTOR and RAS/MEK/ERK signaling more effectively than either treatment alone, but also prevents the cross activation of the other pathway that occurs with single treatments in both sorafenib sensitive and resistant lines. PD0325901 exhibited a stronger synergic effect with sorafenib than MK-2206 2HCL. Sorafenib-resistant cell lines were characterized by activation of both of the two pathways, as indicated by multidrug resistance 1 gene expression profiles and pathway activity analysis.
Conclusions
Our studies have showed that both inhibitors of PI3K/mTOR and RAS/ERK signaling are potentially effective antihepatocellular carcinoma drugs especially in treating sorafenib-resistant hepatocellular carcinoma.
Hepatocellular carcinoma (HCC) is one of the male-dominant liver diseases with poor prognosis, although treatments for HCC have been progressing in the past decades. Androgen receptor (AR) is a member of the nuclear receptor superfamily. Previous studies reported that AR was expressed in human HCC and non-HCC tissues. AR is activated both ligand-dependently and ligand-independently. The latter is associated with a mitogen-activated protein kinase–, v-akt murine thymoma viral oncogene homolog 1–, or signal-transducer and activator of transcription–signaling pathway, which has been implicated in the development of HCC. It has been reported that more than 200 RNA expression levels are altered by androgen treatment. In the liver, androgen-responsive genes are cytochrome P450s, transforming growth factor β, vascular endothelial growth factor, and glucose-regulated protein 78 kDa, which are also associated with human hepatocarcinogenesis. Recent studies also revealed that AR plays a role in cell migration and metastasis. It is possible that cross-talk among AR-signaling, endoplasmic reticulum stress, and innate immune response is important for human hepatocarcinogenesis and HCC development. This review shows that AR could play a potential role in human HCC and represent one of the important target molecules for the treatment of HCC.
Background and aim:
Transforming growth factor-β (TGF-β) has been shown to play a central role in the promotion of cell motility, but its functional mechanism has remained unclear. With the aim of investigating the diagnostic and treatment modalities for patients with hepatocellular carcinoma (HCC), the signaling pathway that may contribute to TGF-β-mediated cell invasion in hepatoma cells was evaluated.
Methods:
Three hepatoma cell lines, HepG2, PLC/PRF/5, and HLF, were treated with TGF-β, and the involvement of the non-canonical TGF-β pathway was analyzed by cell migration assays. HepG2 cells were treated with a p21-activated kinase-2 (PAK2)-targeting small interfering RNA and analyzed for their cell motility. The relationships between the PAK2 status and the clinicopathological characteristics of 62 HCC patients were also analyzed.
Results:
The cell migration assays showed that Akt is a critical regulator of TGF-β-mediated cell migration. Western blotting analyses showed that TGF-β stimulated Akt and PAK2 in all three hepatoma cell lines, and phosphorylated PAK2 was blocked by Akt inhibitor. Suppression of PAK2 expression by small interfering RNA resulted in increased focal adhesions with significantly repressed cell migration in the presence of TGF-β. Clinicopathological analyses showed that the phosphorylation level of PAK2 was closely associated with tumor progression, metastasis, and early recurrence of HCC.
Conclusions:
PAK2 may be a critical mediator of TGF-β-mediated hepatoma cell migration, and may represent a potential target for the treatment of HCC.
The ataxia-telangiectasia mutated (ATM) protein plays a central role in DNA damage response and cell cycle checkpoints, and may be a promising target for cancer therapy if normal tissue toxicity could be avoided. Our strategy to target ATM for breast cancer therapy involves the use of liposomal-encapsulated, gene-specific ATM small interfering RNA (siRNA) delivered with a well-characterized porous silicon-based multistage vector (MSV) delivery system (MSV/ATM). Here we have shown that biweekly treatment of MSV/ATM suppressed ATM expression in tumor tissues, and consequently inhibited growth of MDA-MB-231 orthotopic tumor in nude mice. At the therapeutic dosage, neither free liposomal ATM siRNA nor MSV/ATM triggered acute immune response in BALB/c mice, including changes in serum cytokines, chemokines or colony-stimulating factors. Weekly treatments of mice with free liposomal ATM siRNA or MSV/ATM for 4 weeks did not cause significant changes in body weight, hematology, blood biochemistry, or major organ histology. These results indicate that MSV/ATM is biocompatible and efficacious in inhibiting tumor growth, and that further preclinical evaluation is warranted for the development of MSV/ATM as a potential therapeutic agent.
Citation Format: Rong Xu, Yi Huang, Jun Hua Mai, Guo Dong Zhang, Xiao Jing Guo, Xiao Jun Xia, Eugene J Koay, Xue Wu Liu, Mauro Ferrari, Hai Fa Shen. Multistage vectored siRNA targeting ataxia-telangiectasia mutated for breast cancer therapy. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3206. doi:10.1158/1538-7445.AM2013-3206
Because Ataxia Telangiectasia Mutated (ATM)-deficient cells are hypersensitive to ionizing irradiation and DNA-damaging agents, ATM kinase inhibition is thought to enhance radiochemotherapy efficacy. In this study, we investigated the roles of autophagy and reactive oxygen species (ROS) in modulating cytotoxicity induced by suppression of ATM kinase in head and neck cancer cells.
We use KU55933 to inhibit ATM kinase activity. The cell viability was determined by MTT assays. Autophagy was examined by Western blot for LC3-II and microscopy for acidic vesicles and EGFP-LC3 punctate formation. DCF-DA staining and flow cytometry were used for analyzing ROS generation.
we found that KU55933 reduced cell viability in several head and neck cancer cell lines. KU55933-treated cells showed increased cytoplasmic vesicles, LC3-II accumulation, and EGFP-LC3 punctate formation, indicating that autophagy was induced. KU55933 also increased ROS generation, which was required for autophagy induction because the ROS scavenger N-acetyl-L-cysteine could reduce LC3-II accumulation. KU55933-induced autophagy played a cytoprotective role against ROS-mediated cytotoxicity because autophagy inhibition by chloroquine augmented KU55933's cytotoxicity. In addition, KU55933 reduced cisplatin-resistant head and neck cancer cell viabilities, and induced LC3-II accumulation in these cells.
Together, these results shed light on KU55933's therapeutic values as well as autophagy inhibitors in treating primary and cisplatin-resistant head and neck cancers.
No effective systemic therapy exists for patients with advanced hepatocellular carcinoma. A preliminary study suggested that sorafenib, an oral multikinase inhibitor of the vascular endothelial growth factor receptor, the platelet-derived growth factor receptor, and Raf may be effective in hepatocellular carcinoma.
In this multicenter, phase 3, double-blind, placebo-controlled trial, we randomly assigned 602 patients with advanced hepatocellular carcinoma who had not received previous systemic treatment to receive either sorafenib (at a dose of 400 mg twice daily) or placebo. Primary outcomes were overall survival and the time to symptomatic progression. Secondary outcomes included the time to radiologic progression and safety.
At the second planned interim analysis, 321 deaths had occurred, and the study was stopped. Median overall survival was 10.7 months in the sorafenib group and 7.9 months in the placebo group (hazard ratio in the sorafenib group, 0.69; 95% confidence interval, 0.55 to 0.87; P<0.001). There was no significant difference between the two groups in the median time to symptomatic progression (4.1 months vs. 4.9 months, respectively, P=0.77). The median time to radiologic progression was 5.5 months in the sorafenib group and 2.8 months in the placebo group (P<0.001). Seven patients in the sorafenib group (2%) and two patients in the placebo group (1%) had a partial response; no patients had a complete response. Diarrhea, weight loss, hand-foot skin reaction, and hypophosphatemia were more frequent in the sorafenib group.
In patients with advanced hepatocellular carcinoma, median survival and the time to radiologic progression were nearly 3 months longer for patients treated with sorafenib than for those given placebo. (ClinicalTrials.gov number, NCT00105443.)
Aberrant Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study reports how sorafenib (a multi-kinase inhibitor) and PI-103 (a dual PI3K/mTOR inhibitor) alone and in combination inhibit the proliferation of the HCC cell line, Huh7.
Huh7 proliferation was assayed by 3H-thymidine incorporation and by MTT assay. Western blot was used to detect phosphorylation of the key enzymes in the Ras/Raf and PI3K pathways.
