Activity In Human Cancer Cells Research Articles

Secondary Metabolites from a Culture of the Fungus Neosartorya pseudofischeri and Their In Vitro Cytostatic Activity in Human Cancer Cells

Four known (1, 2, 3, and 6) and three new compounds including a 1,4-diacetyl-2,5-dibenzylpiperazine derivative (4), a quinazolinone-containing indole derivative (5), and a new ester of 2,4-dihydroxy-6-methylbenzoic acid (7) were isolated from the fungus Neosartorya pseudofischeri S. W. Peterson. Compound 2 displayed in vitro growth inhibitory activity that ranged between the activities of etoposide and carboplatin, chosen as reference compounds, in six distinct cancer cell lines. Compound 1 displayed less activity than 2. Computer-assisted phase-contrast microscopy-related analysis revealed that 2 displayed cytostatic, not cytotoxic, effects in human U373 glioblastoma and A549 non-small cell lung cancer apoptosis-resistant cells with marked inhibition of mitotic rates. Cancer cells in the remaining phases of the cell cycle were unchanged. Flow cytometry analysis further confirmed that 2 does not induce apoptotic features in U373 or A549 cancer cells. Thus, 2 represents a novel chemical scaffold from which derivatives for anticancer cytostatic compounds can be derived.

Druggability of the CK2 inhibitor CX-4945 as an anticancer drug and beyond

Casein kinase 2 (CK2) is involved in multiple cellular processes such as proliferation, apoptosis, and cell cycle. In particular, its over-expression in human cancers is associated with angiogenesis and tumor progression. As a first orally bioavailable small molecule inhibitor of CK2, CX-4945 exerts anti-proliferative activity in human cancer cells by inhibiting the cell cycle and the PI3K/Akt signaling pathway. Additionally, CX-4945 reduces angiogenesis via blockade of hypoxia-inducible factor-1α transcription and suppresses the inflammatory interleukin-6 production in human breast cancer cells. These effects are supported by results from mouse xenograft model investigations. Here, we discuss the druggability of CX-4945 and its potential to be developed as an anti-cancer drug in clinical trials.

The hTERT Promoter Enhances the Antitumor Activity of an Oncolytic Adenovirus under a Hypoxic Microenvironment

Hypoxia is a microenvironmental factor that contributes to the invasion, progression and metastasis of tumor cells. Hypoxic tumor cells often show more resistance to conventional chemoradiotherapy than normoxic tumor cells, suggesting the requirement of novel antitumor therapies to efficiently eliminate the hypoxic tumor cells. We previously generated a tumor-specific replication-competent oncolytic adenovirus (OBP-301: Telomelysin), in which the human telomerase reverse transcriptase (hTERT) promoter drives viral E1 expression. Since the promoter activity of the hTERT gene has been shown to be upregulated by hypoxia, we hypothesized that, under hypoxic conditions, the antitumor effect of OBP-301 with the hTERT promoter would be more efficient than that of the wild-type adenovirus 5 (Ad5). In this study, we investigated the antitumor effects of OBP-301 and Ad5 against human cancer cells under a normoxic (20% oxygen) or a hypoxic (1% oxygen) condition. Hypoxic condition induced nuclear accumulation of the hypoxia-inducible factor-1α and upregulation of hTERT promoter activity in human cancer cells. The cytopathic activity of OBP-301 was significantly higher than that of Ad5 under hypoxic condition. Consistent with their cytopathic activity, the replication of OBP-301 was significantly higher than that of Ad5 under the hypoxic condition. OBP-301-mediated E1A was expressed within hypoxic areas of human xenograft tumors in mice. These results suggest that the cytopathic activity of OBP-301 against hypoxic tumor cells is mediated through hypoxia-mediated activation of the hTERT promoter. Regulation of oncolytic adenoviruses by the hTERT promoter is a promising antitumor strategy, not only for induction of tumor-specific oncolysis, but also for efficient elimination of hypoxic tumor cells.

