Satya S. Pathi

Betulinic acid inhibits colon cancer cell and tumor growth and induces proteasome- dependent and -independent downregulation of specificity proteins (Sp) transcription factors

BackgroundBetulinic acid (BA) inhibits growth of several cancer cell lines and tumors and the effects of BA have been attributed to its mitochondriotoxicity and inhibition of multiple pro-oncogenic factors. Previous studies show that BA induces proteasome-dependent degradation of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 in prostate cancer cells and this study focused on the mechanism of action of BA in colon cancer cells.MethodsThe effects of BA on colon cancer cell proliferation and apoptosis and tumor growth in vivo were determined using standardized assays. The effects of BA on Sp proteins and Sp-regulated gene products were analyzed by western blots, and real time PCR was used to determine microRNA-27a (miR-27a) and ZBTB10 mRNA expression.ResultsBA inhibited growth and induced apoptosis in RKO and SW480 colon cancer cells and inhibited tumor growth in athymic nude mice bearing RKO cells as xenograft. BA also decreased expression of Sp1, Sp3 and Sp4 transcription factors which are overexpressed in colon cancer cells and decreased levels of several Sp-regulated genes including survivin, vascular endothelial growth factor, p65 sub-unit of NFκB, epidermal growth factor receptor, cyclin D1, and pituitary tumor transforming gene-1. The mechanism of action of BA was dependent on cell context, since BA induced proteasome-dependent and proteasome-independent downregulation of Sp1, Sp3 and Sp4 in SW480 and RKO cells, respectively. In RKO cells, the mechanism of BA-induced repression of Sp1, Sp3 and Sp4 was due to induction of reactive oxygen species (ROS), ROS-mediated repression of microRNA-27a, and induction of the Sp repressor gene ZBTB10.ConclusionsThese results suggest that the anticancer activity of BA in colon cancer cells is due, in part, to downregulation of Sp1, Sp3 and Sp4 transcription factors; however, the mechanism of this response is cell context-dependent.

GT-094, a NO-NSAID, Inhibits Colon Cancer Cell Growth by Activation of a Reactive Oxygen Species-MicroRNA-27a: ZBTB10-Specificity Protein Pathway

Ethyl 2-((2,3-bis(nitrooxy)propyl)disulfanyl)benzoate (GT-094) is a novel nitric oxide (NO) chimera containing an nonsteroidal anti-inflammatory drug (NSAID) and NO moieties and also a disulfide pharmacophore that in itself exhibits cancer chemopreventive activity. In this study, the effects and mechanism of action of GT-094 were investigated in RKO and SW480 colon cancer cells. GT-094 inhibited cell proliferation and induced apoptosis in both cell lines and this was accompanied by decreased mitochondrial membrane potential (MMP) and induction of reactive oxygen species (ROS), and these responses were reversed after cotreatment with the antioxidant glutathione. GT-094 also downregulated genes associated with cell growth [cyclin D1, hepatocyte growth factor receptor (c-Met), epidermal growth factor receptor (EGFR)], survival (bcl-2, survivin), and angiogenesis [VEGF and its receptors (VEGFR1 and VEGFR2)]. Results of previous RNA interference studies in this laboratory has shown that these genes are regulated, in part, by specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 that are overexpressed in colon and other cancer cell lines and not surprisingly, GT-094 also decreased Sp1, Sp3, and Sp4 in colon cancer cells. GT-094–mediated repression of Sp and Sp-regulated gene products was due to downregulation of microRNA-27a (miR-27a) and induction of ZBTB10, an Sp repressor that is regulated by miR-27a in colon cancer cells. Moreover, the effects of GT-094 on Sp1, Sp3, Sp4, miR-27a, and ZBTB10 were also inhibited by glutathione suggesting that the anticancer activity of GT-094 in colon cancer cells is due, in part, to activation of an ROS-miR-27a:ZBTB10-Sp transcription factor pathway. Mol Cancer Res; 9(2); 195–205. ©2010 AACR.

Aspirin Inhibits Colon Cancer Cell and Tumor Growth and Downregulates Specificity Protein (Sp) Transcription Factors

Acetylsalicylic acid (aspirin) is highly effective for treating colon cancer patients postdiagnosis; however, the mechanisms of action of aspirin in colon cancer are not well defined. Aspirin and its major metabolite sodium salicylate induced apoptosis and decreased colon cancer cell growth and the sodium salt of aspirin also inhibited tumor growth in an athymic nude mouse xenograft model. Colon cancer cell growth inhibition was accompanied by downregulation of Sp1, Sp3 and Sp4 proteins and decreased expression of Sp-regulated gene products including bcl-2, survivin, VEGF, VEGFR1, cyclin D1, c-MET and p65 (NFκB). Moreover, we also showed by RNA interference that β-catenin, an important target of aspirin in some studies, is an Sp-regulated gene. Aspirin induced nuclear caspase-dependent cleavage of Sp1, Sp3 and Sp4 proteins and this response was related to sequestration of zinc ions since addition of zinc sulfate blocked aspirin-mediated apoptosis and repression of Sp proteins. The results demonstrate an important underlying mechanism of action of aspirin as an anticancer agent and, based on the rapid metabolism of aspirin to salicylate in humans and the high salicylate/aspirin ratios in serum, it is likely that the anticancer activity of aspirin is also due to the salicylate metabolite.