Sorafenib and PI-103, as single agents inhibited Huh7 proliferation and epidermal growth factor (EGF)-stimulated Huh7 proliferation in a dose-dependent fashion; the combination of sorafenib and PI-103 produced synergistic effects. EGF increased phosphorylation of MEK and ERK, key Ras/Raf downstream signaling proteins; this activation was inhibited by sorafenib. However, sorafenib as a single agent increased AKT(Ser473) and mTOR phosphorylation. EGF-stimulated activation of PI3K/AKT/mTOR pathway components was inhibited by PI-103. PI-103 is a potent inhibitor of AKT(Ser473) phosphorylation; in contrast, rapamycin stimulated AKT(Ser473) phosphorylation. It was found that PI-103, as a single agent, stimulated MEK and ERK phosphorylation. However, the combination of sorafenib and PI-103 caused inhibition of all the tested kinases in the Ras/Raf and PI3K pathways.
The combination of sorafenib and PI-103 can significantly inhibit EGF-stimulated Huh7 proliferation by blocking both Ras/Raf/MAPK and PI3K/AKT/mTOR pathways.
Targeted therapy approaches have been successfully introduced into the treatment of several cancers. The multikinase inhibitor Sorafenib has antitumor activity in solid tumors and its effects on acute lymphoblastic leukemia (ALL) cells are still unclear.
ALL cell lines (SEM, RS4;11 and Jurkat) were treated with Sorafenib alone or in combination with cytarabine, doxorubicin or RAD001. Cell count, apoptosis and necrosis rates, cell cycle distribution, protein phosphorylation and metabolic activity were determined.
Sorafenib inhibited the proliferation of ALL cells by cell cycle arrest accompanied by down-regulation of CyclinD3 and CDK4. Furthermore, Sorafenib initiated apoptosis by cleavage of caspases 3, 7 and PARP. Apoptosis and necrosis rates increased significantly with most pronounced effects after 96 h. Antiproliferative effects of Sorafenib were associated with a decreased phosphorylation of Akt (Ser473 and Thr308), FoxO3A (Thr32) and 4EBP-1 (Ser65 and Thr70) as early as 0.5 h after treatment. Synergistic effects were seen when Sorafenib was combined with other cytotoxic drugs or a mTOR inhibitor emphasizing the Sorafenib effect.
Sorafenib displays significant antileukemic activity in vitro by inducing cell cycle arrest and apoptosis. Furthermore, it influences PI3K/Akt/mTOR signaling in ALL cells.
Ataxia-telangiectasia mutated (ATM) is a cellular damage sensor that coordinates the cell cycle with damage-response checkpoints and DNA repair to preserve genomic integrity. However, ATM also has been implicated in metabolic regulation, and ATM deficiency is associated with elevated reactive oxygen species (ROS). ROS has a central role in many physiological and pathophysiological processes including inflammation and chronic diseases such as atherosclerosis and cancer, underscoring the importance of cellular pathways involved in redox homeostasis. We have identified a cytoplasmic function for ATM that participates in the cellular damage response to ROS. We show that in response to elevated ROS, ATM activates the TSC2 tumor suppressor via the LKB1/AMPK metabolic pathway in the cytoplasm to repress mTORC1 and induce autophagy. Importantly, elevated ROS and dysregulation of mTORC1 in ATM-deficient cells is inhibited by rapamycin, which also rescues lymphomagenesis in Atm-deficient mice. Our results identify a cytoplasmic pathway for ROS-induced ATM activation of TSC2 to regulate mTORC1 signaling and autophagy, identifying an integration node for the cellular damage response with key pathways involved in metabolism, protein synthesis, and cell survival.
Multiple myeloma is characterized by increased bone marrow neovascularization driven in part by vascular endothelial growth factor (VEGF). In addition, the Ras/Raf/MEK/ERK pathway is critical for the proliferation of myeloma cells and is often upregulated. Sorafenib (Nexavar) is a novel multi-kinase inhibitor that acts predominantly through inhibition of Raf-kinase and VEGF receptor 2, offering the potential for targeting two important aspects of disease biology. In in vitro studies, sorafenib-induced cytotoxicity in MM cell lines as well as freshly isolated patient myeloma cells. It retained its activity against MM cells in co-culture with stromal cells or with interleukin-6, VEGF or IGF; conditions mimicking tumor microenvironment. Examination of cellular signaling pathways showed downregulation of Mcl1 as well as decreased phosphorylation of the STAT3 and MEK/ERK, as potential mechanisms of its anti-tumor effect. Sorafenib induces reciprocal upregulation of Akt phosphorylation; and simultaneous inhibition of downstream mTOR with rapamycin leads to synergistic effects. Sorafenib also synergizes with drugs such as proteasome inhibitors and steroids. In a human in vitro angiogenesis assay, sorafenib showed potent anti-angiogenic activity. Sorafenib, through multiple mechanisms exerts potent anti-myeloma activity and these results favor further clinical evaluation and development of novel sorafenib combinations.
Gastric cancer is a deadly disease for which current therapeutic options are extremely limited. Vascular endothelial growth factor receptors and platelet-derived growth factor receptors regulate gastric cancer cell proliferation, invasion, and tumor angiogenesis. In the present study, we report that sorafenib therapy effectively inhibited tumor growth and angiogenesis in tumor xenografts. These were associated with reduction in the phosphorylation of vascular endothelial growth factor receptor-2 Tyr951, c-Kit Tyr568/570, platelet-derived growth factor receptor-beta Tyr1021, and Akt Ser473 and Thr308, down-regulation of positive cell cycle regulators, increased apoptosis, and up-regulation of p27. Sorafenib treatment also caused up-regulation of p-c-Raf Ser338 and p-extracellular signal-regulated kinase (ERK) Thr202/Tyr204 in gastric cancer xenografts. The combination of sorafenib and MAP/ERK kinase inhibitor AZD6244 enhances the effectiveness of each compound alone. Potential effect of sorafenib/AZD6244 included increase in proapoptotic Bim. Our data show that MAP/ERK kinase inhibition enhances the antitumor activity of sorafenib in vivo, supporting a rationale for multitargeted suppression of the angiogenesis and ERK signaling network in gastric cancer therapy.
ATM and ATR protein kinases play a crucial role in cellular DNA damage responses. The inhibition of ATM and ATR can lead to
the abolition of the function of cell cycle checkpoints. In this regard, it is expected that checkpoint inhibitors can serve
as sensitizing agents for anti-cancer chemo/radiotherapy. Although several ATM inhibitors have been reported, there are no
ATR-specific inhibitors currently available. Here, we report the inhibitory effect of schisandrin B (SchB), an active ingredient
of Fructus schisandrae, on ATR activity in DNA damage response. SchB treatment significantly decreased the viability of A549 adenocarcinoma cells
after UV exposure. Importantly, SchB treatment inhibited both the phosphorylation levels of ATM and ATR substrates, as well
as the activity of the G2/M checkpoint in UV-exposed cells. The protein kinase activity of immunoaffinity-purified ATR was
dose-dependently decreased by SchB in vitro (IC50: 7.25 μM), but the inhibitory effect was not observed in ATM, Chk1, PI3K, DNA-PK, and mTOR. The extent of UV-induced phosphorylation
of p53 and Chk1 was markedly reduced by SchB in ATM-deficient but not siATR-treated cells. Taken together, our demonstration
of the ability of SchB to inhibit ATR protein kinase activity following DNA damage in cells has clinical implications in anti-cancer
therapy.
Sorafenib, a vascular endothelial growth factor (VEGF) receptor-2 and RAF kinase inhibitor, commonly causes skin toxicity. We retrospectively analyzed dermatologic toxicity in patients receiving combined antiangiogenic therapy involving sorafenib and bevacizumab.
Castration-resistant prostate cancer and metastatic non-small cell lung cancer patients were accrued to phase II studies, receiving sorafenib 400 mg twice daily. A phase I study explored sorafenib 200 to 400 mg twice daily with bevacizumab 5 to 10 mg/kg every 2 weeks in patients with advanced solid tumors. The probability of development of maximum grade of dermatologic toxicity as a function of the cumulative dose of sorafenib was determined. Additional analyses compared extent of toxicity, pharmacokinetics, and patient risk factors.
