[Show abstract][Hide abstract] ABSTRACT: The ability of the retinoid X receptors (RXRs) specific agonists (targretin [TRG] and UAB30) to alter rat liver gene and protein expression was determined using Affymetrix Exon arrays and high-performance liquid chromatography – tandem mass spectrometry (LC-MS/MS). TRG profoundly increases triglycerides levels while UAB30 does not. The expression patterns of transcripts or proteins from rat liver treated with TRG or UAB-30 were different from controls and each other. There were six times more gene transcripts identified than proteins. Differentially expressed RNAs or proteins were mapped into known gene ontology (GO) categories and GeneGo Metacore (KEGG) pathway maps. The GO categories which were highly overrepresented with differentially expressed RNAs (P < 10−16) were also overrepresented at the protein level. This high concordance of GO Terms was achieved despite the fact that typically ≤1/3 of the elements identified by gene expression were identified by proteomics. Within these GO categories, the magnitude of alterations induced by RXR agonists at the transcript and protein levels were correlated. When GO categories with moderate overrepresentation (10−5 < P < 10−9) were examined, there was greater discordance between the transcript and protein data. Examination of KEGG pathway maps with highly significant changes at both the protein and the RNA levels showed that the individual proteins/genes altered were often the same and changes were of similar magnitude; while KEGG pathways showed limited statistical significance and exhibited minimal overlap. Finally, metabolomics analysis of liver and serum identified altered expression of metabolites related to fatty acid oxidation and bile acid metabolism that were consistent with transcript/protein changes. We observed significant concordance between genomics and proteomics implying either can identify pathways modulated and can indirectly predict resulting physiologic effects.
Pharmacology Research & Perspectives. 12/2014; 2(6).
[Show abstract][Hide abstract] ABSTRACT: Honokiol is an important bioactive compound found in the bark of Magnolia tree. It is a non-adipogenic PPARγ agonist, and capable of inhibiting the growth of a variety of tumor types both in vitro and in xenograft models. However, to fully appreciate the potential chemopreventive activity of honokiol, a less artificial model system is required. To that end, this study examined the chemopreventive efficacy of honokiol in an initiation model of squamous cell lung cancer (SCC). This model system uses the carcinogen N-nitroso-trischloroethylurea (NTCU) which is applied topically, reliably triggering the development of SCC within 24-26 weeks. Administration of honokiol significantly reduced the percentage of bronchial that exhibit abnormal lung SCC histology from 24.4% bronchial in control to 11.0% bronchial in honokiol treated group (p= 0.01) while protecting normal bronchial histology (present in 20.5% of bronchial in control group and 38.5% of bronchial in honokiol treated group (p= 0.004)). P63 staining at the SCC site confirmed the lung SCCs phenotype. In vitro studies revealed that honokiol inhibited lung SCC cells proliferation, arrested cells at the G1/S cell cycle checkpoint, while also leading to increased apoptosis. Our study showed that interfering with mitochondrial respiration is a novel mechanism by which honokiol increased generation of reactive oxygen species (ROS) in the mitochondria, triggered apoptosis, and finally leads to the inhibition of lung SCC. This novel mechanism of targeting mitochondrial suggests honokiol as a potential lung SCC chemopreventive agent.
Cancer Prevention Research 09/2014; · 5.27 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Urinary bladder cancer prevention studies were performed with the nonsteroidal anti-inflammatory drugs (NSAIDs) naproxen (standard NSAID with a good cardiovascular profile), sulindac, and their nitric oxide (NO) derivatives. Additionally, the effects of the ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), alone or combined with a suboptimal dose of naproxen or sulindac was examined. Agents were evaluated at their human equivalent doses (HEDs), as well as at lower doses. In the hydroxybutyl(butyl)nitrosamine (OH-BBN) model of urinary bladder cancer, naproxen (400 or 75 ppm) and sulindac (400 ppm) reduced the incidence of large bladder cancers by 82, 68 and 44%, respectively, when the agents were initially given 3 months after the final dose of the carcinogen; microscopic cancers already existed. NO-naproxen was highly effective, while NO-sulindac was inactive. To further compare naproxen and NO-naproxen, we examined their effects on gene expression in rat livers following a 7 day exposure. Limited, but similar, gene expression changes in the liver were induced by both agents, implying that the primary effects of both are mediated by the parent NSAID. When agents were initiated 2 weeks after the last administration of OH-BBN, DFMO at 1000 ppm had limited activity, a low dose of naproxen (75 ppm) and sulindac (150 ppm) were highly and marginally effective. Combining DFMO with suboptimal doses of naproxen had minimal effects whereas the combination of DMFO and sulindac was more active than either agent alone. Thus, naproxen and NO-naproxen were highly effective, while sulindac was moderately effective in the OH-BBN model at their HEDs.
