Research Items (44)
Tumor hypoxia is a common microenvironmental factor that adversely influences tumor phenotype and treatment response. Cellular adaptation to hypoxia occurs through multiple mechanisms, including activation of the unfolded protein response (UPR). Recent reports have indicated that hypoxia activates a lysosomal degradation pathway known as autophagy, and here we show that the UPR enhances the capacity of hypoxic tumor cells to carry out autophagy, and that this promotes their survival. In several human cancer cell lines, hypoxia increased transcription of the essential autophagy genes microtubule-associated protein 1 light chain 3beta (MAP1LC3B) and autophagy-related gene 5 (ATG5) through the transcription factors ATF4 and CHOP, respectively, which are regulated by PKR-like ER kinase (PERK, also known as EIF2AK3). MAP1LC3B and ATG5 are not required for initiation of autophagy but mediate phagophore expansion and autophagosome formation. We observed that transcriptional induction of MAP1LC3B replenished MAP1LC3B protein that was turned over during extensive hypoxia-induced autophagy. Correspondingly, cells deficient in PERK signaling failed to transcriptionally induce MAP1LC3B and became rapidly depleted of MAP1LC3B protein during hypoxia. Consistent with these data, autophagy and MAP1LC3B induction occurred preferentially in hypoxic regions of human tumor xenografts. Furthermore, pharmacological inhibition of autophagy sensitized human tumor cells to hypoxia, reduced the fraction of viable hypoxic tumor cells, and sensitized xenografted human tumors to irradiation. Our data therefore demonstrate that the UPR is an important mediator of the hypoxic tumor microenvironment and that it contributes to resistance to treatment through its ability to facilitate autophagy.
Human tumors are characterized by the presence of cells that experience periodic episodes of hypoxia followed by reoxygenation. These cells are exposed to reactive oxygen species (ROS) upon reoxygenation and require adaptation to this stress by lowering ROS production or enhancing ROS-clearance for their survival. We hypothesized that autophagy, a lysosomal degradation pathway, may be involved in reducing ROS during periodic hypoxia through removal of ROS producing species. Human tumor cells (MCF-7, HT29, U373) were exposed to cycles of hypoxia (O(2)<0.02%) and reoxygenation in the absence or presence of the autophagy inhibitor chloroquine (CQ). Clonogenic survival, ROS production and mitochondrial-DNA content were assessed. In addition, A549 cells overexpressing wild-type or K63-mutated ubiquitin (K63R) were analyzed for ROS production. Our data indicate that CQ treatment sensitizes cells to cycling hypoxia, due to increased production of ROS, associated with an incapacity to reduce mitochondrial content. Addition of the ROS-scavenger N-acetyl-cysteine increased cell viability and neutralized CQ-effects. Additionally, genetic prevention of K63-linked ubiquitin chains that are required for the removal of toxic protein aggregates by autophagy, resulted in increased ROS production. Inhibition of autophagy substantially increases cell death induced by cycling hypoxia through increased ROS production, providing an opportunity to decrease the hypoxic fraction within tumors and enhance tumor therapy.
Hypoxia is a common feature of tumors and an important contributor to malignancy and treatment resistance. The ability of tumor cells to survive hypoxic stress is mediated in part by hypoxia-inducible factor (HIF)-dependent transcriptional responses. More severe hypoxia activates endoplasmatic reticulum stress responses, including the double-stranded RNA-activated protein kinase (PKR)-like endoplasmic reticulum kinase (PERK)/eukaryotic initiation factor 2α (eIF2α)-dependent arm of the unfolded protein response (UPR). Although several studies implicate important roles for HIF and UPR in adaption to hypoxia, their importance for hypoxic cells responsible for therapy resistance in tumors is unknown. By using isogenic models, we find that HIF and eIF2α signaling contribute to the survival of hypoxic cells in vitro and in vivo. However, the eIF2α-dependent arm of the UPR is uniquely required for the survival of a subset of hypoxic cells that determine tumor radioresistance. We demonstrate that eIF2α signaling induces uptake of cysteine, glutathione synthesis, and protection against reactive oxygen species produced during periods of cycling hypoxia. Together these data imply that eIF2α signaling is a critical contributor to the tolerance of therapy-resistant cells that arise as a consequence of transient changes in oxygenation in solid tumors and thus a therapeutic target in curative treatments for solid cancers.
- Apr 2019
T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive cancer arising from T-cell progenitors. Although current treatments, including chemotherapy and glucocorticoids, have significantly improved survival, T-ALL remains a fatal disease and new treatment options are needed. Since more than 60% of T-ALL cases bear oncogenic NOTCH1 mutations, small molecule inhibitors of NOTCH1 signalling; γ-secretase inhibitors (GSI), are being actively investigated for the treatment of T-ALL. Unfortunately, GSI have shown limited clinical efficacy and dose-limiting toxicities. We hypothesized that by combining known drugs, blocking NOTCH activity through another mechanism, may synergize with GSI enabling equal efficacy at a lower concentration. Here, we show that the clinically used anti-malarial drug chloroquine (CQ), an inhibitor of lysosomal function and autophagy, decreases T-ALL cell viability and proliferation. This effect of CQ was not observed in GSI-resistant T-ALL cell lines. Mechanistically, CQ impairs the redox balance, induces ds DNA breaks and activates the DNA damage response. CQ also interferes with intracellular trafficking and processing of oncogenic NOTCH1. Interestingly, we show for the first time that the addition of CQ to γ-secretase inhibition has a synergistic therapeutic effect on T-ALL and reduces the concentration of GSI required to obtain a reduction in cell viability and a block of proliferation. Overall, our results suggest that CQ may be a promising repurposed drug in the treatment of T-ALL, as a single treatment or in combination with GSI, increasing the therapeutic ratio.
Introduction Rosmarinus officinalis L., commonly called rosemary, is one of the most widely used and commercialised plant for obtaining botanical extracts which are not only used as a culinary herb for flavouring but also as an antioxidant ingredient in processed foods and cosmetics. This plant has been traditionally used as an herbal medicine for a large variety of disorders. Its beneficial effects have been attributed to the high content in bioactive compounds, such as phenolic diterpenes, flavonoids and other phenolic compounds. Material and methods In this work, a RE obtained using supercritical CO2 extraction has been assayed for the antitumor activity in three colon cancer cell lines (HGUE-C-1, HT-29 and SW480). The effects of ER on different aspects of cell proliferation and migration were determined using RTCA DP instrument (Real Time Cell Analysis Proliferation Assay), wound healing and colony formation assays. Results and discussions Results showed that RE inhibited cell proliferation in a concentration dependent manner in all cell lines. HGUE-C-1 and SW480 cells demonstrated a higher sensitivity against RE on RTCA assay, causing a complete reduction of cell proliferation at 20 and 40 µg/mL. Wound healing experiments showed a significant anti-migrative activity for RE in all the cell lines, without relevant differences between cell lines. Finally, RE treatment also suppressed colony formation in all cell lines. Conclusion Our results support that RE extract enriched in diterpenes and triterpenes shows inhibited cell proliferation, decreased cell migration and colony formation in all the colorectal cancer cell lines tested. We hypothesised that all these effects may contribute to the anticancer and anti-invasive properties of rosemary extract.