Natural resistance to ascorbic acid induced oxidative stress is mainly mediated by catalase activity in human cancer cells and catalase-silencing sensitizes to oxidative stress

BackgroundAscorbic acid demonstrates a cytotoxic effect by generating hydrogen peroxide, a reactive oxygen species (ROS) involved in oxidative cell stress. A panel of eleven human cancer cell lines, glioblastoma and carcinoma, were exposed to serial dilutions of ascorbic acid (5-100 mmol/L). The purpose of this study was to analyse the impact of catalase, an important hydrogen peroxide-detoxifying enzyme, on the resistance of cancer cells to ascorbic acid mediated oxidative stress.MethodsEffective concentration (EC50) values, which indicate the concentration of ascorbic acid that reduced the number of viable cells by 50%, were detected with the crystal violet assay. The level of intracellular catalase protein and enzyme activity was determined. Expression of catalase was silenced by catalase-specific short hairpin RNA (sh-RNA) in BT-20 breast carcinoma cells. Oxidative cell stress induced apoptosis was measured by a caspase luminescent assay.ResultsThe tested human cancer cell lines demonstrated obvious differences in their resistance to ascorbic acid mediated oxidative cell stress. Forty-five percent of the cell lines had an EC50 > 20 mmol/L and fifty-five percent had an EC50 < 20 mmol/L. With an EC50 of 2.6–5.5 mmol/L, glioblastoma cells were the most susceptible cancer cell lines analysed in this study. A correlation between catalase activity and the susceptibility to ascorbic acid was observed. To study the possible protective role of catalase on the resistance of cancer cells to oxidative cell stress, the expression of catalase in the breast carcinoma cell line BT-20, which cells were highly resistant to the exposure to ascorbic acid (EC50: 94,9 mmol/L), was silenced with specific sh-RNA. The effect was that catalase-silenced BT-20 cells (BT-20 KD-CAT) became more susceptible to high concentrations of ascorbic acid (50 and 100 mmol/L).ConclusionsFifty-five percent of the human cancer cell lines tested were unable to protect themselves against oxidative stress mediated by ascorbic acid induced hydrogen peroxide production. The antioxidative enzyme catalase is important to protect cancer cells against cytotoxic hydrogen peroxide. Silenced catalase expression increased the susceptibility of the formerly resistant cancer cell line BT-20 to oxidative stress.

Genetically engineered oncolytic adenovirus induces autophagic cell death through an E2F1‐microRNA‐7‐epidermal growth factor receptor axis

Autophagy is known to have a cytoprotective role under various cellular stresses; however, it also results in robust cell death as an important safeguard mechanism that protects the organism against invading pathogens and unwanted cancer cells. Autophagy is regulated by cell signalling including microRNA (miRNA), a post-transcriptional regulator of gene expression. Here, we show that genetically engineered telomerase-specific oncolytic adenovirus induced miR-7 expression, which is significantly associated with its cytopathic activity in human cancer cells. Virus-mediated miR-7 upregulation depended on enhanced expression of the E2F1 protein. Ectopic expression of miR-7 suppressed cell viability and induced autophagy by inhibiting epidermal growth factor receptor (EGFR) expression. Our results suggest that oncolytic adenovirus induces autophagic cell death through an E2F1-miR-7-EGFR pathway in human cancer cells, providing a novel insight into the molecular mechanism of an anticancer virotherapy.

P120RasGAP-Mediated Activation of c-Src Is Critical for Oncogenic Ras to Induce Tumor Invasion

Ras genes are the most common targets for somatic gain-of-function mutations in human cancers. In this study, we found a high incidence of correlation between Ras oncogenic mutations and c-Src activation in human cancer cells. We showed that oncogenic Ras induces c-Src activation mainly on the Golgi complex and endoplasmic reticulum. Moreover, we identified p120RasGAP as an effector for oncogenic Ras to activate c-Src. The recruitment of p120RasGAP to the Golgi complex by oncogenic Ras facilitated its interaction with c-Src, thereby leading to c-Src activation, and this p120RasGAP-mediated activation of c-Src was important for tumor invasion induced by oncogenic Ras. Collectively, our findings unveil a relationship between oncogenic Ras, p120RasGAP, and c-Src, suggesting a critical role for c-Src in cancers evoked by oncogenic mutations in Ras genes.