Mechanism of Action of Phenethylisothiocyanate and Other Reactive Oxygen Species-Inducing Anticancer Agents

ABSTRACT Reactive oxygen species (ROS)-inducing anticancer agents such as phenethylisothiocyanate (PEITC) activate stress pathways for killing cancer cells. Here we demonstrate that PEITC-induced ROS decreased expression of microRNA 27a (miR-27a)/miR-20a:miR-17-5p and induced miR-regulated ZBTB10/ZBTB4 and ZBTB34 transcriptional repressors, which, in turn, downregulate specificity protein (Sp) transcription factors (TFs) Sp1, Sp3, and Sp4 in pancreatic cancer cells. Decreased expression of miR-27a/miR-20a:miR-17-5p by PEITC-induced ROS is a key step in triggering the miR-ZBTB Sp cascade leading to downregulation of Sp TFs, and this is due to ROS-dependent epigenetic effects associated with genome-wide shifts in repressor complexes, resulting in decreased expression of Myc and the Myc-regulated miRs. Knockdown of Sp1 alone by RNA interference also induced apoptosis and decreased pancreatic cancer cell growth and invasion, indicating that downregulation of Sp transcription factors is an important common mechanism of action for PEITC and other ROS-inducing anticancer agents.

Metformin inhibits pancreatic cancer cell and tumor growth and downregulates Sp transcription factors

Metformin is a widely used antidiabetic drug, and epidemiology studies for pancreatic and other cancers indicate that metformin exhibits both chemopreventive and chemotherapeutic activities. Several metformin-induced responses and genes are similar to those observed after knockdown of specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 by RNA interference, and we hypothesized that the mechanism of action of metformin in pancreatic cancer cells was due, in part, to downregulation of Sp transcription factors. Treatment of Panc1, L3.6pL and Panc28 pancreatic cancer cells with metformin downregulated Sp1, Sp3 and Sp4 proteins and several pro-oncogenic Sp-regulated genes including bcl-2, survivin, cyclin D1, vascular endothelial growth factor and its receptor, and fatty acid synthase. Metformin induced proteasome-dependent degradation of Sps in L3.6pL and Panc28 cells, whereas in Panc1 cells metformin decreased microRNA-27a and induced the Sp repressor, ZBTB10, and disruption of miR-27a:ZBTB10 by metformin was phosphatase dependent. Metformin also inhibited pancreatic tumor growth and downregulated Sp1, Sp3 and Sp4 in tumors in an orthotopic model where L3.6pL cells were injected directly into the pancreas. The results demonstrate for the first time that the anticancer activities of metformin are also due, in part, to downregulation of Sp transcription factors and Sp-regulated genes.

PHARMACOLOGIC DOSES OF ASCORBIC ACID REPRESS SPECIFICITY PROTEIN (Sp) TRANSCRIPTION FACTORS AND Sp-REGULATED GENES IN COLON CANCER CELLS

Ascorbic acid (vitamin C) inhibits cancer cell growth, and there is a controversy regarding the cancer chemoprotective effects of pharmacologic doses of this compound that exhibits prooxidant activity. We hypothesized that the anticancer activity of pharmacologic doses of ascorbic acid (<5 mM) is due, in part, to reactive oxygen species-dependent downregulation of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4 and Sp-regulated genes. In this study, ascorbic acid (1–3 mM) decreased RKO and SW480 colon cancer cell proliferation and induced apoptosis and necrosis, and this was accompanied by downregulation of Sp1, Sp3, and Sp4 proteins. In addition, ascorbic acid decreased expression of several Sp-regulated genes that are involved in cancer cell proliferation [hepatocyte growth factor receptor (c-Met), epidermal growth factor receptor and cyclin D1], survival (survivin and bcl-2), and angiogenesis [vascular endothelial growth factor (VEGF) and its receptors (VEGFR1 and VEGFR2)]. Other prooxidants such as hydrogen peroxide exhibited similar activities in colon cancer cells, and cotreatment with glutathione inhibited these responses. This study demonstrates for the first time that the anticancer activities of ascorbic acid are due, in part, to ROS-dependent repression of Sp transcription factors.