Abstract The hypoxic areas of solid cancers represent a negative prognostic factor irrespective of which treatment modality is chosen for the patient. Still, after almost 80 years of focus on the problems created by hypoxia in solid tumours, we still largely lack methods to deal efficiently with these treatment-resistant cells. The consequences of this lack may be serious for many patients: Not only is there a negative correlation between the hypoxic fraction in tumours and the outcome of radiotherapy as well as many types of chemotherapy, a correlation has been shown between the hypoxic fraction in tumours and cancer metastasis. Thus, on a fundamental basis the great variety of problems related to hypoxia in cancer treatment has to do with the broad range of functions oxygen (and lack of oxygen) have in cells and tissues. Therefore, activation-deactivation of oxygen-regulated cascades related to metabolism or external signalling are important areas for the identification of mechanisms as potential targets for hypoxia-specific treatment. Also the chemistry related to reactive oxygen radicals (ROS) and the biological handling of ROS are part of the problem complex. The problem is further complicated by the great variety in oxygen concentrations found in tissues. For tumour hypoxia to be used as a marker for individualisation of treatment there is a need for non-invasive methods to measure oxygen routinely in patient tumours. A large-scale collaborative EU-financed project 2009-2014 denoted METOXIA has studied all the mentioned aspects of hypoxia with the aim of selecting potential targets for new hypoxia-specific therapy and develop the first stage of tests for this therapy. A new non-invasive PET-imaging method based on the 2-nitroimidazole [(18)F]-HX4 was found to be promising in a clinical trial on NSCLC patients. New preclinical models for testing of the metastatic potential of cells were developed, both in vitro (2D as well as 3D models) and in mice (orthotopic grafting). Low density quantitative real-time polymerase chain reaction (qPCR)-based assays were developed measuring multiple hypoxia-responsive markers in parallel to identify tumour hypoxia-related patterns of gene expression. As possible targets for new therapy two main regulatory cascades were prioritised: The hypoxia-inducible-factor (HIF)-regulated cascades operating at moderate to weak hypoxia (<1% O2), and the unfolded protein response (UPR) activated by endoplasmatic reticulum (ER) stress and operating at more severe hypoxia (<0.2%). The prioritised targets were the HIF-regulated proteins carbonic anhydrase IX (CAIX), the lactate transporter MCT4 and the PERK/eIF2α/ATF4-arm of the UPR. The METOXIA project has developed patented compounds targeting CAIX with a preclinical documented effect. Since hypoxia-specific treatments alone are not curative they will have to be combined with traditional anti-cancer therapy to eradicate the aerobic cancer cell population as well.
Conditions of poor oxygenation (hypoxia) are present in many human tumors including cervix cancer, and are associated with increased risk of metastasis and poor prognosis. Hypoxia is a potent activator of the PERK/eIF2α signaling pathway, a component of the unfolded protein response (UPR) and an important mediator of hypoxia tolerance and tumor growth. Here, the importance of this pathway in the metastasis of human cervix carcinoma was investigated. Amplification and expression of LAMP3, a UPR metastasis-associated gene, was examined using FISH and immunofluorescence in a cohort of human cervix tumors from patients who had received oxygen needle electrode tumor oxygenation measurements. To evaluate the importance of this pathway in metastasis in vivo, we constructed a series of inducible cell lines to interfere with PERK signaling during hypoxia and used these in an orthotopic cervix cancer model of hypoxia-driven metastasis. We demonstrate that LAMP3 expression in human cervix tumors is augmented both by gene copy number alterations, and by hypoxia. Induced disruption of PERK signaling in established orthotopic xenografts resulted in complete inhibition of hypoxia-induced metastasis to the lymph nodes. This is due in part to a direct influence of the UPR pathway on hypoxia tolerance. However, we also find that LAMP3 is a key mediator of hypoxia-driven nodal metastasis, through its ability to promote metastatic properties including cell migration. These data suggest that the association between hypoxia, metastasis and poor prognosis is due in part to hypoxic activation of the UPR and expression of LAMP3.
CD44 family members are cell surface glycoproteins, which are expressed on tubular epithelial cells (TEC) solely upon kidney injury and are involved in renal fibrosis development. Renal interstitial fibrosis is the final manifestation of chronic kidney diseases and is regulated by a complex network of cytokines, including the pro-fibrotic factor TGF-β1 and the two anti-fibrotic cytokines BMP-7 and HGF. The present study investigates the potential role of CD44 standard (CD44s) and CD44v3-v10 (CD44v3) isoforms as modulators of the balance between TGF-β1 and HGF/BMP-7. CD44s is the shortest and most common isoform. CD44v3-v10 (CD44v3) has heparan sulfate moieties, which enable the binding to HGF/BMP-7, and hence, might exert reno-protective effects. Using transgenic mice overexpressing either CD44s or CD44v3 specifically on proximal TEC, we found that in vitro the overexpression of CD44v3 on primary TEC renders cells less susceptible to TGF-β1 pro-fibrotic actions and more sensitive to BMP-7 and HGF in comparison to TEC overexpressing CD44s. One day after unilateral ureteric obstruction, obstructed kidneys from CD44v3 transgenic mice showed less tubular damage and myofibroblasts accumulation, which was associated with decreased TGF-β1 signaling and increased BMP-7 synthesis and signaling compared to kidneys from WT and CD44s transgenic mice. These data suggest that CD44v3 plays a renoprotective role in early stage of chronic obstructive nephropathy.