Abstract 256: Involvement of ERK and ROS in CLEFMA-induced apoptosis in lung cancer cells

Abstract CLEFMA (4-[3,5-bis(2-chlorobenzylidene-4-oxo-piperidine-1-yl)-4-oxo-2-butenoic acid) is novel synthetic molecule with potent growth inhibitory activity in human cancer cells. Our microarray gene expression analysis suggested that it induces oxidative stress in lung adenocarcinoma H441 cells but not in normal lung fibroblasts. That oxidative stress is a triggering mechanism for both apoptotic and autophagic cell death has been supported by several recent reports. Compared to normal cells, the cancer cells are more vulnerable to oxidative stress-induced cell death because of their high metabolic activity, increased likelihood of electron escape during oxidative phosphorylation and overburdened antioxidant system. In this study, we tested our hypothesis that CLEFMA-induced apoptotic cell death was dependent on the accompanying increase in oxidative stress in H441cells. Methods: Inhibition of cell growth by CLEFMA (10 µM) was estimated by MTT or hexosaminidase assay. ROS production, cell cycle and apoptotic cell population were assessed by flow cytometry. The phosphorylation levels of extracellular signal-regulated kinase (ERK1/2), p38 and JNK1/2 were examined by Western blotting. N-acetylcysteine (NAC, 5 mM) and PD98059 (5 mM) were used as ROS scavenger and ERK1/2 inhibitors, respectively. The molecular markers of apoptosis were analyzed by RT-PCR and immunobloting. Results: NCI-DTP program confirmed anticancer activity of CLEFMA in 60 human cancer cell lines. CLEFMA-treated H441 cells showed typical molecular signatures of apoptosis- upregulated pro-apoptotic and downregulated anti-apoptotic genes. There was time-dependent activation of caspases 3/7, PARP cleavage and BAX; the expression of anti-apoptotic XIAP1 and BCL-xL was reduced, whereas pro-apoptotic p53 was increased. Since BID was not activated, it appears that CLEFMA works through intrinsic apoptosis pathway. CLEFMA also induced sustained activation of ERK, p38 and JNK. The pharmacologic inhibition of ERK, but not that of p38 and JNK, resulted in decreased anti-proliferative activity of CLEFMA. ERK inhibition with PD98059 abrogated CLEFMA-induced apoptosis. The sustained ERK activation was found to be associated with the inhibition of dual specificity phosphatase, DUSP6, expression. Treatment with ROS scavenger NAC abrogated CLEFMA-induced cell death, ERK activation and pro-apoptotic biomarkers, and reinstated DUSP6 expression. Conclusion: From the results of this study, we conclude that CLEFMA-induces apoptosis by enhancing oxidative stress in the cancer cells, and that the accompanying prolonged ERK activation is required for CLEFMA-induced H441 cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 256. doi:1538-7445.AM2012-256

Inhibition of fatty acid synthase by ginkgolic acids from the leaves of Ginkgo biloba and their cytotoxic activity

Fatty acid synthase (FAS) has been proposed to be a new drug target for the development of anticancer agents because of the significant difference in expression of FAS between normal and tumour cells. Since a n-hexane-soluble extract from Ginkgo biloba was demonstrated to inhibit FAS activity in our preliminary test, we isolated active compounds from the n-hexane-soluble extract and evaluated their cytotoxic activity in human cancer cells. Three ginkgolic acids 1–3 isolated from the n-hexane-soluble extract inhibited the enzyme with IC50 values 17.1, 9.2 and 10.5 µM, respectively, and they showed cytotoxic activity against MCF-7 (human breast adenocarcinoma), A549 (human lung adenocarcinoma) and HL-60 (human leukaemia) cells. Our findings suggest that alkylphenol derivatives might be a new type of FAS inhibitor with cytotoxic activity.