Cancer Prevention Research 12/2013; · 4.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Naproxen ((S)-6-methoxy-α-methyl-2-naphthaleneacetic acid) is a potent nonsteroidal anti-inflammatory drug that inhibits both COX-1 and COX-2 and is widely used as an over-the-counter medication. Naproxen exhibits analgesic, anti-pyretic, and anti-inflammatory activities. Naproxen, as well as other NSAIDS, has been reported to be effective in the prevention of urinary bladder cancer in rodents. However, potential targets other than the COX isozymes have not been reported. We examined potential additional targets in urinary bladder cancer cells and in rat bladder cancers. Computer kinase profiling results suggested that phosphatidylinositol 3-kinase (PI3-K) is a potential target for naproxen. In vitro kinase assay data revealed that naproxen interacts with PI3-K and inhibits its kinase activity. Pull-down binding assay data confirmed that PI3-K directly binds with naproxen in vitro and ex vivo. Western blot data showed that naproxen decreased phosphorylation of Akt, and subsequently decreased Akt signaling in UM-UC-5 and UMUC-14 urinary bladder cancer cells. Furthermore, naproxen suppressed anchorage-independent cell growth and decreased cell viability by targeting PI3-K in both cell lines. Naproxen caused an accumulation of cells at the G1 phase mediated through CDK4, cyclin D1 and p21. Moreover, naproxen induced significant apoptosis, accompanied with increased levels of cleaved caspase 3, caspase 7, and poly (ADP-ribose) polymerase (PARP) in both cell types. Naproxen-induced cell death was mainly due to apoptosis in which a prominent down-regulation of Bcl-2 and up-regulation of Bax were involved. Naproxen also caused apoptosis and inhibited Akt phosphorylation in rat urinary bladder cancers induced by N-butyl-N-(4-hydroxybutyl)-nitrosamine (OH-BBN).
Cancer Prevention Research 12/2013; · 4.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent clinical trials raised concerns regarding the cardiovascular toxicity of selective cyclooxygenase-2 (COX-2) inhibitors and COX-1 is now being reconsidered as a target for chemoprevention. Our aims were to determine whether selective COX-1 inhibition could delay or prevent cancer development and also clarify the underlying mechanisms. Data clearly showed that COX-1 was required for maintenance of malignant characteristics of colon cancer cells or tumor promoter-induced transformation of pre-neoplastic cells. We also successfully applied a ligand docking computational method to identify a novel selective COX-1 inhibitor, 6-C-(E-phenylethenyl)-naringenin (designated herein as 6CEPN). 6CEPN could bind to COX-1 and specifically inhibited its activity both in vitro and ex vivo. In colorectal cancer cells, it potently suppressed anchorage-independent growth by inhibiting COX-1 activity. 6CEPN also effectively suppressed tumor growth in a 28-day colon cancer xenograft model without any obvious systemic toxicity. Taken together, COX-1 plays a critical role in human colorectal carcinogenesis, and this specific COX-1 inhibitor merits further investigation as a potential preventive agent against colorectal cancer.