Ninety-six patients were enrolled: 54 received sorafenib and 42 received bevacizumab/sorafenib. Hand-foot skin reaction (HFSR) was observed in 50 of 96 (52%) patients. Grade 2 to 3 HFSR developed in 16 of 54 (30%) sorafenib patients and 24 of 42 (57%) bevacizumab/sorafenib patients (P=0.012) and was associated with cumulative sorafenib exposure (P=0.0008). Twenty-four of 42 phase I patients randomized to start with bevacizumab had increased risk of grade 2 to 3 HFSR than those starting with sorafenib (P=0.013) after adjusting for association between HFSR risk and hypertension (P=0.01), which was the only toxicity associated with HFSR. There was no association between HFSR and baseline history of neuropathy, prior taxane/platinum treatment, or systemic sorafenib levels.
Sorafenib-related HFSR is associated with increasing cumulative sorafenib dose. HFSR is increased in patients treated with bevacizumab/sorafenib combination anti-VEGF therapy, and this finding is not explained by pharmacokinetic interaction between the two agents. Our results suggest that the pathophysiology of HFSR may be related to VEGF inhibition.
The anti-metabolite 5-fluorouracil (5-FU) is employed clinically to manage solid tumors including colorectal and breast cancer. Intracellular metabolites of 5-FU can exert cytotoxic effects via inhibition of thymidylate synthetase, or through incorporation into RNA and DNA, events that ultimately activate apoptosis. In this review, we cover the current data implicating DNA repair processes in cellular responsiveness to 5-FU treatment. Evidence points to roles for base excision repair (BER) and mismatch repair (MMR). However, mechanistic details remain unexplained, and other pathways have not been exhaustively interrogated. Homologous recombination is of particular interest, because it resolves unrepaired DNA intermediates not properly dealt with by BER or MMR. Furthermore, crosstalk among DNA repair pathways and S-phase checkpoint signaling has not been examined. Ongoing efforts aim to design approaches and reagents that (i) approximate repair capacity and (ii) mediate strategic regulation of DNA repair in order to improve the efficacy of current anticancer treatments.
Although patients with advanced refractory solid tumors have poor prognosis, the clinical development of targeted protein kinase inhibitors offers hope for the future treatment of many cancers. In vivo and in vitro studies have shown that the oral multikinase inhibitor, sorafenib, inhibits tumor growth and disrupts tumor microvasculature through antiproliferative, antiangiogenic, and/or proapoptotic effects. Sorafenib has shown antitumor activity in phase II/III trials involving patients with advanced renal cell carcinoma and hepatocellular carcinoma. The multiple molecular targets of sorafenib (the serine/threonine kinase Raf and receptor tyrosine kinases) may explain its broad preclinical and clinical activity. This review highlights the antitumor activity of sorafenib across a variety of tumor types, including renal cell, hepatocellular, breast, and colorectal carcinomas in the preclinical setting. In particular, preclinical evidence that supports the different mechanisms of action of sorafenib is discussed.
The RAS/RAF signaling pathway is an important mediator of tumor cell proliferation and angiogenesis. The novel bi-aryl urea BAY 43-9006 is a potent inhibitor of Raf-1, a member of the RAF/MEK/ERK signaling pathway. Additional characterization showed that BAY 43-9006 suppresses both wild-type and V599E mutant BRAF activity in vitro. In addition, BAY 43-9006 demonstrated significant activity against several receptor tyrosine kinases involved in neovascularization and tumor progression, including vascular endothelial growth factor receptor (VEGFR)-2, VEGFR-3, platelet-derived growth factor receptor beta, Flt-3, and c-KIT. In cellular mechanistic assays, BAY 43-9006 demonstrated inhibition of the mitogen-activated protein kinase pathway in colon, pancreatic, and breast tumor cell lines expressing mutant KRAS or wild-type or mutant BRAF, whereas non-small-cell lung cancer cell lines expressing mutant KRAS were insensitive to inhibition of the mitogen-activated protein kinase pathway by BAY 43-9006. Potent inhibition of VEGFR-2, platelet-derived growth factor receptor beta, and VEGFR-3 cellular receptor autophosphorylation was also observed for BAY 43-9006. Once daily oral dosing of BAY 43-9006 demonstrated broad-spectrum antitumor activity in colon, breast, and non-small-cell lung cancer xenograft models. Immunohistochemistry demonstrated a close association between inhibition of tumor growth and inhibition of the extracellular signal-regulated kinases (ERKs) 1/2 phosphorylation in two of three xenograft models examined, consistent with inhibition of the RAF/MEK/ERK pathway in some but not all models. Additional analyses of microvessel density and microvessel area in the same tumor sections using antimurine CD31 antibodies demonstrated significant inhibition of neovascularization in all three of the xenograft models. These data demonstrate that BAY 43-9006 is a novel dual action RAF kinase and VEGFR inhibitor that targets tumor cell proliferation and tumor angiogenesis.
The gene mutated in ataxia telangiectasia, ATM, has been implicated in several cell functions such as cell cycle control and response to DNA damage and insulin. PKB/Akt
has also been implicated in the cellular response to insulin, γ-radiation, and cell cycle control. Interestingly, lack of
PKB/Akt function in vivo is able to mimic some phenotypic abnormalities associated with ataxia telangiectasia (AT). Here we show that ATM is a major
determinant of full PKB/Akt activation in response to insulin or γ-radiation. This effect is mediated through the phosphatidylinositol
3-kinase domain of ATM that specifically affects Akt serine 473 phosphorylation. This conclusion was inferred from the results
obtained in transient transfection assays using exogenous PKB/Akt and ATM in Cos cells. Moreover, the use of ATM inhibitors
or small interfering RNA confirmed our observation. Further supporting these results, we also observed that biological responses
tightly regulated by Akt, such as transcription factor of the forkhead family activity after insulin treatment or γ-radiation
response, were altered in cell lines derived from AT patients and knockout mice for ATM in which phosphorylation in serine
473 was almost abolished. This study proposes new clues in the search of the unknown PDK2 and new explanations for the radiosensitivity
or insulin intolerance described more than 30 years ago in AT patients.
The serine/threonine protein kinase ATM signals to cell cycle and DNA repair components by phosphorylating downstream targets such as p53, CHK2, NBS1, and BRCA1. Mutation of ATM occurs in the human autosomal recessive disorder ataxia-telangiectasia, which is characterized by hypersensitivity to ionizing radiation and a failure of cells to arrest the cell cycle after the induction of DNA double-strand breaks. It has thus been proposed that ATM inhibition would cause cellular radio- and chemosensitization. Through screening a small molecule compound library developed for the phosphatidylinositol 3'-kinase-like kinase family, we identified an ATP-competitive inhibitor, 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (KU-55933), that inhibits ATM with an IC(50) of 13 nmol/L and a Ki of 2.2 nmol/L. KU-55933 shows specificity with respect to inhibition of other phosphatidylinositol 3'-kinase-like kinases. Cellular inhibition of ATM by KU-55933 was demonstrated by the ablation of ionizing radiation-dependent phosphorylation of a range of ATM targets, including p53, gammaH2AX, NBS1, and SMC1. KU-55933 did not show inhibition of UV light DNA damage induced cellular phosphorylation events. Exposure of cells to KU-55933 resulted in a significant sensitization to the cytotoxic effects of ionizing radiation and to the DNA double-strand break-inducing chemotherapeutic agents, etoposide, doxorubicin, and camptothecin. Inhibition of ATM by KU-55933 also caused a loss of ionizing radiation-induced cell cycle arrest. By contrast, KU-55933 did not potentiate the cytotoxic effects of ionizing radiation on ataxia-telangiectasia cells, nor did it affect their cell cycle profile after DNA damage. We conclude that KU-55933 is a novel, specific, and potent inhibitor of the ATM kinase.