Synthesis and Evaluation of the Cytotoxicities of Tetraindoles: Observation that the 5-Hydroxy Tetraindole (SK228) Induces G2Arrest and Apoptosis in Human Breast Cancer Cells

Current chemical and biological interest in indole-3-carbinol (I3C) and its metabolites has resulted in the discovery of new biologically active indoles. As part of a program aimed at the development of indole analogues, tetraindoles 1-15 were prepared and their antiproliferative effects on human breast cancer cells were evaluated. The results show that the 5-hydroxy-tetraindole 8 (SK228) has optimum antiproliferative activity against breast adenocarcinoma (MCF 7 and MDA-MB-231) cells and that this activity involves G(2)-phase arrest of the cell cycle with a distinctive increase in the expression of cyclin B1 and phospho-cdc2. Further observations suggest that 5-hydroxy-tetraindole 8 induces apoptosis through externalization of membrane phosphatidylserine, DNA fragmentation, and activation of caspase-3. Given the fact that I3C and its metabolites have been shown to improve therapeutic efficacy and to have a broad range of antitumor activities in human cancer cells, the current findings have important pharmacological relevance as they open a promising route to the development of a potential chemotherapeutic application of tetraindoles as agents for the treatment of breast cancer.

Study of the cytotoxicity and particle action in human cancer cells of titanocene-functionalized materials with potential application against tumors

Titanocene dichloride [Ti(η 5-C 5H 5) 2Cl 2] ( 1), has been grafted onto dehydrated hydroxyapatite (HAP), Al 2O 3 and two mesoporous silicas MSU-2 (Michigan State University Silica type 2) and HMS (Hexagonal Mesoporous Silica), to give the novel materials HAP/[Ti(η 5-C 5H 5) 2Cl 2] ( S1) (1.01 wt.% Ti), Al 2O 3/[Ti(η 5-C 5H 5) 2Cl 2] ( S2) (2.36 wt.% Ti), HMS/[Ti(η 5-C 5H 5) 2Cl 2] ( S3) (0.75 wt.% Ti) and MSU-2/[Ti(η 5-C 5H 5) 2Cl 2] ( S4) (0.74 wt.% Ti), which have been characterized by powder X-ray diffraction, X-ray fluorescence, nitrogen gas sorption, multinuclear magic angle spinning NMR spectroscopy, IR spectroscopy, thermogravimetry analysis, UV spectroscopy, scanning electronic microscopy and transmission electronic microscopy. The cytotoxicity of the titanocene-functionalized materials toward human cancer cell lines from five different histogenic origins: 8505 C (anaplastic thyroid cancer), A253 (head and neck cancer), A549 (lung carcinoma), A2780 (ovarian cancer) and DLD-1 (colon cancer) has been determined. M 50 values (quantity of material needed to inhibit normal cell growth by 50%) and Ti-M 50 values (quantity of anchored titanium needed to inhibit normal cell growth by 50%) indicate that the activity of S1– S4 against studied human cancer cells depended on the surface type as well as on the cell line. In addition, studies on the titanocene release and the interaction of the materials S1– S4 with DNA show that the cytotoxic activity may be due to particle action, because no release of titanium complexes has been observed in physiological conditions, while electrostatic interactions of titanocene-functionalized particles with DNA have been observed.

Abstract B14: Attacking the Achilles' heel in the oncogenic K-RAS signaling pathway to block tumorigenesis and metastasis in human cancers