[Show abstract][Hide abstract] ABSTRACT: Recent clinical trials raised concerns regarding the cardiovascular toxicity of selective cyclooxygenase-2 (COX-2) inhibitors. Many active dietary factors are reported to suppress carcinogenesis by targeting COX-2. A major question was accordingly raised: why has the lifelong use of phytochemicals that likely inhibit COX-2 presumably not been associated with adverse cardiovascular side effects. To answer this question, we selected a library of dietary-derived phytochemicals and evaluated their potential cardiovascular toxicity in human umbilical vein endothelial cells. Our data indicated that the possibility of cardiovascular toxicity of these dietary phytochemicals was low. Further mechanistic studies revealed that the actions of these phytochemicals were similar to aspirin in that they mainly inhibited COX-1 rather than COX-2, especially at low doses.
PLoS ONE 10/2013; 8(10):e76452. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aromatase inhibitors(AIs)are effective in therapy/prevention of ER+ breast cancers. Rats bearing methylnitrosourea(MNU)-induced ER+ mammary cancers were treated with the AI vorozole (1.25 mg/kg BW/day) for 5 days. RNA expression showed 162 down-regulated and 180 up-regulated (p < 0.05 and fold change >1.5) genes. Genes modulated by vorozole were compared with published data from four clinical neoadjuvant trials employing AIs (anastrozole or letrozole). More than thirty genes and multiple pathways exhibited synchronous changes in animal and human data sets. Cell cycle genes related to chromosome condensation in prometaphase (APC pathway, including Aurora-A kinase, BUBR1B, TOP2, Cyclin A, Cyclin B CDC2 and TPX-2) were down-regulated in animal and human studies reflecting the strong anti-proliferative effects of AIs. Comparisons of rat arrays with a cell culture study where estrogen was removed from MCF-7 cells showed decreased expression of E2F1-modulated genes as a major altered pathway. Alterations of the cell cycle and E2F related genes were confirmed in a large independent set of human samples (81 pairs baseline and 2 weeks anastrozole treatment). Decreases in proliferation related genes were confirmed at the protein level for Cyclin A2, BuRB1, cdc2, Pttg and TPX-2. Interestingly, the proteins down-regulated in tumors were similarly down-regulated in vorozole treated normal rat mammary epithelium. Finally, decreased expression of known estrogen responsive genes (including TFF 1,3, progesterone receptor, etc.) were decreased in the animal model. These studies demonstrate that gene expression changes (pathways and individual genes) are similar in humans and the rat model.
Cancer Prevention Research 09/2013; · 4.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The UVB component of sunlight, which causes DNA damage and inflammation, is the major cause of nonmelanoma skin cancer (NMSC), the most prevalent of all cancers. Nonsteroidal anti-inflammatory drugs (NSAIDs) and coxibs have been shown to be effective chemoprevention agents in multiple preclinical trials, including NMSC, colon and urinary bladder cancer. NSAIDs, however, cause gastrointestinal irritation, which led to the recent development of nitric oxide (NO) derivatives that may partially ameliorate this toxicity. This study compared the efficacy of several NSAIDs and NO-NSAIDs on UV-induced NMSC in SKH-1 hairless mice and determined whether various short-term biomarkers were predictive of long-term tumor outcome with these agents. Naproxen at 100 (p>.05) and 400 ppm (p<.01) in the diet reduced tumor multiplicity by 26 and 63% respectively. The NO-naproxen at slightly lower molar doses shows similar activities. Aspirin at 60 or 750 ppm in the diet reduced tumor multiplicity by 19 and 50%; while the equivalent doses (108 and 1350 ppm) were slightly less effective. Sulindac at 25 and 150 ppm in the diet doses far below the Human Equivalent Dose, was the most potent NSAID with reductions of 50 and 94% respectively. In testing short-term biomarkers we found that agents that reduce UV-induced prostaglandin E2 synthesis and/or inhibit UV-induced keratinocyte proliferation yielded long-term tumor efficacy.