The phosphatidylinositol 3-kinase/AKT pathway is activated frequently in human cancer, and it has been implicated in tumor cell proliferation, survival, and chemoresistance. In this study, we addressed the role of AKT in cellular responses to the therapeutic methylating agent temozolomide (TMZ), and we investigated the possible link between TMZ-induced modulation of AKT function and activation of ataxia-telangiectasia and Rad3-related (ATR)- and ataxia telangiectasia mutated (ATM)-dependent signaling pathways. We found that clinically relevant concentrations of TMZ caused activation of endogenous AKT in lymphoblastoid cells, and in colon and breast cancer cells, and that this molecular event required a functional mismatch repair system. Transfection of a dominant-negative kinase-dead form of AKT1 into breast cancer cells abrogated TMZ-induced activation of endogenous AKT, and it markedly enhanced cell sensitivity to the drug. Likewise, exposure of the MMR-proficient cell lines to the AKT inhibitor D-3-deoxy-2-O-methyl-myo inositol 1-[(R)-2-methoxy-3-(octadecyloxy)-propyl hydrogen phosphate] (SH-5) impaired AKT phosphorylation in response to TMZ, and it significantly increased cell chemosensitivity. Furthermore, small interfering RNA (siRNA)-mediated reduction of AKT1 expression in colon cancer cells potentiated the growth inhibitory effects of TMZ. Inhibition of ATM expression in colon cancer cells by siRNA did not impair TMZ-induced activation of AKT, whereas siRNA-mediated inhibition of ATR prevented AKT activation in response to the drug and increased cell chemosensitivity. These results strongly support the hypothesis that clinical benefit could be obtained by combining TMZ with inhibitors of the AKT pathway. Moreover, they provide the first evidence of a novel function of ATR as an upstream activator of AKT in response to DNA damage induced by O(6)-guanine-methylating agents.
We investigated the effects of methylxanthines on enzymatic activity of phosphoinositide 3-kinases (PI3Ks). We found that caffeine inhibits the in vitro lipid kinase of class I PI3Ks (IC(50) = 75 microm for p110 delta, 400 microm for p110 alpha and p110 beta, and 1 mm for p110 gamma), and theophylline has similar effects (IC(50) = 75 microm for p110 delta, 300 microm for p110 alpha, and 800 microm for p110 beta and p110 gamma) and also inhibits the alpha isoform of class II PI3K (PI3K-C2 alpha) (IC(50) approximately 400 microm). However, four other xanthine derivatives tested (3-isobutyl-1-methylxanthine, 3-propylxanthine, alloxazine, and PD116948 (8-cyclopentyl-1,3-dipropylxanthine)) were an order of magnitude less effective. Surprisingly the triazoloquinazoline CGS15943 (9-chloro-2-(2-furyl)(1,2,d)triazolo(1,5-c)quinazolin-5-amine) also selectively inhibits p110 delta (IC(50) < 10 microm). Caffeine and theophylline also inhibit the intrinsic protein kinase activity of the class IA PI3Ks and DNA-dependent protein kinase, although with a much lower potency than that for the lipid kinase (IC(50) approximately 10 mm for p110 alpha, 3 mm for p110 beta, and 10 mm for DNA-dependent protein kinase). In CHO-IR cells and rat soleus muscle, theophylline and caffeine block the ability of insulin to stimulate protein kinase B with IC(50) values similar to those for inhibition of PI3K activity, whereas insulin stimulation of ERK1 or ERK2 was not inhibited at concentrations up to 10 mm. Theophylline and caffeine also blocked insulin stimulation of glucose transport in CHO-IR cells. These results demonstrate that these methylxanthines are direct inhibitors of PI3K lipid kinase activity but are distinctly less effective against serine kinase activity and thus could be of potential use in dissecting these two distinct kinase activities. Theophylline, caffeine, and CGS15943 may be of particular use in dissecting the specific role of the p110 delta lipid kinase. Finally, we conclude that inhibition of PI3K (p110 delta in particular) is likely explain some of the physiological and pharmacological properties of caffeine and theophylline.
Caffeine is a major biologically active constituent in coffee and tea. Because caffeine has been reported to inhibit carcinogenesis in UVB-exposed mice, the cancer-preventing effect of caffeine has attracted considerable attention. In the present study, the effect of caffeine in quiescent (G0 phase) cells was investigated. Pretreatment with caffeine suppressed cell proliferation in a dose-dependent manner 36 h after addition of fetal bovine serum as a cell growth stimulator. Analysis by flow cytometry showed that caffeine suppressed cell cycle progression at the G0/G1 phase, i.e., 18 h after addition of fetal bovine serum, the percentages of cells in G0/G1 phase in 1 mm caffeine-treated cells and in caffeine-untreated cells were 61.7 and 29.0, respectively. The percentage of cells in G0/G1 phase at 0 h was 75.5. Caffeine inhibited phosphorylation of retinoblastoma protein at Ser780 and Ser807/Ser811, the sites where retinoblastoma protein has been reported to be phosphorylated by cyclin-dependent kinase 4 (cdk4). Furthermore, caffeine inhibited the activation of the cyclin D1-cdk4 complex in a dose-dependent manner. However this compound did not directly inhibit the activity of this complex. In addition, caffeine did not affect p16INK4 or p27Kip1 protein levels, but inhibited the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3β. Our results showed that caffeine suppressed the progression of quiescent cells into the cell cycle. The inhibitory mechanism may be due to the inhibition of cell growth signal-induced activation of cdk4, which may be involved in the inhibition of carcinogenesis in vivo.
Activation of the protein p70s6k by mitogens leads to increased translation of a family of messenger RNAs that encode essential components of the protein
synthetic apparatus. Activation of the kinase requires hierarchical phosphorylation at multiple sites, culminating in the
phosphorylation of the threonine in position 229 (Thr229), in the catalytic domain. The homologous site in protein kinase B (PKB), Thr308, has been shown to be phosphorylated by the phosphoinositide-dependent protein kinase PDK1. A regulatory link between p70s6k and PKB was demonstrated, as PDK1 was found to selectively phosphorylate p70s6k at Thr229. More importantly, PDK1 activated p70s6k in vitro and in vivo, whereas the catalytically inactive PDK1 blocked insulin-induced activation of p70s6k.
Hepatocellular carcinoma (HCC) has an aggressive clinical course with frequent recurrence and metastasis. Orthotopic liver transplantation has been the only curative tool for unresectable HCC; therefore, recent advances in molecular targeted therapy may improve the prognosis of HCC. The multiple kinase inhibitor sorafenib and the macrolide antibiotic rapamycin are currently the most promising agents for treating unresectable HCC. A large population-based clinical trial revealed that sorafenib significantly prolonged the overall survival of HCC patients. However, subsequent clinical studies showed that sorafenib rarely reduced tumor volume and inadequately prolonged survival of patients with severe liver damage. To improve its therapeutic effect, the development of a predictive biomarker and a sorafenib-based combination is awaited. Another molecular targeting agent, rapamycin, has now been considered as a putative agent for preventing tumor recurrence in post-liver transplantation HCC patients, because it not only has immunosuppressive activity but also exerts an anti-tumor effect. In the near future, a combination of molecular targeting agents, such as sorafenib and rapamycin, may become a standard protocol for treating unresectable HCC. For specifying cases with more effective and less harmful modalities, further investigation in clinical and basic research to identify unexpected effects are needed.
Hepatocellular carcinoma (HCC) is one of the most common potentially lethal human malignancies worldwide. Sorafenib, a tyrosine kinase inhibitor, was recently approved by the United States Food and Drug Administration for HCC. In this study, we established two sorafenib-resistant HCC cell lines from Huh7, a human HCC cell line, by long-term exposure of cells to sorafenib. Sorafenib induced significant apoptosis in Huh7 cells; however, Huh7-R1 and Huh7-R2 showed significant resistance to sorafenib-induced apoptosis at the clinical relevant concentrations (up to 10 μM). Thorough comparisons of the molecular changes between Huh7 and resistant cells showed that the phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway played a significant role in mediating acquired resistance to sorafenib in Huh7-R1 and Huh7-R2 cells. Phospho-Akt and p85 (a regulatory subunit of PI3K) were up-regulated, whereas tumor suppressor phosphatase and tensin homolog were down-regulated in these resistant cells. In addition, ectopic expression of constitutive Akt in Huh7 demonstrated similar resistance to sorafenib. The knockdown of Akt by RNA interference reversed resistance to sorafenib in Huh7-R1 cells, indicating the importance of Akt in drug sensitivity. Furthermore, the combination of 8-[4-(1-aminocyclobutyl)phenyl]-9-phenyl-1,2,4-triazolo[3,4-f][1,6]naphthyridin-3(2H)-one dihydrochloride (MK-2206), a novel allosteric Akt inhibitor, and sorafenib restored the sensitivity of resistant cells to sorafenib-induced apoptosis. In conclusion, activation of PI3K/Akt signaling pathway mediates acquired resistance to sorafenib in HCC, and the combination of sorafenib and MK-2206, an Akt inhibitor, overcomes the resistance at clinical achievable concentrations.