Abstract Hyperactive K-RAS signaling is a major menace that drives aggressive tumor growth and metastasis in human cancer. Currently, there are no effective ways to treat human cancers that have oncogenic K-RAS mutations that confer drug resistance, aggressive tumor growth, systemic metastasis, and poor clinical outcome. Therefore, finding novel approaches and new drug targets to inhibit oncogenic K-RAS is an urgent goal and the major challenge in cancer therapy and treatment. Instead of targeting an upstream signaling module such as EGFR/HER2/KRAS/B-RAF/MEK/MAPK/AKT/mT0R, we targeted the most downstream signaling module in the EGFR/HER2/K-RAS pathway called the SIAH-dependent proteolytic machinery. SIAHs are the human homologs of seven-in-absentia (SINA), an evolutionarily conserved RING E3 ligase, an essential downstream signaling module, and a critical gatekeeper required for proper ERBB/RAS/RAF signal transduction. Guided by the insights and principles learned from Drosophila RAS signaling pathway, we conducted preclinical studies to dissect SIAH function in the context of ERBB/K-RAS-mediated tumorigenesis and metastasis in human cancer cells and tumor specimens. We found that: 1) SIAH is a novel biomarker for human cancer, i.e., SIAH specifically decorates proliferating tumor cells (not stromal or immune cells in the tumor microenvironment). SIAH expression is progressively and markedly upregulated as human cancer progresses to the advanced stages in human pancreatic, lung, breast, and prostate cancers; and 2) inhibiting SIAH function is highly effective to abolish tumorigenesis and metastasis in multiple of the most aggressive human cancer cell lines known in the literature that include pancreatic cancer cells (MiaPaCa and Panc-1), lung cancer cells (A549, H727, and UMC11), invasive breast cancer cells (MDA-MB-231, MDA-MB-468, and MDA-MB-435), colorectal cancer cells (SW620 and HCT116) and hormonal-refractory prostate cancer cells (PC-3 and DU-145) in our preclinical studies. These findings demonstrate that SIAH is an attractive and logical new therapeutic target for novel anti-K-RAS and anticancer therapy in human cancer. Through our work, SIAH has emerged as a new and effective drug target against oncogenic K-RAS activation in human cancer cells. Using anti-SIAH molecules to block K-RAS signaling in human cancer is an excellent example of science going from the bench (basic research in fruit flies) to the bedside (preclinical studies and ultimately clinical trials). As a highly evolutionarily conserved E3 ligase and the most downstream gatekeeper of EGFR/HER2/K-RAS/B-RAF signaling pathway, SIAH is uniquely and logically positioned to become a next-generation anti-K-RAS drug target. Our preclinical studies have demonstrated that SIAH-dependent proteolysis is an Achilles' heel for human cancer cells. Albeit at an early preclinical stage, knowledge gained from our study has great promise that has generated many excitements with immediate translational values. Anti-SIAH-based small molecule inhibitors may aid in expanding our arsenal of novel anticancer therapies. By attacking the oncogenic ERBB/K-RAS/B-RAF pathway using multipronged synergistic inhibitions at upstream (EGFR/HER2 membrane receptors), midstream (K-RAS/B-RAF/MEK/mTOR), and downstream (SIAH E3 ligase) signaling modules in parallel, we will be in a great position to halt the genesis, progression, and metastasis of the most aggressive and the deadliest forms of human cancers. If successful, we hope to translate our findings to the cancer clinics to treat many cancer patients with metastatic diseases in the near future. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B14.

Readthrough of Premature Termination Codons in the Adenomatous Polyposis Coli Gene Restores Its Biological Activity in Human Cancer Cells

The APC tumor suppressor gene is frequently mutated in human colorectal cancer, with nonsense mutations accounting for 30% of all mutations in this gene. Reintroduction of the WT APC gene into cancer cells generally reduces tumorigenicity or induces apoptosis. In this study, we explored the possibility of using drugs to induce premature termination codon (PTC) readthrough (aminoglycosides, negamycin), as a means of reactivating endogenous APC. By quantifying the readthrough of 11 nonsense mutations in APC, we were able to identify those giving the highest levels of readthrough after treatment. For these mutations, we demonstrated that aminoglycoside or negamycin treatment led to a recovery of the biological activity of APC in cancer cell lines, and showed that the level of APC activity was proportional to the level of induced readthrough. These findings show that treatment with readthrough inducers should be considered as a potential strategy for treating cancers caused by nonsense mutations APC gene. They also provide a rational basis for identifying mutations responsive to readthrough inducers.