The epidermal growth factor receptor (EGFR) is overexpressed, amplified or mutated in various human epithelial tumors, and is associated with tumor aggressiveness and therapy resistance. Autophagy activation provides a survival advantage for cells in the tumor microenvironment. In the current study, we assessed the potential of autophagy inhibition (using chloroquine (CQ)) in treatment of EGFR expressing tumors. Quantitative PCR, immunohistochemistry, clonogenic survival, proliferation assays and in vivo tumor growth were used to assess this potential. We show that EGFR overexpressing xenografts are sensitive to CQ treatment and are sensitized to irradiation by autophagy inhibition. In HNSSC xenografts, a correlation between EGFR and expression of the autophagy marker LC3b is observed, suggesting a role for autophagy in EGFR expressing tumors. This observation was substantiated in cell lines, showing high EGFR expressing cells to be more sensitive to CQ addition as reflected by decreased proliferation and survival. Surprisingly high EGFR expressing cells display a lower autophagic flux. The EGFR high expressing cells and tumors investigated in this study are highly dependent on autophagy for growth and survival. Inhibition of autophagy may therefore provide a novel treatment opportunity for EGFR overexpressing tumors.
Oxygen sensing in mammalian cells is a conserved signaling pathway regulated by hypoxia inducible factor type 1 (HIF-1). Inadequate oxygen supply (hypoxia) is common to many pathological disorders where autophagy plays an import role. The aim of this study was the identification and characterization of novel HIF-1 target genes that promote autophagy during hypoxia. Whole genome Chromatin Immune Precipitation from hypoxic HeLa cells was used to identify novel HIF-1 target genes. Hypoxia induced expression and transcription regulation was studied in wild type and HIF-deficient cells. siRNA silencing of candidate genes was used to establish their role during autophagy. Recombinant protein was used for screening immobilized glycosylated lipids to identify potential ligands. We identified the Nucleotide Oligomerization Domain 2 (NOD2/CARD15) as a novel HIF-1 target and 3-O-sulfo-galactoceramide (sulfatide) and Mycobacterium sp. specific sulfolipid-1 as the first NOD2 ligands that both compete for binding to NOD2. Loss of NOD2 function impaired autophagy upstream of the autophagy inhibitor chloroquine by reducing the number of acidic vesicles. Inhibition of sulfatide synthesis elicited defects in autophagy similar to the NOD2 loss of function but did not influence NOD2-mediated NF-kB signaling. Our findings suggest that the interaction of NOD2 with sulfatide may mediate the balance between autophagy and inflammation in hypoxic cells. These findings may lead to a better understanding of complex inflammatory pathologies like Crohn's disease and tuberculosis where both NOD2 and hypoxia are implicated.
Tumor hypoxia is associated with therapy resistance and malignancy. Previously we demonstrated that activation of autophagy and the unfolded protein response (UPR) promote hypoxia tolerance. Here we explored the importance of ULK1 in hypoxia tolerance, autophagy induction and its prognostic value for recurrence after treatment. Hypoxic regulation of ULK1 mRNA and protein was assessed in vitro and in primary human head and neck squamous cell carcinoma (HNSCC) xenografts. Its importance in autophagy induction, mitochondrial homeostasis and tolerance to chronic and acute hypoxia was evaluated in ULK1 knockdown cells. The prognostic value of ULK1 mRNA expression was assessed in 82 HNSCC patients. ULK1 enrichment was observed in hypoxic tumor regions. High enrichment was associated with a high hypoxic fraction. In line with these findings, high ULK1 expression in HNSCC patients appeared associated with poor local control. Exposure of cells to hypoxia induced ULK1 mRNA in a UPR and HIF1α dependent manner. ULK1 knockdown decreased autophagy activation, increased mitochondrial mass and ROS exposure and sensitized cells to acute and chronic hypoxia. We demonstrate that ULK1 is a hypoxia regulated gene and is associated with hypoxia tolerance and a worse clinical outcome.
Multiple Myeloma (MM) is an incurable plasma cell malignancy residing within the bone marrow (BM). We aim to develop allogeneic Natural Killer (NK) cell immunotherapy for MM. As the BM contains hypoxic regions and the tumor environment can be immunosuppressive, we hypothesized that hypoxia inhibits NK cell anti-MM responses. NK cells were isolated from healthy donors by negative selection and NK cell function and phenotype were examined at oxygen levels representative of hypoxic BM using flowcytometry. Additionally, NK cells were activated with IL-2 to enhance NK cell cytotoxicity under hypoxia. Hypoxia reduced NK cell killing of MM cell lines in an oxygen dependent manner. Under hypoxia, NK cells maintained their ability to degranulate in response to target cells, though, the percentage of degranulating NK cells was slightly reduced. Adaptation of NK- or MM cells to hypoxia was not required, hence, the oxygen level during the killing process was critical. Hypoxia did not alter surface expression of NK cell ligands (HLA-ABC, -E, MICA/B and ULBP1-2) and receptors (KIR, NKG2A/C, DNAM-1, NCRs and 2B4). It did, however, decrease expression of the activating NKG2D receptor and of intracellular perforin and granzyme B. Pre-activation of NK cells by IL-2 abrogated the detrimental effects of hypoxia and increased NKG2D expression. This emphasized that activated NK cells can mediate anti-MM effects, even under hypoxic conditions. Hypoxia abolishes the killing potential of NK cells against multiple myeloma, which can be restored by IL-2 activation. Our study shows that for the design of NK cell-based immunotherapy it is necessary to study biological interactions between NK- and tumor cells also under hypoxic conditions.
Hypoxia in solid tumors is associated with treatment resistance, resulting in poor prognosis. Tribbles homolog 3 (TRIB3) is induced during hypoxia and is involved in multiple cellular pathways involved in cell survival. Here, we investigated the role of TRIB3 in breast cancer. TRIB3 mRNA expression was measured in breast tumor tissue from 247 patients and correlated with clinicopathological parameters and clinical outcome. Furthermore, we studied TRIB3 expression regulation in cell lines, xenografts tissues and human breast cancer material using Reverse transcriptase, quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining. Finally, the effect of small interfering RNA (siRNA) mediated TRIB3 knockdown on hypoxia tolerance was assessed. Breast cancer patients with low, intermediate or high TRIB3 expression exhibited a mean disease free survival (DFS) of 80 (95% confidence interval [CI] = 74 to 86), 74 (CI = 67 to 81), and 63 (CI = 55 to 71) months respectively (P = .002, Mantel-Cox log-rank). The prognostic value of TRIB3 was limited to those patients that had received radiotherapy as part of their primary treatment (n = 179, P = .005) and remained statistically significant after correction for other clinicopathological parameters (DFS, Hazard Ratio = 1.90, CI = 1.17 to 3.08, P = .009). In breast cell lines TRIB3 expression was induced by hypoxia, nutrient starvation, and endoplasmic reticulum stress in an hypoxia inducible factor 1 (HIF-1) independent manner. TRIB3 induction after hypoxia did not increase with decreasing oxygen levels. In breast tumor xenografts and human breast cancer tissues TRIB3 co-localized with the hypoxic cell marker pimonidazole. The induction of TRIB3 by hypoxia was shown to be regulated via the PERK/ATF4/CHOP pathway of the unfolded protein response and knockdown of TRIB3 resulted in a dose-dependent increase in hypoxia sensitivity. TRIB3 is independently associated with poor prognosis of breast cancer patients, possibly through its association with tumor cell hypoxia.