In a randomized phase 3 trial, 400 mg of sorafenib twice daily prolonged overall survival of patients with advanced hepatocellular carcinoma (HCC) and Child-Pugh A disease. In a phase 1 study, sorafenib combined with doxorubicin, 60 mg/m(2), was well tolerated by patients with refractory solid tumors. The combination of sorafenib and doxorubicin in patients with advanced HCC has not been evaluated in a phase 2 or 3 trial.
To evaluate the efficacy and safety of doxorubicin plus sorafenib compared with doxorubicin alone in patients with advanced HCC and Child-Pugh A disease.
In a double-blind phase 2 multinational study, conducted from April 2005 to October 2006, 96 patients (76% male; median age, 65 years [range, 38-82 years]) with advanced HCC, Eastern Cooperative Oncology Group performance status 0 to 2, Child-Pugh A status, and no prior systemic therapy were randomly assigned to receive 60 mg/m(2) of doxorubicin intravenously every 21 days plus either 400 mg of sorafenib or placebo orally twice a day. The date of the last patient's follow-up was April 2008.
Time to progression as determined by independent review.
Following complete accrual, an unplanned early analysis for efficacy was performed by the independent data monitoring committee, so the trial was halted. The 2 patients remaining in the placebo group at that time were offered sorafenib. Based on 51 progressions, 63 deaths, and 70 events for progression-free survival, median time to progression was 6.4 months in the sorafenib-doxorubicin group (95% confidence interval [CI], 4.8-9.2), and 2.8 months (95% CI, 1.6-5) in the doxorubicin-placebo monotherapy group (P = .02). Median overall survival was 13.7 months (95% CI, 8.9--not reached) and 6.5 months (95% CI, 4.5-9.9; P = .006), and progression-free survival was 6.0 months (95% CI, 4.6-8.6) and 2.7 months (95% CI, 1.4-2.8) in these groups, respectively (P = .006). Toxicity profiles were similar to those for the single agents.
Among patients with advanced HCC, treatment with sorafenib plus doxorubicin compared with doxorubicin monotherapy resulted in greater median time to progression, overall survival, and progression-free survival. The degree to which this improvement may represent synergism between sorafenib and doxorubicin remains to be defined. The combination of sorafenib and doxorubicin is not yet indicated for routine clinical use.
clinicaltrials.gov Identifier: NCT00108953.
Sorafenib is an inhibitor of several intracellular signalling kinases with anti-proliferative, anti-angiogenic and pro-apoptotic effects in tumour cells. Sorafenib is used in the therapy of advanced renal cell carcinoma, and several phase II clinical trials are being carried out in patients with urothelial carcinomas.
Using a panel of human bladder cancer cell lines (RT4, T24, J82), we characterized systematically the effects of sorafenib on intracellular signalling, migration, proliferation and apoptosis.
We demonstrated that at low concentrations (<1 microM), sorafenib is capable of significantly stimulating migration and proliferation of the bladder cancer cells. We hypothesize that these stimulatory effects on tumour cell functions might be explained by an activation of the Ras/ERK-1/2 signal transduction pathway. In addition, the comparison of different bladder cancer cell lines not only revealed a different biology (e.g. cell migration), but also a differential susceptibility to the anti-apoptotic effects of sorafenib. Finally, we confirmed in different bladder cancer cell lines the known inhibitory actions of sorafenib in pharmacological concentrations (> or =3 microM) on ERK-1/2 phosphorylation, migration and proliferation, as well as the pro-apoptotic effects of the compound.
Taken together, these findings suggest that although sorafenib has the potential to be used in the treatment of urothelial carcinoma, this compound might also activate bladder cancer cells at low concentrations. This should be relevant for dosing regiments to optimize the treatment with this promising anti-tumour drug.
Hepatocellular carcinoma (HCC) is a highly prevalent, treatment-resistant malignancy with a multifaceted molecular pathogenesis. Current evidence indicates that during hepatocarcinogenesis, two main pathogenic mechanisms prevail: (1) cirrhosis associated with hepatic regeneration after tissue damage caused by hepatitis infection, toxins (for example, alcohol or aflatoxin) or metabolic influences, and (2) mutations occurring in single or multiple oncogenes or tumor suppressor genes. Both mechanisms have been linked with alterations in several important cellular signaling pathways. These pathways are of interest from a therapeutic perspective, because targeting them may help to reverse, delay or prevent tumorigenesis. In this review, we explore some of the major pathways implicated in HCC. These include the RAF/MEK/ERK pathway, phosphatidylinositol-3 kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, WNT/beta-catenin pathway, insulin-like growth factor pathway, hepatocyte growth factor/c-MET pathway and growth factor-regulated angiogenic signaling. We focus on the role of these pathways in hepatocarcinogenesis, how they are altered, and the consequences of these abnormalities. In addition, we also review the latest preclinical and clinical data on the rationally designed targeted agents that are now being directed against these pathways, with early evidence of success.
Hepatocellular carcinoma (HCC) is a global health problem, although developing countries are disproportionally affected: over 80% of HCCs occur in such regions. About three-quarters of HCCs are attributed to chronic HBV and HCV infections. In areas endemic for HCV and HBV, viral transmission occurs at an early age, and infected individuals develop HCC in mid-adulthood. As these are their most productive years of life, HCC accounts for a substantial burden on the health-care system and drain of productive capacity in the low-income and middle-income countries most affected by HCV and HBV infections. Environments with disparate resource levels require different strategies for the optimal management of HCC. In high-resource environments, guidelines from the American Association for the Study of Liver Diseases or European Association for the Study of the Liver should be applied. In intermediate-resource or low-resource environments, the fundamental focus should be on primary prevention of HCC, through universal HBV vaccination, taking appropriate precautions and antiviral treatments. In intermediate-resource and low-resource environments, the infrastructure and capacity for abdominal ultrasonography, percutaneous ethanol injection, radiofrequency ablation and surgical resection should be established. Programs to provide targeted therapy at low cost, similar to the approach used for HIV therapy in the developing world, should be pursued.
Sorafenib is a multikinase inhibitor that has been reported to induce cell growth inhibition through the Raf-MAPK signaling pathway. We now report that Sorafenib treatment of Hep3B and PLC/PRF/5 human hepatoma cells also results in morphological changes and cell detachment in culture. Actin cytoskeletal analysis of Sorafenib-exposed Hep3B cells showed a loss of polymerized F-actin and a concomitant increase in unpolymerized G-actin, implying that Sorafenib-induced cell shape changes may be related to actin cytoskeletal rearrangement by inhibiting actin polymerization. Cofilin, an actin depolymerization factor, was found to be dephosphorylated and thus activated by Sorafenib, consistent with the observed increase in unpolymerized G-actin. In examining likely mechanisms, we found that Sorafenib induced activation of the cofilin phosphatase Slingshot 1 (SSH-1), since endogenous SSH-1 from Sorafenib-treated Hep3B cells was able to dephosphorylate cofilin in a concentration dependent manner. The activation of SSH-1 by Sorafenib is probably regulated by the PI3K pathway, since Sorafenib can induce PI3K and its substrate Akt phosphorylation, and both PI3K inhibitors Ly294002 and wortmannin antagonized Sorafenib-mediated cofilin dephosphorylation. Furthermore, we found that Sorafenib induced c-Met phosphorylation at Tyr-1349 but not Tyr-1234, which is probably mediated by inhibition of receptor tyrosine phosphatase density enhanced phosphatase-1 (DEP-1). Our data provide evidence that besides inhibition of the Raf-MAPK pathway, Sorafenib might also regulate hepatoma cell growth via alteration of receptor-mediated cytoskeletal rearrangement.