Cancer Prevention Research 05/2013; · 4.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The phosphatidylinositol 3-kinase (PI3-K)/Akt and mammalian target of rapamycin (mTOR) signaling pathway plays a critical role in cell survival and proliferation and is often aberrantly activated in many types of cancer. The mTOR kinase protein, one of the key molecules in this pathway, has been shown to be an important target for cancer therapy. In the present study, a ligand docking method was used to screen for novel scaffold mTOR inhibitors. Sixty thousand compounds in the Natural Product Database (NPD) were screened against the mTOR homologous structure and 13 commercially available compounds listed in the top-ranked 100 compounds were selected for further examination. Compound (E)-3-(4-(benzo[d][1,3]dioxol-5-yl)-2-oxobut-3-en-1-yl)- 3-hydroxyindolin-2-one; designated herein as 3HOI-BA-01) was then selected for further study of its antitumor activity. An in vitro study demonstrated that 3HOI-BA-01 inhibited mTOR kinase activity in a dose-dependent manner by directly binding with mTOR. In a panel of NSCLC cells, the compound also attenuated mTOR downstream signaling, including the phosphorylation of p70S6K, S6, and Akt, resulting in G1 cell cycle arrest and growth inhibition. Results of an in vivo study demonstrated that i.p. injection of 3HOI-BA-01 in A549 lung tumor-bearing mice effectively suppressed cancer growth without affecting the body weight of the mice. The expression of downstream signaling molecules in the mTOR pathway in tumor tissues was also reduced after 3HOI-BA-01 treatment. Taken together, we identified 3HOI-BA-01 as a novel and effective mTOR inhibitor.
Molecular Cancer Therapeutics 03/2013; · 5.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: EGFR inhibitors are employed in therapy of lung and pancreatic cancers, and effectively prevent cancers in multiple animal models. Although daily dosing with erlotinib is effective, weekly dosing may reduce toxicity and have advantages, particularly for prevention. We tested alternative dosing regimens for preventive/therapeutic efficacy in a rat mammary cancer model. For prevention, erlotinib was administered by gavage beginning 5 days after MNU. For therapy and biomarker studies, rats with palpable mammary cancers were treated for six weeks or for 6 days, respectively. Prevention. Experiment A. Erlotinib (6 mg/Kg BW/day, i.g.): daily (7x/week); one day on/one day off; and two days on/two days off. All regimens decreased tumor incidence, increased tumor latency, and decreased cancer multiplicity vs controls (P<.01). However, intermittent dosing was less effective than daily dosing (P<.05). Experiment B. Erlotinib (6 mg/kg BW/day) daily or two days on/two days off; or 1x/week at 42 mg/kg BW. All regimens reduced cancer incidence and multiplicity vs controls (P<.01). Interestingly, daily and weekly dosing were equally effective (P>0.5). Experiment C. Erlotinib administered at 42 or 21 mg/kg BW, 1x/week, decreased tumor incidence and multiplicity (P<.01). Pharmacokinetics. Erlotinib had a serum half-life of ≤8 hours, and weekly treatment yielded effective serum levels for <48 hours. Therapy. Daily or weekly treatment of cancer bearing rats reduced mammary tumor size 25-35%, while control cancers increased >250%. Biomarkers. Levels of phosphorylated ERK were strongly decreased in rats treated daily/weekly with erlotinib. Thus, altering the dosing of erlotinib retained most of its preventive and therapeutic efficacy.
Cancer Prevention Research 03/2013; · 4.89 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Examining three RXR agonists [Targretin (TRG), UAB30, and 4-methyl-UAB30 (4-Me-UAB30)], all inhibited mammary cancer in rodents and two (TRG and 4-Me-UAB30) strikingly increased serum triglycerides levels. Agents were administered in diets to female Sprague-Dawley rats. Liver RNA was isolated and microarrayed on the Affymetrix GeneChip Rat Exon 1.0 ST array. Statistical tests identified genes that exhibited differential expression and fell into groups, or modules, with differential expression among agonists. Genes in specific modules were changed by one two or all three agonists. An interactome analysis assessed the effects on genes which heterodimerize with known nuclear receptors. For PPARα/RXR activated genes, the strongest response was TRG > 4-Me-UAB30>UAB30. Many LXR/RXR related genes (e.g., SCD-1 and SREBP-1c which are associated with increased triglycerides) were highly expressed in TRG and 4-Me-UAB30 but not UAB30 treated livers. There was minimal expression changes associated with RAR or VDR heterodimers by any of the agonists. UAB30 unexpectedly and uniquely activated genes associated with the Ah receptor (Cyp1a1, Cyp1a2, Cyp1b1 and NQO1). Based on the Ah receptor activation, UAB30 was tested for its ability to prevent DMBA-induced mammary cancers, presumably by inhibiting DMBA activation, and was highly effective. Gene expression changes were determined by RT-PCR in rat livers treated with Targretin for 2.3, 7 and 21 days. These showed similar gene expression changes at all three time points; arguing some steady state effect. Different patterns of gene expression among the agonists provided insight into molecular differences, and allowed one to predict certain physiologic consequences of agonist treatment.