Phospholipase D Mediates Nutrient Input to Mammalian Target of Rapamycin Complex 1 (mTORC1)

The mammalian target of rapamycin (mTOR) is a critical sensor of nutritional sufficiency. Although much is known about the regulation of mTOR in response to growth factors, much less is known about the regulation of mTOR in response to nutrients. Amino acids have no impact on the signals that regulate Rheb, a GTPase required for the activation of mTOR complex 1 (mTORC1). Phospholipase D (PLD) generates a metabolite, phosphatidic acid, that facilitates association between mTOR and the mTORC1 co-factor Raptor. We report here that elevated PLD activity in human cancer cells is dependent on both amino acids and glucose and that amino acid- and glucose-induced increases in mTORC1 activity are dependent on PLD. Amino acid- and glucose-induced PLD and mTORC1 activity were also dependent on the GTPases RalA and ARF6 and the type III phosphatidylinositol-3-kinase hVps34. Thus, a key stimulatory event for mTORC1 activation in response to nutrients is the generation of phosphatidic acid by PLD.

Preparation and Characterization of Amino-Linked Heterocyclic Carbene Palladium, Gold, and Silver Complexes and Their Use as Anticancer Agents That Act by Triggering Apoptotic Cell Death

Transition metal complexes bearing amino linked N-heterocyclic carbenes (NHC) were prepared and evaluated for their antiproliferative activities in human cancer cells. The optimum antiproliferative activity, observed for the gold complex 3 in U-87 MG cells, was found to involve S-phase arrest of the cell cycle. The results indicate that 3 induces apoptosis through a p53-bak pathway, a finding that could serve as a new strategy to reduce the resistance of cancer cells to p53-induced apoptosis.

Abstract 2335: Oncogenic K-RAS activation promotes cell motility, invasion, tumor growth and cancer metastasis via its most downstream signaling module, the SIAH2-dependent proteolytic machinery, in the highly aggressive and metastatic human cancer cells

Abstract Hyperactivation of EGFR/HER2/K-RAS is often associated with reduced cell/focal adhesion, altered cell junctions, increased cell motility, and enhanced invasion/metastasis in a large percentage of human cancers. Finding novel measures that inhibit activated ERBB/K-RAS signals and, thus, reverse tumorigenesis and metastasis remains an important goal in cancer biology. Instead of targeting an upstream component in the EGFR/HER2/K-RAS pathway, we have targeted the most downstream signaling module, called the SIAH-dependent proteolytic machinery. SIAH/SINA are evolutionarily conserved RING E3 ligases that act as an essential “gatekeeper” signaling module in the RAS signal transduction pathway. We have demonstrated that proper SIAH function is required for K-RAS signal transduction. SIAH is a novel and tumor-specific biomarker for proliferating cells in human cancer. Furthermore, we have shown that SIAH2 inhibition can successfully block both tumorigenesis and metastasis in eleven of the most aggressive human cancer cell lines known in athymic nude mice. These observations highlight the importance of SIAH2 in K-RAS-dependent tumorigenesis and metastasis in human cancer. To elucidate the molecular mechanism of SIAH2 action in human cancer cells, a proteomic approach was taken to identify novel SIAH2-interacting proteins in three highly aggressive human cancer cell lines carrying K-RAS mutations (MiaPaCa, A549 and MDA-MB-435). From these studies, three focal adhesion LIM domain proteins (LPXN, TRIP6 and FHL2) were found to interact with SIAH2 endogenously in these human cancer cells. Here, we provide extensive biochemical evidence demonstrating that SIAH2 interacts with, ubiquitinates and degrades TRIP6/FHL2/LPXN in a dose-dependent fashion, both exogenously and endogenously. This study clearly demonstrates that LPXN, TRIP6 and FHL2 are bona fide SIAH2 substrates in vivo. Ectopic expression of LPXN/TRIP6/FHL2 successfully rescued the defects in cell motility and tumor growth observed in these SIAH2-deficient human cancer cells. These focal adhesion LIM domain proteins may, therefore, represent a novel class of SIAH2 substrates (logical RAS targets) that function downstream of SIAH2 and modulate focal adhesion, increased cell motility, invasion, tumorigenesis and metastasis in response to active ERBB/K-RAS signals. The identification of SIAH2 as an important regulator of focal adhesion and cell motility downstream of K-RAS pathway may help to explain the well-documented changes in cell adhesion, aggressive tumor growth, invasion and metastasis in response to K-RAS pathway activation in the most lethal forms of human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2335. doi:10.1158/1538-7445.AM2011-2335