Aberrant activation of Akt plays a pivotal role in cancer development. ATM, a protein deficient in patients with ataxia-telangiectasia disease, is traditionally considered as a nuclear protein kinase that functions as a signal transducer in response to DNA damage. It has recently been shown that ATM is also a cytoplasmic protein that mediates the full activation of Akt in response to insulin. Our study shows that a specific ATM inhibitor, KU-55933, blocks the phosphorylation of Akt induced by insulin and insulin-like growth factor I in cancer cells that exhibit abnormal Akt activity. Moreover, KU-55933 inhibits cancer cell proliferation by inducing G(1) cell cycle arrest. It does so through the downregulation of the synthesis of cyclin D1, a protein known to be elevated in a variety of tumors. In addition, KU-55933 treatment during serum starvation triggers apoptosis in these cancer cells. Our results suggest that KU-55933 may be a novel chemotherapeutic agent targeting cancer resistant to traditional chemotherapy or immunotherapy due to aberrant activation of Akt. Furthermore, KU-55933 completely abrogates rapamycin-induced feedback activation of Akt. Combination of KU-55933 and rapamycin not only induces apoptosis, which is not seen in cancer cells treated only with rapamycin, but also shows better efficacy in inhibiting cancer cell proliferation than each drug alone. Therefore, combining KU-55933 with rapamycin may provide a highly effective approach for improving mammalian target of rapamycin-targeted anticancer therapy that is currently hindered by rapamycin-induced feedback activation of Akt.
Sorafenib (Nexavar, BAY43-9006), a bi-arylurea, is a newly established anti-
cancer drug and its functional attribute of cytotoxicity is based on the multi-
kinase inhibitory action. Here, we report yet another novel pathway in which
sorafenib can induce apoptotic cell death preferentially and efficaciously on an
experimentally proven drug- and radio-resistant human Hep G2 cells via a
mitochondria-dependent oxidative stress mechanism. A real time confocal
imaging assay revealed that sorafenib could rapidly provoke the production of
ROS plethorically, mainly concentrating in the mitochondria, albeit substantial
amounts of ROS could also be detected in cytosol and nucleus. The rapid
production of ROS could simultaneously induce intracellular glutathione (iGSH)
depletion. A nearly 90 % of iGSH was found to be depleted in one-hour period
after the cells received the drug treatment. Besides mitochondria, iGSH
depletion could also be detected in other cellular compartment including
cytoplasm and nucleus. Interestingly, we also demonstrated that sorafenib
could trigger mitochondrial Ca2+ overload. All these events compoundedly
serve as the final arbitrator to initiate lethal apoptotic process through the
release of cytochrome c and caspase 3 / 7 activation. Collectively, we provide
first evidence here that sorafenib can provoke an alternative pathway for
apoptosis induction of Hep G2 cells through a mitochondria-dependent
oxidative stress mechanism which is independent of original kinase inhibitory
attribute of the drug action. Most importantly, we also demonstrate that
sorafenib can effectively eradicate a highly drug- and radio-resistant HCC
cells. Thus, our data can provide the basis for a potential applicability of
sorafenib in a combined treatment modality.
The importance of tumor angiogenesis in tumor biology is now widely accepted. Hepatocellular carcinoma (HCC) is a highly vascular tumor, and angiogenesis is believed to play a considerable role in its development and progression. The authors reviewed the role of circulating vascular endothelial growth factor (VEGF) in screening for HCC and in risk stratification and treatment monitoring. They searched the world medical literature by accessing MEDLINE and PubMed for articles on: 1) the utility of circulating VEGF for HCC screening in patients with cirrhosis; 2) the role of circulating VEGF as a predictor of the invasive potential of HCC; and 3) monitoring anti-HCC treatment effects by serial measurements of circulating VEGF. They found evidence to support a potential role for VEGF in screening and surveillance of HCC. They also found support for developing the use of VEGF in the monitoring of treatment outcomes. Several studies suggested that the circulating VEGF level may be an independent prognostic marker in HCC. Further studies are needed to determine the utility of circulating VEGF in screening of patients with cirrhosis and to determine its potential role as a prognostic and predictive biomarker in patients with HCC. Cancer 2009. (c) 2009 American Cancer Society.
Radiotherapy is one of the major therapeutic modalities for eradicating malignant tumors. However, the existence of radioresistant cells remains one of the most critical obstacles in radiotherapy and radiochemotherapy. Standard radiotherapy for tumor treatment consists of approximately 2 Gy once a day, 5 days a week, over a period of 5-8 weeks. To understand the characteristics of radioresistant cells and to develop more effective radiotherapy, we established a novel radioresistant cell line, HepG2-8960-R with clinical relevance from parental HepG2 cells by long-term fractionated exposure to 2 Gy of X-rays. HepG2-8960-R cells continued to proliferate with daily exposure to 2 Gy X-rays for more than 30 days, while all parental HepG2 cells ceased. After exposure to fractionated 2 Gy X-rays, induction frequencies of micronuclei and remaining foci of gamma-H2AX in HepG2-8960-R were less than those in HepG2. Flow cytometric analysis revealed that the proportion of cells in S- and G2/M-phase of the cell cycle was higher in HepG2-8960-R than in HepG2. These suggest that the response of clinically relevant radioresistant (CRR) cells to fractionated radiation is not merely an accumulated response to each fractionated radiation. This is the first report on the establishment of a CRR cell line from an isogenic parental cell line.
Most cases of hepatocellular carcinoma occur in the Asia-Pacific region, where chronic hepatitis B infection is an important aetiological factor. Assessing the efficacy and safety of new therapeutic options in an Asia-Pacific population is thus important. We did a multinational phase III, randomised, double-blind, placebo-controlled trial to assess the efficacy and safety of sorafenib in patients from the Asia-Pacific region with advanced (unresectable or metastatic) hepatocellular carcinoma.
Between Sept 20, 2005, and Jan 31, 2007, patients with hepatocellular carcinoma who had not received previous systemic therapy and had Child-Pugh liver function class A, were randomly assigned to receive either oral sorafenib (400 mg) or placebo twice daily in 6-week cycles, with efficacy measured at the end of each 6-week period. Eligible patients were stratified by the presence or absence of macroscopic vascular invasion or extrahepatic spread (or both), Eastern Cooperative Oncology Group performance status, and geographical region. Randomisation was done centrally and in a 2:1 ratio by means of an interactive voice-response system. There was no predefined primary endpoint; overall survival, time to progression (TTP), time to symptomatic progression (TTSP), disease control rate (DCR), and safety were assessed. Efficacy analyses were done by intention to treat. This trial is registered with ClinicalTrials.gov, number NCT00492752.
271 patients from 23 centres in China, South Korea, and Taiwan were enrolled in the study. Of these, 226 patients were randomly assigned to the experimental group (n=150) or to the placebo group (n=76). Median overall survival was 6.5 months (95% CI 5.56-7.56) in patients treated with sorafenib, compared with 4.2 months (3.75-5.46) in those who received placebo (hazard ratio [HR] 0.68 [95% CI 0.50-0.93]; p=0.014). Median TTP was 2.8 months (2.63-3.58) in the sorafenib group compared with 1.4 months (1.35-1.55) in the placebo group (HR 0.57 [0.42-0.79]; p=0.0005). The most frequently reported grade 3/4 drug-related adverse events in the 149 assessable patients treated with sorafenib were hand-foot skin reaction (HFSR; 16 patients [10.7%]), diarrhoea (nine patients [6.0%]), and fatigue (five patients [3.4%]). The most common adverse events resulting in dose reductions were HFSR (17 patients [11.4%]) and diarrhoea (11 patients [7.4%]); these adverse events rarely led to discontinuation.
Sorafenib is effective for the treatment of advanced hepatocellular carcinoma in patients from the Asia-Pacific region, and is well tolerated. Taken together with data from the Sorafenib Hepatocellular Carcinoma Assessment Randomised Protocol (SHARP) trial, sorafenib seems to be an appropriate option for the treatment of advanced hepatocellular carcinoma.
First described over 80 years ago, ataxia-telangiectasia (A-T) was defined as a clinical entity 50 years ago. Although not encountered by most clinicians, it is a paradigm for cancer predisposition and neurodegenerative disorders and has a central role in our understanding of the DNA-damage response, signal transduction and cell-cycle control. The discovery of the protein A-T mutated (ATM) that is deficient in A-T paved the way for rapid progress on understanding how ATM functions with a host of other proteins to protect against genome instability and reduce the risk of cancer and other pathologies.