- Jun 2011
Tumour hypoxia is an important limiting factor in the successful treatment of cancer. Adaptation to hypoxia includes inhibition of mTOR, causing scavenging of eukaryotic initiation factor 4E (eIF4E), the rate-limiting factor for cap-dependent translation. The aim of this study was to determine the effect of preventing mTOR-dependent translation inhibition on hypoxic cell survival and tumour sensitivity towards irradiation. The effect of eIF4E-overexpression on cell proliferation, hypoxia-tolerance, and radiation sensitivity was assessed using isogenic, inducible U373 and HCT116 cells. We found that eIF4E-overexpression significantly enhanced proliferation of cells under normal conditions, but not during hypoxia, caused by increased cell death during hypoxia. Furthermore, eIF4E-overexpression stimulated overall rates of tumour growth, but resulted in selective loss of hypoxic cells in established tumours and increased levels of necrosis. This markedly increased overall tumour sensitivity to irradiation. Our results demonstrate that hypoxia induced inhibition of translational control through regulation of eIF4E is an important mediator of hypoxia tolerance and radioresistance of tumours. These data also demonstrate that deregulation of metabolic pathways such as mTOR can influence the proliferation and survival of tumour cells experiencing metabolic stress in opposite ways of nutrient replete cells.
- Oct 2010
In the intestine, Paneth cells participate in the innate immune response. Their highly secretory function makes them susceptible to environmental conditions that cause endoplasmic reticulum (ER) stress. We investigated whether intestinal ischemia/reperfusion (I/R) induces ER stress, thereby activating the unfolded protein response (UPR), and whether excessive UPR activation affects Paneth cells. In addition, we investigated the consequences of Paneth cell compromise during physical barrier damage. Jejunal I/R was studied using a human experimental model (n = 30 patients). Activation of the UPR was assessed using immunofluorescence for binding protein and quantitative polymerase chain reaction analyses for C/EBP homologous protein (CHOP), growth arrest and DNA-damage inducible protein 34 (GADD34), and X-box binding protein 1 (XBP1) splicing. Paneth cell apoptosis was assessed by double staining for lysozyme and M30. Male Sprague-Dawley rats underwent either intestinal I/R to investigate UPR activation and Paneth cell apoptosis, or hemorrhagic shock with or without intraperitoneal administration of dithizone, to study consequences of Paneth cell compromise during physical intestinal damage. In these animals, bacterial translocation and circulating tumor necrosis factor-α and interleukin-6 levels were assessed. In jejunum samples from humans and rats, I/R activated the UPR and resulted in Paneth cell apoptosis. Apoptotic Paneth cells showed signs of ER stress, and Paneth cell apoptosis correlated with the extent of ER stress. Apoptotic Paneth cells were shed into the crypt lumen, significantly lowering their numbers. In rats, Paneth cell compromise increased bacterial translocation and inflammation during physical intestinal damage. ER stress-induced Paneth cell apoptosis contributes to intestinal I/R-induced bacterial translocation and systemic inflammation.
- Jun 2010
BNIP3 is a hypoxia-inducible BH3-only member of the Bcl-2 family of proteins that regulate apoptosis and autophagy. However the role of BNIP3 in the hypoxia response has proved difficult to define and remains controversial. In this study we show that in cancer cells, knockdown or forced expression of BNIP3 fails to modulate cell survival under hypoxic or normoxic conditions. However, we demonstrate that BNIP3 is regulated post-translationally, existing as multiple monomeric and dimeric phosphorylated forms. Upon treatment with microtubule inhibitors, but not other classes of chemotherapeutics, BNIP3 becomes hyperphosphorylated. We demonstrate that the phosphorylation of BNIP3 occurs in synchrony with phosphorylation of its binding partners Bcl-2 and Bcl-xL. Microtubule inhibitor-induced phosphorylation of these proteins occurs independently of the AKT/mTor and JNK kinase pathways and requires Mps1 mitotic checkpoint kinase activity. Inhibition of mitotic arrest in the presence of paclitaxel blocks the phosphorylation of BNIP3, Bcl-2 and Bcl-xL, demonstrating that these proteins are phosphorylated by a mitochondrially active mitotic kinase. We show that phosphorylation increases the stability of BNIP3 and that BNIP3 predominantly interacts with the phosphorylated form of Bcl-2. This study provides new insight into the post-translational functional control of these Bcl-2 family members.
- Sep 2009
Tumour hypoxia contributes to failure of cancer treatment through its ability to protect against therapy and adversely influence tumour biology. In particular, several studies suggest that hypoxia promotes metastasis. Hypoxia-induced cellular changes are mediated by oxygen-sensitive signaling pathways that activate downstream transcription factors. We have investigated the induction and transcriptional regulation of a novel metastasis-associated gene, LAMP3 during hypoxia. Microarray, quantitative PCR, Western blot analysis and immunohistochemistry were used to investigate hypoxic regulation of LAMP3. The mechanism for LAMP3 induction was investigated using transient RNAi and stable shRNA targeting components of the hypoxic response. Endoplasmic reticulum stress inducing agents, including proteasome inhibitors were assessed for their ability to regulate LAMP3. LAMP3 is strongly induced by hypoxia at both the mRNA and protein levels in a large panel of human tumour cell lines. Induction of LAMP3 occurs as a consequence of the activation of the PERK/eIF2alpha/ATF4 arm of the unfolded protein response (UPR) and is independent of HIF-1alpha. LAMP3 is expressed heterogeneously within the microenvironment of tumours, overexpressed in breast cancer, and increases in tumours treated with avastin. These data identify LAMP3 as a novel hypoxia-inducible gene regulated by the UPR. LAMP3 is a new candidate biomarker of UPR activation by hypoxia in tumours and is a potential mediator of hypoxia-induced metastasis.