[Show abstract][Hide abstract] ABSTRACT: The sigma-2 receptor is expressed in higher density in proliferating (P) tumor cells versus quiescent (Q) tumor cells, thus providing an attractive target for imaging the proliferative status (i.e., P:Q ratio) of solid tumors. Here we evaluate the utility of the sigma-2 receptor ligand 2-(2-[(18)F]fluoroethoxy)-N-(4-(3,4-dihydro-6,7-dimethoxyisoquinolin-2(1H)-yl)butyl)-5-methyl-benzamide, [(18)F]ISO-1, in two different rodent models of breast cancer. In the first study, small animal Positron Emission Tomography (PET) imaging studies were conducted with [(18)F]ISO-1 and (18)FDG in xenografts of mouse mammary tumor 66 and tracer uptake was correlated with the in vivo P:Q ratio determined by flow cytometric measures of BrdU-labeled tumor cells. The second model utilized a chemically-induced (N-methyl-N-nitrosourea [MNU]) model of rat mammary carcinoma to correlate measures of [(18)F]ISO-1 and FDG uptake with MR-based volumetric measures of tumor growth. In addition, [(18)F]ISO-1 and FDG were used to assess the response of MNU-induced tumors to bexarotene and Vorozole therapy. In the mouse mammary 66 tumors, a strong linear correlation was observed between the [(18)F]ISO-1 tumor: background ratio and the proliferative status (P:Q ratio) of the tumor (R = 0.87). Similarly, measures of [(18)F]ISO-1 uptake in MNU-induced tumors significantly correlated (R = 0.68, P<0.003) with changes in tumor volume between consecutive MR imaging sessions. Our data suggest that PET studies of [(18)F]ISO-1 provide a measure of both the proliferative status and tumor growth rate, which would be valuable in designing an appropriate treatment strategy.
PLoS ONE 01/2013; 8(9):e74188. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction: In a variety of cancers there is evidence that specific regimens can prevent or significantly delay the development of cancer. Thus, for breast cancer (ER+) use of SERMs or aromatase inhibitors can substantially decrease tumor incidence. For cervical cancer, HPV vaccination will inhibit long term cancer incidence. For colon cancer, the second greatest cancer killer, administration of aspirin and other NSAIDs decreases advanced colon adenomas in Phase II trials and epidemiologic data support their ability to prevent colon cancer. To date prevention trials in the area of lung cancer have shown minimal efficacy. Areas covered: The paper examines and discusses in greater detail certain promising agents which the authors have tested either preclinically and or in early phase clinical trials. These agents include RXR agonists, EGFr inhibitors, NSAIDs and Triterpenoids. Other agents including glucocorticoids, pioglitazone and iloprost are briefly mentioned. In addition, the paper presents various types of potential Phase II lung cancer prevention trials and describes their strengths and weaknesses. The potential use of various biomarkers as endpoints in trials e.g. histopathology, non-specific biomarkers (e.g., Ki67, cyclin D expression, apoptosis) and molecular biomarkers (e.g. specific phosphorylated proteins, gene expression etc.) is presented. Finally, we examine at least one approach, the use of aerosols, which may diminish the systemic toxicity associated with certain of these agents. Expert Opinion: The manuscript presents: a) a number of promising agents which appear applicable to further Phase II prevention trials; b) approaches to defining potential preventive agents as well; c) approaches which might mitigate the side effects associated with potential agents most specifically the use of aerosols. Finally, we discuss biomarker studies both preclinical and clinical which might help support potential Phase II trials. The particular appeal to the preclinical studies is that they can be followed to a tumor endpoint. We hope that this will give the reader further background and allow one to appreciate the potential and some of the hurdles associated with lung cancer chemoprevention.