Caffeine and other methylxanthines produce multiple physiologic effects throughout the human body, many of these effects could potentially modulate the activity of anticancer therapy. Caffeine may directly interfere with drug transport to tumor cells by formation of mixed stacking complexes with polyaromatic drugs. If formed in cells, these complexes may also prevent of intercalating drugs from DNA binding and, in this way, lower their antitumor activity. Since many of potent carcinogens are polyaromatic compounds, formation of stacking complexes with carcinogens could be associated with anti-genotoxic activity of caffeine and its use in cancer chemoprevention. Caffeine has also been reported to inhibit ATM and ATR kinases which leads to the disruption of multiple DNA damage-responsive cell cycle checkpoints and greatly sensitizes tumor cells to antitumor agents which induce genotoxic stress. Caffeine may inhibit repair of DNA lesions through a direct interference with DNA-PK activity and other repair enzymes. A number of in vitro and in vivo studies demonstrated that caffeine modulates both innate and adaptive immune responses via inhibition of cyclic adenosine monophosphate (cAMP)-phosphodiesterase. Finally, another group of effects induced by caffeine is mediated through its inhibitory action on adenosine receptors. This may modulate the stability of HIF1 alpha as well as VEGF and interleukin-8 expression in tumor cells, which could have a direct impact on neovascularization of human tumors. In this review, we present different molecular mechanisms by which caffeine and other methylxanthines may directly or indirectly modulate the effect of antitumor treatment in tumor cells and in cancer patients.
Insulin activated endogenous protein kinase B alpha (also known as RAC/Akt kinase) activity 12-fold in L6 myotubes, while after transfection into 293 cells PKBalpha was activated 20- and 50-fold in response to insulin and IGF-1 respectively. In both cells, the activation of PKBalpha was accompanied by its phosphorylation at Thr308 and Ser473 and, like activation, phosphorylation of both of these residues was prevented by the phosphatidylinositol 3-kinase inhibitor wortmannin. Thr308 and/or Ser473 were mutated to Ala or Asp and activities of mutant PKBalpha molecules were analysed after transfection into 293 cells. The activity of wild-type and mutant PKBalpha was also measured in vitro after stoichiometric phosphorylation of Ser473 by MAPKAP kinase-2. These experiments demonstrated that activation of PKBalpha by insulin or insulin-like growth factor-1 (IGF-1) results from phosphorylation of both Thr308 and Ser473, that phosphorylation of both residues is critical to generate a high level of PKBalpha activity and that the phosphorylation of Thr308 in vivo is not dependent on phosphorylation of Ser473 or vice versa. We propose a model whereby PKBalpha becomes phosphorylated and activated in insulin/IGF-1-stimulated cells by an upstream kinase(s).
The activation of p70s6k is accompanied by a complex series of phosphorylation events. In this review we propose a model of activation which divides p70s6k into four functional modules that cooperate in leading to full enzyme activity. In the light of the model, we suggest how candidate effectors of p70s6k activation might function by directing the phosphorylation of specific sites.
Hepatic resection (HX), percutaneous ethanol injection (PEI), and transcatheter arterial embolization (TCAE) have all been used in the treatment of patients with small-sized hepatocellular carcinomas (HCCs). However, the indications for these therapeutic modalities remain unclear. Therefore, the first step to minimize the debate on these indications is to review the standard results from each treatment based on an extensive survey. The participants in this study were patients with HCCs less than 5 cm in diameter who were enrolled in The Liver Cancer Study Group of Japan. The survival rates in the HX (n = 8,010), PEI (n = 4,037), and TCAE (n = 841) groups were calculated in relation to the number of tumors and the clinical stage. In the clinical stage I cases with a solitary tumor less than 2 cm in diameter and in all clinical stages with a solitary tumor greater than 2 cm and in the clinical stage II cases with 2 tumors greater than 2 cm, the HX group showed higher survival rates than the nonsurgical groups. The HX group had a higher male/female ratio and a younger mean age than the PEI or TCAE group. The ratio of HBs antigen-positive cases/hepatitis C virus antibody-positive cases in the PEI group was lower than that in the corresponding HX group. In contrast, the PIVKA-II values in the HX group tended to be higher than in the PEI group. In conclusion, these findings will provide useful information for selection of a therapeutic modality for small-sized HCCs.
Protein kinase B or Akt (PKB/Akt) is a serine/threonine kinase, which in mammals comprises three highly homologous members known as PKBalpha (Akt1), PKBbeta (Akt2), and PKBgamma (Akt3). PKB/Akt is activated in cells exposed to diverse stimuli such as hormones, growth factors, and extracellular matrix components. The activation mechanism remains to be fully characterised but occurs downstream of phosphoinositide 3-kinase (PI-3K). PI-3K generates phosphatidylinositol-3,4,5-trisphosphate (PIP(3)), a lipid second messenger essential for the translocation of PKB/Akt to the plasma membrane where it is phosphorylated and activated by phosphoinositide-dependent kinase-1 (PDK-1) and possibly other kinases. PKB/Akt phosphorylates and regulates the function of many cellular proteins involved in processes that include metabolism, apoptosis, and proliferation. Recent evidence indicates that PKB/Akt is frequently constitutively active in many types of human cancer. Constitutive PKB/Akt activation can occur due to amplification of PKB/Akt genes or as a result of mutations in components of the signalling pathway that activates PKB/Akt. Although the mechanisms have not yet been fully characterised, constitutive PKB/Akt signalling is believed to promote proliferation and increased cell survival and thereby contributing to cancer progression. This review surveys recent developments in understanding the mechanisms and consequences of PKB/Akt activation in human malignancy.
We investigated the effects of methylxanthines on enzymatic activity of phosphoinositide 3-kinases (PI3Ks). We found that caffeine inhibits the in vitro lipid kinase of class I PI3Ks (IC(50) = 75 microm for p110 delta, 400 microm for p110 alpha and p110 beta, and 1 mm for p110 gamma), and theophylline has similar effects (IC(50) = 75 microm for p110 delta, 300 microm for p110 alpha, and 800 microm for p110 beta and p110 gamma) and also inhibits the alpha isoform of class II PI3K (PI3K-C2 alpha) (IC(50) approximately 400 microm). However, four other xanthine derivatives tested (3-isobutyl-1-methylxanthine, 3-propylxanthine, alloxazine, and PD116948 (8-cyclopentyl-1,3-dipropylxanthine)) were an order of magnitude less effective. Surprisingly the triazoloquinazoline CGS15943 (9-chloro-2-(2-furyl)(1,2,d)triazolo(1,5-c)quinazolin-5-amine) also selectively inhibits p110 delta (IC(50) < 10 microm). Caffeine and theophylline also inhibit the intrinsic protein kinase activity of the class IA PI3Ks and DNA-dependent protein kinase, although with a much lower potency than that for the lipid kinase (IC(50) approximately 10 mm for p110 alpha, 3 mm for p110 beta, and 10 mm for DNA-dependent protein kinase). In CHO-IR cells and rat soleus muscle, theophylline and caffeine block the ability of insulin to stimulate protein kinase B with IC(50) values similar to those for inhibition of PI3K activity, whereas insulin stimulation of ERK1 or ERK2 was not inhibited at concentrations up to 10 mm. Theophylline and caffeine also blocked insulin stimulation of glucose transport in CHO-IR cells. These results demonstrate that these methylxanthines are direct inhibitors of PI3K lipid kinase activity but are distinctly less effective against serine kinase activity and thus could be of potential use in dissecting these two distinct kinase activities. Theophylline, caffeine, and CGS15943 may be of particular use in dissecting the specific role of the p110 delta lipid kinase. Finally, we conclude that inhibition of PI3K (p110 delta in particular) is likely explain some of the physiological and pharmacological properties of caffeine and theophylline.
Hepatocellular carcinoma (HCC) is the fifth most common cause of cancer, and its incidence is increasing worldwide because of the dissemination of hepatitis B and C virus infection. Patients with cirrhosis are at the highest risk and should be monitored every 6 months. Surveillance can lead to diagnosis at early stages, when the tumour might be curable by resection, liver transplantation, or percutaneous treatment. In the West and Japan, these treatments can be applied to 30% of patients, and result in 5-year survival rates higher than 50%. Resection is indicated among patients who have one tumour and well-preserved liver function. Liver transplantation benefits patients who have decompensated cirrhosis and one tumour smaller than 5 cm or three nodules smaller than 3 cm, but donor shortage greatly limits its applicability. This difficulty might be overcome by living donation. Most HCC patients are diagnosed at advanced stages and receive palliative treatments, which have been assessed in the setting of 63 randomised controlled trials during the past 25 years. Meta-analysis shows that only chemoembolisation improves survival in well-selected patients with unresectable HCC.