Carbonic anhydrase (CA) 9 expression is induced under hypoxic conditions. Recently we discovered that hypoxia-induced CA9 expression requires an intact unfolded protein response (UPR) pathway. The objective of this study was to evaluate the effect of reduced CA9 expression in UPR-impaired tumor cells on pH regulation and survival under acidic conditions. CA9 expression was assessed in wild-type or UPR-defective U373-MG cells by quantitative-PCR, Western blot, immunofluorescence and flow cytometry. Proliferation and cell survival were measured under acidic conditions in the absence of bicarbonate under ambient CO(2) levels. Cell-surface CA9 expression was significantly reduced in UPR-defective U373 cell lines compared to parental U373 cells. This resulted in decreased CA9 activity and extracellular acidification. Differences in CA9 expression and activity were correlated with the ability to tolerate acidic conditions. In addition we observed a strong synergistic effect of anoxia and low pH on cell survival. Our data indicate that UPR-impaired U373 cell lines have a defect in pH regulation due to reduced CA9 expression that sensitizes to acidic conditions. We speculate that this defect may contribute to the poor growth of UPR-defective tumors.
The epidermal growth factor receptor (EGFR) is overexpressed or mutated in many tumour types. The truncated, constitutively active EGFRvIII variant has not been detected in normal tissues but is found in many malignancies. In the current study, we have investigated the hypothesis that EGFRvIII contributes to a growth and survival advantage under tumour microenvironment-related stress conditions. U373MG doxycycline-regulated isogenic cells expressing EGFRwt or EGFRvIII were created and validated using Western blot, FACS and qRT-PCR. In vitro proliferation was evaluated with standard growth assays. Cell survival was assayed using clonogenic survival. Animal experiments were performed using NMRI-nu-xenografted mice. Inducible isogenic cell lines were created and showed high induction of EGFRwt and EGFRvIII upon doxycycline addition. Overexpression of EGFRvIII but not of EGFRwt in this model resulted in a growth and survival advantage upon different tumour microenvironment-related stress conditions in vitro. Induction of EGFRvIII increased tumour growth in vivo, which was reversible upon loss of expression. Under conditions where nutrients are limited and stress is apparent, as in the tumour microenvironment, expression of EGFRvIII leads to a growth and survival advantage. These data indicate a potential selection of EGFRvIII-expressing tumour cells under such stress conditions.
Adaptation to tumor hypoxia is mediated in large part by changes in protein expression. These are driven by multiple pathways, including activation of the hypoxia inducible factor-1 (HIF-1) transcription factor and the PKR-like endoplasmic reticulum kinase PERK, a component of the unfolded protein response. Through gene expression profiling we discovered that induction of the HIF-1 target gene CA9 was defective in mouse embryo fibroblasts derived from mice harboring an eIF2α S51A knock-in mutation. This finding was confirmed in two isogenic human cell lines with an engineered defect in eIF2α phosphorylation. We show that impaired CA9 expression was not due to changes in HIF activity or CA9 mRNA stability. Using chromatin immunoprecipitation we show that the eIF2α-dependent translationally regulated gene ATF4 binds directly to the CA9 promoter and is associated with loss of the transcriptional repressive histone 3 lysine 27 tri-methylation mark. Loss or overexpression of ATF4 confirmed its role in CA9 induction during hypoxia. Our data indicate that expression of CA9 is regulated through both the HIF-1 and unfolded protein response hypoxia response pathways in vitro and in vivo.
- Jun 2009
The poorly developed vasculature in solid human tumors is responsible for a profound level of intra- and inter-tumor heterogeneity in oxygen concentration. High levels of hypoxia are associated with poor patient prognosis due in part to hypoxia-induced changes in cell metabolism, angiogenesis, invasiveness and resistance to therapy. Over the past decade several distinct oxygen sensing pathways that regulate the cellular response to hypoxia have been defined. These include transcriptional and translational responses initiated by oxygen-dependent stabilization of the HIF-1 transcription factor, activation of the unfolded protein response (UPR) and inhibition of the mTOR (mammalian target of rapamycin) kinase signaling pathway. Variations in the duration and severity of hypoxic stress differentially activate these responses and lead to substantial phenotypic variation amongst otherwise identical tumor cells. Nevertheless, several studies have provided links between HIF-, UPR- and mTOR-mediated signaling and the induction of autophagy. This process facilitates survival during metabolic stress and may also be an important mechanism for the removal of potentially toxic damaged proteins and organelles. We propose that overlapping mechanisms of autophagy regulation by HIF, mTOR and the UPR function to coordinately promote hypoxia tolerance and tumor cell survival.
The p53 tumor suppressor protein negatively regulates hypoxia-inducible factor 1α (HIF-1α). Here, we show that induction of p53 by the small-molecule RITA (reactivation of p53 and induction of tumor cell apoptosis) [2,5-bis(5-hydroxymethyl-2-thienyl) furan] (NSC-652287) inhibits HIF-1α and vascular endothelial growth factor expression in vivo and induces significant tumor cell apoptosis in normoxia and hypoxia in p53-positive cells. RITA has been proposed to stabilize p53 by inhibiting the p53-HDM2 interaction. However, induction of p53 alone was insufficient to block HIF-1α induced in hypoxia and has previously been shown to require additional stimuli, such as DNA damage. Here, we identify a new mechanism of action for RITA: RITA activates a DNA damage response, resulting in phosphorylation of p53 and γH2AX in vivo. Unlike other DNA damage response-inducing agents, RITA treatment of cells induced a p53-dependent increase in phosphorylation of the α subunit of eukaryotic initiation factor 2, requiring PKR-like endoplasmic reticulum kinase activity, and led to the subsequent downregulation of HIF-1α and p53 target proteins, including HDM2 and p21. Through the identification of a new mechanism of action for RITA, our study uncovers a novel link between the DNA damage response-p53 pathway and the protein translational machinery.
Acute pyelonephritis is one of the most common bacterial infections. Tissue-type plasminogen activator (tPA) is a potent fibrinolytic agent, but can play a role in inflammatory processes as well. We induced pyelonephritis in tPA(-/-) and C57BL/6 wild-type (WT) mice by intravesical inoculation with 10(10) CFU uropathogenic Escherichia coli 1677. The mice were killed after 24 and 48 h, after which bacterial outgrowth and cytokine levels in kidney homogenates were determined. Influx of neutrophils was quantified by myeloperoxidase-ELISA. Neutrophil phagocytosis and oxidative burst were measured. The tPA(-/-) kidneys contained significantly higher numbers of E. coli CFU, accompanied by higher levels of interleukin-1beta (IL-1beta) and tumour necrosis factor-alpha (TNF-alpha). The number of infiltrating neutrophils was similar in tPA(-/-) and WT mice at both time points, suggesting that tPA(-/-) neutrophils have a lower ability to eliminate E. coli. Phagocytosis of E. coli organisms was not diminished in tPA(-/-) neutrophils. Interestingly, tPA(-/-) neutrophils showed a significantly lower ability to generate an oxidative burst reaction upon stimulation with E. coli than WT neutrophils. Incubation with recombinant tPA reversed this effect completely. These results show that deletion of the tPA-gene in mice leads to lower bactericidal potential of tPA(-/-) neutrophils, which results in significantly more bacterial outgrowth during experimental pyelonephritis.