Expert Opinion on Investigational Drugs 11/2012; · 4.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. In the present study, we used a ligand-docking method to explore the novel scaffold of potential Aurora B inhibitors. One thousand compounds from our in-house compound library were screened against the Aurora B structure and one compound, (E)-3-((E)-4-(benzo[d][1,3]dioxol-5-yl)-2-oxobut-3-en-1-ylidene)indolin-2-one (designated herein as HOI-07) was selected for further study. HOI-07 potently inhibited in vitro Aurora B kinase activity in a dose-dependent manner, without obvious inhibition of another 49 kinases, including Aurora A. This compound suppressed Aurora B kinase activity in lung cancer cells, evidenced by the inhibition of the phosphorylation of histone H3 on Ser10 in a dose- and time-dependent manner. This inhibition resulted in apoptosis induction, G2/M arrest, polyploidy cells, and attenuation of cancer cell anchorage-independent growth. Moreover, knocking down the expression of Aurora B effectively reduced the sensitivity of cancer cells to HOI-07. Results of an in vivo xenograft mouse study showed that HOI-07 treatment effectively suppressed the growth of A549 xenografts, without affecting the body weight of mice. The expression of phospho-histone H3, phospho-Aurora B, and Ki-67 was also suppressed in the HOI-07 treatment group. Taken together, we identified HOI-07 as a specific Aurora B inhibitor, which deserves further investigation.
[Show abstract][Hide abstract] ABSTRACT: Ceftriaxone, an FDA-approved third-generation cephalosporin antibiotic, has antimicrobial activity against both gram-positive and gram-negative organisms. Generally, ceftriaxone is used for a variety of infections such as community-acquired pneumonia, meningitis and gonorrhea. Its primary molecular targets are the penicillin-binding proteins. However, other activities of ceftriaxone remain unknown. Herein, we report for the first time that ceftriaxone has antitumor activity in vitro and in vivo. Kinase profiling results predicted that Aurora B might be a potential 'off' target of ceftriaxone. Pull-down assay data confirmed that ceftriaxone could bind with Aurora B in vitro and in A549 cells. Furthermore, ceftriaxone (500 µM) suppressed anchorage-independent cell growth by targeting Aurora B in A549, H520 and H1650 lung cancer cells. Importantly, in vivo xenograft animal model results showed that ceftriaxone effectively suppressed A549 and H520 lung tumor growth by inhibiting Aurora B. These data suggest the anticancer efficacy of ceftriaxone for the treatment of lung cancers through its inhibition of Aurora B.
[Show abstract][Hide abstract] ABSTRACT: N-(4-hydroxyphenyl) retinamide (4HPR, fenretinide) is a synthetic retinoid that has been tested in clinical trials as a cancer therapeutic and chemopreventive agent. Although 4HPR has been shown to be cytotoxic to many kinds of cancer cells, the underlying molecular mechanisms are only partially understood. Until now, no direct cancer-related molecular target has been reported to be involved in the antitumor activities of 4HPR. Herein, we found that 4HPR inhibited mammalian target of rapamycin (mTOR) kinase activity by directly binding with mTOR, which suppressed the activities of both the mTORC1 and the mTORC2 complexes. The predicted binding mode of 4HPR with mTOR was based on a homology computer model, which showed that 4HPR could bind in the ATP-binding pocket of the mTOR protein through hydrogen bonds and hydrophobic interactions. In vitro studies also showed that 4HPR attenuated mTOR downstream signaling in a panel of non-small-cell lung cancer cells, resulting in growth inhibition. Moreover, knockdown of mTOR in cancer cells decreased their sensitivity to 4HPR. Results of an in vivo study demonstrated that i.p. injection of 4HPR in A549 lung tumor-bearing mice effectively suppressed cancer growth. The expression of mTOR downstream signaling molecules in tumor tissues was also decreased after 4HPR treatment. Taken together, our results are the first to identify mTOR as a direct antitumor target of 4HPR both in vitro and in vivo, providing a valuable rationale for guiding the clinical uses of 4HPR.