Histone Deacetylase Inhibitors Increase Human Arylamine N-Acetyltransferase-1 Expression in Human Tumor Cells

Arylamine N-acetyltransferase-1 (NAT1) has been associated with disorders involving folate metabolism, such as spina bifida, as well as numerous human cancers. As a result, the transcriptional and post-transcriptional regulation of NAT1 activity has been extensively studied. However, little work has been reported on the epigenetic control of NAT1 expression. Here, we demonstrate that the histone deacetylase inhibitor trichostatin A (TSA) increases NAT1 activity in human cancer cells by increasing transcription from the proximal promoter NATb. A specific Sp1 binding site was identified as essential for optimal induction of NAT1 by TSA. However, TSA did not increase the expression of Sp1 in HeLa cells. Instead, TSA increased the acetylation of histones associated with the NATb promoter. This allowed recruitment of Sp1 to the promoter along with acetylated histones. We propose that NAT1 transcription is partially repressed by the local chromatin condensation in the vicinity of NATb and that histone deacetylase inhibition leads to up-regulation of NAT1 expression via a direct change in chromatin conformation.

Targeting Mitochondrial Glutaminase Activity Inhibits Oncogenic Transformation

Rho GTPases impact a number of activities important for oncogenesis. We describe a small molecule inhibitor that blocks oncogenic transformation induced by various Rho GTPases in fibroblasts, and the growth of human breast cancer and B lymphoma cells, without affecting normal cells. We identify the target of this inhibitor to be the metabolic enzyme glutaminase, which catalyzes the hydrolysis of glutamine to glutamate. We show that transformed fibroblasts and breast cancer cells exhibit elevated glutaminase activity that is dependent on Rho GTPases and NF-κB activity, and is blocked by the small molecule inhibitor. These findings highlight a previously unappreciated connection between Rho GTPase activation and cellular metabolism and demonstrate that targeting glutaminase activity can inhibit oncogenic transformation.

Abstract 4808: SIAH2-dependent proteolysis in cell migration, tumor growth, and cancer metastasis

Abstract The dismal prognosis of patients diagnosed with invasive and metastatic cancer points to our limited arsenal of effective anticancer therapies. The central importance of EGFR/HER2/RAS pathway activation has been well established in neoplastic transformation and tumorigenesis. An important goal in cancer biology is to identify novel and effective means to inhibit activated EGFR/HER2/K-RAS signals and reverse malignant transformation, tumor growth and cancer metastasis. SIAHs are the human homologs of Drosophila Seven-In-Absentia (SINA), an evolutionarily conserved RING E3 ligase that is an essential downstream signaling module and critical “gatekeeper” required for RAS signal transduction in human cancer cells. By targeting the most downstream signaling module identified in the RAS pathway - the SIAH2 proteolytic machinery, we are able to block tumorigenesis and metastasis in multiple of the most aggressive forms of human cancer cells known in athymic nude mice. Although we are fully cognizant of the systemic limitation in the xenograft models and incomplete mimicry of the full-spectrum metastatic processes observed in athymic nude mice, we are encouraged by the impressive anti-SIAH2-based tumor suppression in these preclinical studies. In this study, we aim to identify the molecular mechanism(s) of SIAH2 action in human cancer cells. To delineate SIAH2 function in cancer, through a proteomic approach, we identified three endogenous LIM domain proteins, TRIP6/FHL2/LPXN, as the bona fide SIAH2-interacting proteins isolated from the most aggressive human cancer cell lines (A549, MiaPaCa and MDA-MB-435 cells) in parallel. We provide extensive biochemical evidence to show that TRIP6/FHL2/LPXN are bona fide SIAH2 substrates. These new SIAH2 substrates can successfully rescue cell motility and viability defects observed in SIAH2-deficient cancer cells, suggesting that these focal adhesion proteins may represent one of many diverse signaling modules that function downstream of SIAH2 in mediating aspects of RAS-dependent signaling in normal development and cancer. Our results suggest In response to ERBB/RAS activation, SIAH2 E3 ligase modulates cell junction, adhesion and migration by targeting focal adhesion proteins for ubiquitin-mediated proteolysis to promote cell migration, tumorigenesis and metastasis in human cancer cells. The identification of SIAH2-mediated focal adhesion and cell junction modulation may have important implications in cancer biology to explain the well-documented modulations in focal adhesion, cell junction and motility in response to ERBB/RAS activation in human cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4808.