Ischemia-reperfusion injury is the leading cause of acute renal failure and determinant of renal-transplant outcome. Although many experimental studies show decreased injury and preserved renal function after dampening of the inflammatory response, surprisingly little progress has been made in the development of novel therapies.
Hypoxia is a common feature of solid tumors associated with therapy resistance, increased malignancy and poor prognosis. Several approaches have been developed with the hope of identifying patients harboring hypoxic tumors including the use of microarray based gene signatures. However, studies to date have largely ignored the strong time dependency of hypoxia-regulated gene expression. We hypothesized that use of time-dependent patterns of gene expression during hypoxia would enable development of superior prognostic expression signatures. Using published data from the microarray study of Chi et al., we extracted gene signatures correlating with induction during either early or late hypoxic exposure. Gene signatures were derived from in vitro exposed human mammary epithelial cell line (HMEC) under 0% or 2% oxygen. Gene signatures correlating with early and late up-regulation were tested by means of Kaplan-Meier survival, univariate, and multivariate analysis on a patient data set with primary breast cancer treated conventionally (surgery plus on indication radiotherapy and systemic therapy). We found that the two early hypoxia gene signatures extracted from 0% and 2% hypoxia showed significant prognostic power (log-rank test: p=0.004 at 0%, p=0.034 at 2%) in contrast to the late hypoxia signatures. Both early gene signatures were linked to the insulin pathway. From the multivariate Cox-regression analysis, the early hypoxia signature (p=0.254) was found to be the 4th best prognostic factor after lymph node status (p=0.002), tumor size (p=0.016) and Elston grade (p=0.111). On this data set it indeed provided more information than ER status or p53 status. The hypoxic stress elicits a wide panel of temporal responses corresponding to different biological pathways. Early hypoxia signatures were shown to have a significant prognostic power. These data suggest that gene signatures identified from in vitro experiments could contribute to individualized medicine.
Human tumors are characterized by temporal fluctuations in oxygen tension. The biological pathways that respond to the dynamic tumor microenvironment represent potential molecular targets for cancer therapy. Anoxic conditions result in eIF2alpha dependent inhibition of overall mRNA translation, differential gene expression, hypoxia tolerance and tumor growth. The signaling pathway which governs eIF2alpha phosphorylation has therefore emerged as a potential molecular target. In this study, we investigated the role of eIF2alpha in regulating mRNA translation and hypoxia tolerance during moderate hypoxia. Since other molecular pathways that regulate protein synthesis are frequently mutated in cancer, we also assessed mRNA translation in a panel of cell lines from different origins. Immortalized human fibroblast, transformed mouse embryo fibroblasts (MEFs) and cells from six cancer cell lines were exposed to 0.2% or 0.0% oxygen. We assayed global mRNA translation efficiency by polysome analysis, as well as proliferation and clonogenic survival. The role of eIF2alpha was assessed in MEFs harboring a homozygous inactivating mutation (S51A) as well as in U373-MG cells overexpressing GADD34 (C-term) under a tetracycline-dependent promoter. The involvement of eIF4E regulation was investigated in HeLa cells stably expressing a short hairpin RNA (shRNA) targeting 4E-BP1. All cells investigated inhibited mRNA translation severely in response to anoxia and modestly in response to hypoxia. Two independent genetic cell models demonstrated that inhibition of mRNA translation in response to moderate hypoxia was dependent on eIF2alpha phosphorylation. Disruption of eIF2alpha phosphorylation caused sensitivity to hypoxia and anoxia. Disruption of eIF2alpha phosphorylation is a potential target for hypoxia-directed molecular cancer therapy.
- Jun 2007
Human tumors are characterized by large variations in oxygen concentration and hypoxic tumors are associated with poor prognosis. In addition, tumors are subjected to periodic changes in oxygenation characterized by hypoxia followed by reoxygenation. Cellular adaptation to hypoxia is well documented, nevertheless little is known about adaptive mechanisms to reoxygenation. Here, we investigate the changes in protein expression during reoxygenation using proteomics. HeLa cervix carcinoma cells were exposed to 4h of hypoxia (<0.01% O(2)) followed by 1h of reoxygenation. The cellular proteome was examined using 2D gel electrophoresis coupled with mass spectrometry. Validation and investigation of the underlying basis for induced protein expression was investigated using Western blot analysis and quantitative RT-PCR. We identified proteins involved in several cellular processes that are responsible for regulating RNA metabolism, protein synthesis and degradation, including ribosomal protein P0, VCP/p97 and FUSE binding protein 2. Our results suggest that these newly identified proteins function in pathways that may assist in the recovery of ER stress and protein synthesis during reoxygenation. These proteins may thus be important determinants of the behaviour and survival of tumor cells to transient hypoxic exposures.
The urokinase plasminogen activator receptor (uPAR) is expressed at the cell surface of inflammatory cells and plays an important role in neutrophil migration. To investigate the in vivo role of uPAR during urinary tract infection, acute pyelonephritis was induced in uPAR-/- and wild-type (WT) mice by intravesical inoculation with 1 x 10(9) colony-forming units (CFU) of uropathogenic Escherichia coli. Mice were killed after 24 and 48 h, after which bacterial outgrowth and cytokine levels in kidney homogenates were determined. Influx of neutrophils was quantified by myeloperoxidase-enzyme-linked immunosorbent assay. uPAR-/- kidneys had significantly higher numbers of E. coli CFU, accompanied by higher levels of interleukin-1beta (IL-1beta), IL-6, keratinocyte-derived chemokine (KC), macrophage inflammatory protein-2 (MIP-2), and tumor necrosis factor-alpha (TNF-alpha). However, the number of infiltrating neutrophils was similar in uPAR-/- and WT mice at both time points, suggesting that uPAR-/- neutrophils have a lower ability to eliminate E. coli. To further investigate this, neutrophil oxidative burst and phagocytosis was measured. The generation of reactive oxygen species upon stimulation with E. coli was not diminished in uPAR-/- neutrophils compared with WT. Interestingly, uPAR-/- neutrophils displayed significantly impaired phagocytosis of E. coli organisms compared with WT neutrophils. We conclude that uPAR is crucially involved in host defense through phagocytosis during E. coli induced acute pyelonephritis.