Caffeine is a major biologically active constituent in coffee and tea. Because caffeine has been reported to inhibit carcinogenesis in UVB-exposed mice, the cancer-preventing effect of caffeine has attracted considerable attention. In the present study, the effect of caffeine in quiescent (G0 phase) cells was investigated. Pretreatment with caffeine suppressed cell proliferation in a dose-dependent manner 36 h after addition of fetal bovine serum as a cell growth stimulator. Analysis by flow cytometry showed that caffeine suppressed cell cycle progression at the G0/G1 phase, i.e., 18 h after addition of fetal bovine serum, the percentages of cells in G0/G1 phase in 1 mM caffeine-treated cells and in caffeine-untreated cells were 61.7 and 29.0, respectively. The percentage of cells in G0/G1 phase at 0 h was 75.5. Caffeine inhibited phosphorylation of retinoblastoma protein at Ser780 and Ser807/Ser811, the sites where retinoblastoma protein has been reported to be phosphorylated by cyclin-dependent kinase 4 (cdk4). Furthermore, caffeine inhibited the activation of the cyclin D1-cdk4 complex in a dose-dependent manner. However this compound did not directly inhibit the activity of this complex. In addition, caffeine did not affect p16INK4 or p27Kip1 protein levels, but inhibited the phosphorylation of protein kinase B (Akt) and glycogen synthase kinase 3beta. Our results showed that caffeine suppressed the progression of quiescent cells into the cell cycle. The inhibitory mechanism may be due to the inhibition of cell growth signal-induced activation of cdk4, which may be involved in the inhibition of carcinogenesis in vivo.
The phosphatidylinositol 3-kinase (PI3K)/Akt (protein kinase B, PKB) signaling pathway plays a critical role in cell growth and survival. Dysregulation of this pathway has been found in a variety of cancer cells. Recently, constitutively active PI3K/Akt signaling has been firmly established as a major determinant for cell growth and survival in an array of cancers. Blocking the constitutively active PI3K/AKT signaling pathway provides a new strategy for targeted cancer therapy. Thus, inhibitors of this signaling pathway would be potential anticancer agents, particularly for cancer cells whose survival and growth are dominated by constitutively active PI3K/Akt signaling. This review describes the current understanding of small molecule drugs targeting this pathway both in vitro and in vivo. Inhibitors and functions of the upstream and downstream molecular targets of the PI3K/Akt pathway are discussed in the context of using the inhibitors to block this pathway for targeted cancer therapy. Special emphasis is placed on the following targets: receptor tyrosine kinases, PI3K, Akt, and the mammalian target of rapamycin. While the molecular therapeutic strategy holds great promise for the treatment of a variety of cancers, few small molecule inhibitors with potential high therapeutic indexes are available. Thus, new inhibitors with high selectivity, bioavailability, and potency are greatly needed. Novel approaches toward the development of PI3K/Akt pathway inhibitors as anticancer therapeutics are discussed in detail, with emphasis on chemical genetics-based and structure-based drug design.
Mammalian target of rapamycin (mTOR) controls cell growth and proliferation via the raptor-mTOR (TORC1) and rictor-mTOR (TORC2) protein complexes. Recent biochemical studies suggested that TORC2 is the elusive PDK2 for Akt/PKB Ser473 phosphorylation in the hydrophobic motif. Phosphorylation at Ser473, along with Thr308 of its activation loop, is deemed necessary for Akt function, although the regulatory mechanisms and physiological importance of each phosphorylation site remain to be fully understood. Here, we report that SIN1/MIP1 is an essential TORC2/PDK2 subunit. Genetic ablation of sin1 abolished Akt-Ser473 phosphorylation and disrupted rictor-mTOR interaction but maintained Thr308 phosphorylation. Surprisingly, defective Ser473 phosphorylation affected only a subset of Akt targets in vivo, including FoxO1/3a, while other Akt targets, TSC2 and GSK3, and the TORC1 effectors, S6K and 4E-BP1, were unaffected. Our findings reveal that the SIN1-rictor-mTOR function in Akt-Ser473 phosphorylation is required for TORC2 function in cell survival but is dispensable for TORC1 function.
Primary liver cancer, which consists predominantly of hepatocellular carcinoma (HCC), is the fifth most common cancer worldwide and the third most common cause of cancer mortality. HCC has several interesting epidemiologic features including dynamic temporal trends; marked variations among geographic regions, racial and ethnic groups, and between men and women; and the presence of several well-documented environmental potentially preventable risk factors. Moreover, there is a growing understanding on the molecular mechanisms inducing hepatocarcinogenesis, which almost never occurs in healthy liver, but the cancer risk increases sharply in response to chronic liver injury at the cirrhosis stage. A detailed understanding of epidemiologic factors and molecular mechanisms associated with HCC ultimately could improve our current concepts for screening and treatment of this disease.
Caffeine, which has a DNA-repair inhibiting effect, enhances the cytocidal effects of anticancer drugs and radiation. The present study was performed to assess the efficacy of caffeine-potentiated chemotherapy for high-grade soft tissue sarcoma (STS).
A non-randomised prospective clinical trial was initiated for 90 patients with non-metastatic (stages II and III) or metastatic (stage IV) STS. Following doxorubicin or ifosfamide combined with caffeine, with or without radiotherapy, 88 patients were treated surgically. A radiographic and histological response to chemotherapy was assessed. Local-recurrence free, distant-metastasis free and overall survival were analyzed by multivariate analysis.
Radiographic and histological response rates were 57.8% and 42%, respectively. The local recurrence rate was 23.7% in stages II and III and 13.6% in stage IV. Lung metastases newly developed in 21 (35.6%) patients at stages II and III. With a median follow-up period of 52 months, the overall 5-year cumulative survival rate at stages II and III was 80.7%. Local recurrence-free survival for the histological responders and distant metastasis-free survival for the radiographic responders at stages II and III were significantly improved compared to the non-responders (p=0.004 and p=0.034). Overall survival for the radiographic responders at all stages was significant longer than for the non-responders (p=0.009).
Caffeine-potentiated chemotherapy resulted in a favourable radiographic response and prolonged overall survival of the patients at all stages.
Ataxia-telangiectasia (A-T) is an autosomal recessive disorder characterized by cerebellar ataxia and oculocutaneous telangiectasias. Patients with A-T also have high incidences of type 2 diabetes mellitus. The gene mutated in this disease, ATM (A-T, mutated), encodes a protein kinase. Previous studies have demonstrated that cytoplasmic ATM is an insulin-responsive protein and a major upstream activator of Akt following insulin treatment. To further investigate the function of ATM in insulin signal transduction, insulin resistance was induced in rats by feeding them a high-fat diet. Muscle tissue of rats with insulin resistance had both dramatically reduced ATM levels and substantially decreased Akt phosphorylation at Ser473 in comparison to that of regular chow-fed controls. The decreased ATM expression suggests that ATM is involved in the development of insulin resistance through down-regulation of Akt activity. The role of ATM in activation of Akt was further confirmed in mouse embryonic fibroblast (MEF) A29 (ATM+/+) and A38 (ATM-/-) cells. In addition, insulin-mediated Akt phosphorylation in mouse L6 muscle cells was greatly reduced by KU-55933, a specific inhibitor of ATM. A 2-deoxyglucose incorporation assay showed that this inhibitor also caused a significant reduction in insulin-mediated glucose uptake in L6 cells. An immunofluorescence experiment demonstrated that in L6 cells transfected with wild-type (WT) ATM, insulin caused a dramatic increase of the cell surface glucose transporter 4 (GLUT4), while in cells transfected with kinase-dead (KD) ATM, translocation of GLUT4 to the cell surface in response to insulin was markedly inhibited.
A. Trail, BAY 43-9006 exhibits broad spectrum oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis
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Results of surgical and nonsurgical treatment for small-sized hepatocellular carcinomas: a retrospective and nationwide survey in Japan
- Arii
Identification and characterization of a novel and specific inhibitor of the ataxia-telangiectasia mutated kinase ATM
- Hickson