A novel small molecule inhibits STAT3 phosphorylation and DNA binding activity and exhibits potent growth suppressive activity in human cancer cells

BackgroundTargeting Signal Transducer and Activator of Transcription 3 (STAT3) signaling is an attractive therapeutic approach for most types of human cancers with constitutively activated STAT3. A novel small molecular STAT3 inhibitor, FLLL32 was specifically designed from dietary agent, curcumin to inhibit constitutive STAT3 signaling in multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cells.ResultsFLLL32 was found to be a potent inhibitor of STAT3 phosphorylation, STAT3 DNA binding activity, and the expression of STAT3 downstream target genes in vitro, leading to the inhibition of cell proliferation as well as the induction of Caspase-3 and PARP cleavages in human multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cell lines. However, FLLL32 exhibited little inhibition on some tyrosine kinases containing SH2 or both SH2 and SH3 domains, and other protein and lipid kinases using a kinase profile assay. FLLL32 was also more potent than four previously reported JAK2 and STAT3 inhibitors as well as curcumin to inhibit cell viability in these cancer cells. Furthermore, FLLL32 selectively inhibited the induction of STAT3 phosphorylation by Interleukin-6 but not STAT1 phosphorylation by IFN-γ.ConclusionOur findings indicate that FLLL32 exhibits potent inhibitory activity to STAT3 and has potential for targeting multiple myeloma, glioblastoma, liver cancer, and colorectal cancer cells expressing constitutive STAT3 signaling.

A Novel Small Molecule, LLL12, Inhibits STAT3 Phosphorylation and Activities and Exhibits Potent Growth-Suppressive Activity in Human Cancer Cells

Constitutive activation of signal transducer and activator of transcription 3 (STAT3) signaling is frequently detected in cancer, promoting its emergence as a promising target for cancer treatment. Inhibiting constitutive STAT3 signaling represents a potential therapeutic approach. We used structure-based design to develop a nonpeptide, cell-permeable, small molecule, termed as LLL12, which targets STAT3. LLL12 was found to inhibit STAT3 phosphorylation (tyrosine 705) and induce apoptosis as indicated by the increases of cleaved caspase-3 and poly (ADP-ribose) polymerase in various breast, pancreatic, and glioblastoma cancer cell lines expressing elevated levels of STAT3 phosphorylation. LLL12 could also inhibit STAT3 phosphorylation induced by interleukin-6 in MDA-MB-453 breast cancer cells. The inhibition of STAT3 by LLL12 was confirmed by the inhibition of STAT3 DNA binding activity and STAT3-dependent transcriptional luciferase activity. Downstream targets of STAT3, cyclin D1, Bcl-2, and survivin were also downregulated by LLL12 at both protein and messenger RNA levels. LLL12 is a potent inhibitor of cell viability, with half-maximal inhibitory concentrations values ranging between 0.16 and 3.09 microM, which are lower than the reported JAK2 inhibitor WP1066 and STAT3 inhibitor S3I-201 in six cancer cell lines expressing elevated levels of STAT3 phosphorylation. In addition, LLL12 inhibits colony formation and cell migration and works synergistically with doxorubicin and gemcitabine. Furthermore, LLL12 demonstrated a potent inhibitory activity on breast and glioblastoma tumor growth in a mouse xenograft model. Our results indicate that LLL12 may be a potential therapeutic agent for human cancer cells expressing constitutive STAT3 signaling.