Escherichia coli is the most common pathogen found in urinary tract infections (UTIs), mainly affecting children and women. We report that CD44, a hyaluronic acid (HA) binding protein that mediates cell-cell and cell-matrix interactions, facilitates the interaction of E. coli with urothelial cells and thus the infection of the host. We found that CD44 is constitutively expressed on urothelial cells and that HA accumulates in E. coli-induced UTI. In CD44-deficient mice, the bacterial outgrowth was dramatically less compared with wild-type mice despite similar granulocyte influx in the bladder and in the kidney as well as comparable cytokines/chemokines levels in both genotypes. E. coli was able to bind HA, which adhered to CD44-positive tubular epithelial cells. Most importantly, the interaction of CD44 on tubular epithelial cells with HA facilitated the migration of E. coli through the epithelial monolayer. The results provide evidence that CD44 on urothelial cells facilitates E. coli UTI. Disruption of the interaction between CD44 and HA in the bladder may provide a new approach to prevent and to treat UTI.
As CD44 is involved in the activation, proliferation, adhesion, and extravasation of lymphocytes, we hypothesized that CD44 could be involved in the pathogenesis of acute renal allograft rejection. Renal biopsies and plasma were collected from patients suffering an episode of acute renal allograft rejection. CD44 and its ligands, hyaluronic acid (HA) and osteopontin, were analyzed retrospectively by immunohistochemistry and, computer-aided, morphometric analysis. Soluble CD44 (sCD44) and osteopontin in the plasma were determined by enzyme-linked immunosorbent assay. During acute rejection episodes, CD44 and its ligands, HA and osteopontin, were upregulated in the renal allograft. Also, increased sCD44 plasma levels were observed, which correlated with both tubular expression of CD44 and the extent of infiltrate. No differences could be detected between the different pathologic grades of rejection. Upregulation of tubular CD44 and increased levels of circulating sCD44 may reflect a common pathogenic mechanism during acute renal rejection and could be useful markers in the diagnosis of acute renal rejection.
- Apr 2006
CD44 is a glycoprotein that is involved in inflammation and cell-cell/cell-matrix interactions, is upregulated in the kidney upon injury, and leads to fibrosis through enhancement of TGF-beta1 signaling. Absence of CD44 prevents development of renal fibrosis in unilateral ureteral obstruction (UUO). A hallmark of development of renal fibrosis is the degeneration of the capillary network. This study shows that CD44 is upregulated on capillary endothelial cells during UUO. For elucidation of the role of CD44 on peritubular endothelial cells in UUO, capillary network degeneration was compared in CD44+/+ and CD44-/- mice. As expected, degeneration of the capillary network was observed in CD44+/+ mice during UUO, associated with increased endothelial apoptosis. However, in the absence of CD44, degeneration of the network is prevented as a result of a decrease in the rate of apoptosis in endothelial cells. The divergence in endothelial apoptosis is not correlated to differential vascular endothelial growth factor or thrombospondin-1 expression. For further investigation of capillary regression, CD44+/+ and CD44-/- peritubular capillary endothelial cell lines were established. With the use of these cells, it is shown that interaction between CD44 and its ligand hyaluronic acid enhances the proapoptotic effect of TGF-beta1 but not thrombospondin-1 on endothelial cells, contributing to the degeneration of the capillary network. Blocking interaction between hyaluronic acid and CD44 therefore may be a potential therapeutic opportunity to preserve the capillary network and prevent the development of fibrosis in chronic renal disease.
Acute renal failure is often the result of ischemia-reperfusion (I/R) injury. Neutrophil influx is an important damaging event in I/R. Tissue-type plasminogen activator (tPA) not only is a major fibrinolytic agent but also is involved in inflammatory processes. A distinct upregulation of tPA after I/R, with de novo tPA production by proximal renal tubules, was found. For investigating the role of tPA in I/R, renal ischemia was induced in tPA-/- and wild-type (WT) mice by clamping both renal arteries for 35 min followed by reperfusion. Mice were killed 1, 5, and 10 d after reperfusion. After 1 d, tPA-/- mice displayed significantly less neutrophil influx into the interstitial area compared with WT mice. In addition, tPA-/- mice showed quicker recovery of renal function than WT mice. The protocol was repeated after injection of tPA-antisense oligonucleotides into WT mice, leading to even more explicit results: Antisense-treated mice showed less histologic damage, better renal function, and less neutrophil influx than control mice. Surprising, complement C3 concentration, levels of proinflammatory cytokines and chemokines, intercellular adhesion molecule-1 expression, and matrix metalloproteinase activity were similar in WT and tPA-/- mice. Plasmin activity levels in WT and tPA-/- kidneys were also comparable, indicating that tPA influences neutrophil influx into ischemic renal tissue independent from plasmin generation. This study shows that targeting tPA could be of therapeutic importance in treating I/R injury by diminishing neutrophil influx and preserving renal function.
Since CD44 is involved in activation, proliferation, rolling and extravasation of lymphocytes, we hypothesized that it could be involved in the pathophysiology of acute renal allograft rejection. Plasma and peripheral blood mononuclear cells (PBMCs) were collected from patients 24 h prior to transplantation and analysed retrospectively. Soluble CD44, interleukin-2 receptor (IL-2R), intracellular adhesion molecule-1 (ICAM-1) and C-reactive protein (CRP) in plasma were determined by enzyme-linked immunosorbent assay (ELISA). Cellular CD44 expression on peripheral lymphocytes was determined by flow cytometric analysis. Patients who later developed renal allograft rejection had statistically significantly increased soluble CD44 levels, but not soluble ICAM-1, IL-2R or CRP in plasma prior to transplantation. In addition, cellular CD44 on T-lymphocytes was decreased 24 h prior to transplantation in patients that would reject their allograft, compared with patients without rejection. Additionally, plasma CD44 and cellular CD44 revealed an inversely proportional correlation. Lipopolysaccharide (LPS)-induced immune activation did not influence plasma or cellular CD44 levels in healthy volunteers, suggesting that more specific factors influence the shedding of CD44 on T lymphocytes, leading to increased risk of renal allograft rejection. Although the exact mechanism remains to be elucidated and further research is required, soluble CD44 levels and cellular surface CD44 on T lymphocytes prior to transplantation might be useful as predictive markers for the occurrence of acute renal rejection.
TLRs are conserved pattern recognition receptors that detect motifs of pathogens and host material released during injury. For unknown reasons, renal TLR2 mRNA is mainly expressed by tubular cells and is enhanced upon renal ischemia/reperfusion (I/R) injury. We evaluated the role of TLR2 in I/R injury using TLR2-/- and TLR2+/+ mice, TLR2 antisense oligonucleotides, and chimeric mice deficient in leukocyte or renal TLR2. Tubular cells needed TLR2 to produce significant cytokine and chemokine amounts upon ischemia in vitro. TLR2 played a proinflammatory and detrimental role in vivo after I/R injury, as reflected by a reduction in the amount of local cytokines and chemokines, leukocytes, and the level of renal injury and dysfunction in TLR2-/- mice compared with controls. Analysis of chimeric mice suggested that TLR2 expressed on renal parenchyma plays a crucial role in the induction of inflammation and injury. TLR2-antisense treatment protected mice from renal dysfunction, neutrophil influx, and tubular apoptosis after I/R injury compared with nonsense treatment. In summary, we identified renal-associated TLR2 as an important initiator of inflammatory responses leading to renal injury and dysfunction in I/R injury. These data imply that TLR2 blockade could provide a basis for therapeutic strategies to treat or prevent renal ischemic injury.
Inflammation contributes to renal ischemia reperfusion (I/R) injury, potentially causing renal dysfunction. The inflammatory infiltrate mainly consists of neutrophils, which are deleterious for the renal tissue. Because CD44 is expressed by neutrophils and is rapidly upregulated by capillary endothelial cells after I/R injury, it was hypothesized that CD44 might play an important role in the development of I/R injury. This study showed that rapid CD44 upregulation on renal capillary endothelial cells mediates neutrophil recruitment to the postischemic tissue. Hence, CD44 deficiency led to decreased influx of neutrophils regardless of comparable levels in chemotactic factors expressed in the kidney. The reduced influx of neutrophils was associated with preserved renal function and morphology. Adoptive transfer experiments of labeled neutrophils revealed that endothelial CD44 rather than neutrophil CD44 mediates neutrophil migration. Activation of neutrophils increased cell-surface expression of hyaluronic acid (HA). Altogether, a novel mechanism in the recruitment of neutrophils that involves interaction of endothelial CD44 and neutrophil HA was found. Either blocking endothelial CD44 or removal of neutrophil HA decreased rolling and adhesion of neutrophils. Administration of anti-CD44 to mice reduced the influx of neutrophils into the postischemic tissue, associated with renal function preservation. Therefore, anti-CD44-based therapies may contribute to prevent or reduce renal I/R injury.
- Apr 2004
CD44 is a glycoprotein involved in inflammation and cell-cell/cell-matrix interactions. CD44 is upregulated in the kidney upon injury; however, its role in the pathogenesis of renal damage and fibrosis remains largely unknown. The authors show that mice lacking CD44 developed more tubular damage, associated with decreased proliferation and increased apoptosis of tubular epithelial cells, but less renal fibrosis after unilateral ureteral obstruction. In addition, impaired influx of macrophages and decreased accumulation of myofibroblasts was observed in the obstructed kidney of CD44(-/-) mice compared with CD44(+/+) mice. Hepatocyte growth factor (HGF) and transforming growth factor-beta1 (TGF-beta1) exert reciprocal functions in the progression of renal diseases and interact with CD44 in vitro. For the first time, the authors establish diminished HGF-signaling, via its high affinity receptor c-Met, in the absence of CD44 in vivo. In parallel, the signaling of TGF-beta1 reflected by the relative phosphorylation and nuclear translocation of Smad-2 and Smad-3 was reduced in the obstructed kidney of CD44(-/-) mice. In conclusion, CD44 exerts protective effects on tubuli but contributes to renal fibrogenesis at least in part through enhancement of HGF and TGF-beta1 signaling pathway in obstructive nephropathy.
- Nov 2003
Progressive renal fibrosis occurs via common pathophysiologic mechanisms, regardless of the primary underlying disease. This cascade includes release of cytokines/chemokines and toxic molecules, interstitial inflammation, tubular cell damage, accumulation of myofibroblasts, and finally, fibrosis. Hepatocyte growth factor (HGF) and transforming growth factor-beta1 (TGF-beta1) are key molecules in this cascade that, in general, exert opposite actions. Hepatocyte growth factor promotes, to some extent, inflammation, protects tubular epithelial cells, blocks myofibroblast transition, and contributes to tissue remodeling. In contrast, TGF-beta1 has powerful anti-inflammatory actions, promotes apoptosis, induces myofibroblast transition, and is a strong pro-fibrotic agent. The mechanisms which orchestrate the reciprocal actions of HGF and TGF-beta1 are still largely unknown and are probably multiple. One of these mechanisms involves the selective up-regulation of CD44 in damaged kidney. The glomerular and tubular expression of CD44 closely correlates with the degree of renal damage, and CD44 has been shown to facilitate the action of both HGF and TGF-beta1. Moreover, during chronic obstructive nephropathy CD44 knock-out mice display much more tubular damage but develop less fibrosis in the course of the renal disease. These histologic findings are associated with impairment of signaling pathways of both HGF and TGF-beta1. The development of new therapeutic strategies aimed at preventing progression of renal diseases that are based on HGF and/or TGF-beta1 may take in account the pivotal role of CD44 expression in the functions of both molecules.
- Aug 2002
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent inducer of apoptosis in tumor cells but not in healthy cells. Similar to CD95 ligand (CD95L), TRAIL signaling requires ligand-receptor interaction; the downstream signaling molecules, such as Fas-associated death domain and caspase-8, also seem similar. Using cells stably expressing TRAIL and CD95L, we show that both TRAIL and CD95L induce apoptosis in the rat colon carcinoma cell line CC531. The mitochondrial damage (loss of mitochondrial membrane potential (MMP) and release of cytochrome c) observed after co-incubation with TRAIL-expressing cells occurs much earlier than that observed with CD95L-expressing cells. The decrease in MMP induced by both ligands was caspase-8-mediated; no difference in caspase-8 activation by TRAIL and CD95L was found. TRAIL, but not CD95L, induced activation of caspase-10. bcl-2 overexpression could not prevent TRAIL-induced mitochondrial dysfunction, whereas it completely prevented CD95L-mediated loss of MMP and cytochrome c release. The selective effect of TRAIL on tumor cells and the apparent inability of bcl-2 to block TRAIL-induced apoptosis suggest that TRAIL may offer a lead for cancer therapy in the future.