Induction of the Transcriptional Repressor ZBTB4 in Prostate Cancer Cells by Drug-Induced Targeting of MicroRNA-17-92/106b-25 Clusters

Androgen-insensitive DU145 and PC3 human prostate cancer cells express high levels of specificity protein (Sp) transcription factors Sp1, Sp3, and Sp4, and treatment of cells with methyl 2-cyano-3,11-dioxo-18β-olean-1,12-dien-30-oate (CDODA-Me) inhibited cell growth and downregulated Sp1, Sp3, and Sp4 expression. CDODA-Me (15 mg/kg/d) was a potent inhibitor of tumor growth in a mouse xenograft model (PC3 cells) and also decreased expression of Sp transcription factors in tumors. CDODA-Me–mediated downregulation of Sp1, Sp3, and Sp4 was due to induction of the transcriptional repressor ZBTB4, which competitively binds and displaces Sp transcription factors from GC-rich sites in Sp1-, Sp3-, Sp4-, and Sp-regulated gene promoters. ZBTB4 levels are relatively low in DU145 and PC3 cells due to suppression by miR paralogs that are members of the miR-17-92 (miR-20a/17-5p) and miR-106b-25 (miR-106b/93) clusters. Examination of publically available prostate cancer patient array data showed an inverse relationship between ZBTB4 and miRs-20a/17-5p/106b/93 expression, and increased ZBTB4 in patients with prostate cancer was a prognostic factor for increased survival. CDODA-Me induces ZBTB4 in prostate cancer cells through disruption of miR–ZBTB4 interactions, and this results in downregulation of pro-oncogenic Sp transcription factors and Sp-regulated genes. Mol Cancer Ther; 11(9); 1852–62. ©2012 AACR.

Inhibition of rhabdomyosarcoma cell and tumor growth by targeting specificity protein (Sp) transcription factors

Specificity protein (Sp) transcription factors Sp1, Sp3 and Sp4 are highly expressed in rhabdomyosarcoma (RMS) cells. In tissue arrays of RMS tumor cores from 71 patients, 80% of RMS patients expressed high levels of Sp1 protein, whereas low expression of Sp1 was detected in normal muscle tissue. The non‐steroidal anti‐inflammatory drug (NSAID) tolfenamic acid (TA) inhibited growth and migration of RD and RH30 RMS cell lines and also inhibited tumor growth in vivo using a mouse xenograft (RH30 cells) model. The effects of TA were accompanied by downregulation of Sp1, Sp3, Sp4 and Sp‐regulated genes in RMS cells and tumors, and the role of Sp protein downregulation in mediating inhibition of RD and RH30 cell growth and migration was confirmed by individual and combined knockdown of Sp1, Sp3 and Sp4 proteins by RNA interference. TA treatment and Sp knockdown in RD and RH30 cells also showed that four genes that are emerging as individual drug targets for treating RMS, namely c‐MET, insulin‐like growth factor receptor (IGFR), PDGFRα and CXCR4, are also Sp‐regulated genes. These results suggest that NSAIDs such as TA may have potential clinical efficacy in drug combinations for treating RMS patients.

Tolfenamic acid inhibits colon cancer cell and tumor growth and induces degradation of specificity protein (Sp) transcription factors

Tolfenamic acid (TA) is a non‐steroidal anti‐inflammatory drug (NSAID) that inhibits lung, esophageal, breast and pancreatic cancer cell and tumor growth, and this study investigated the anticancer activity of TA in colon cancer. TA inhibited growth and induced apoptosis in RKO, SW480, HT‐29, and HCT‐116 colon cancer cells, and TA (50 mg/kg/d) also inhibited tumor growth in athymic nude mice bearing RKO cells as xenografts. TA downregulated expression of Sp proteins (Sp1, Sp3, and Sp4) in colon cancer cells and this was accompanied by decreased expression of several Sp‐regulated growth promoting (cyclin D1, hepatocyte growth factor receptor), angiogenic (vascular endothelial growth factor (VEGF) and its receptor 1), survival (survivin and bcl‐2), and inflammatory (NFκBp65/p50) gene products. The mechanism of TA‐mediated effects on Sp proteins was due to activation of caspases. These results now extend the number of NSAIDs that may have clinical potential for colon cancer chemotherapy and show that the anticancer activity of TA is due, in part, to targeting Sp transcription factors.

MicroRNA-101 regulated transcriptional modulator SUB1 plays a role in prostate cancer

MicroRNA-101, a tumor suppressor microRNA (miR), is often downregulated in cancer and is known to target multiple oncogenes. Some of the genes that are negatively regulated by miR-101 expression include histone methyltransferase EZH2 (enhancer of zeste homolog 2), COX2 (cyclooxygenase-2), POMP (proteasome maturation protein), CERS6, STMN1, MCL-1 and ROCK2, among others. In the present study, we show that miR-101 targets transcriptional coactivator SUB1 homolog (Saccharomyces cerevisiae)/PC4 (positive cofactor 4) and regulates its expression. SUB1 is known to have diverse role in vital cell processes such as DNA replication, repair and heterochromatinization. SUB1 is known to modulate transcription and acts as a mediator between the upstream activators and general transcription machinery. Expression profiling in several cancers revealed SUB1 overexpression, suggesting a potential role in tumorigenesis. However, detailed regulation and function of SUB1 has not been elucidated. In this study, we show elevated expression of SUB1 in aggressive prostate cancer. Knockdown of SUB1 in prostate cancer cells resulted in reduced cell proliferation, invasion and migration in vitro, and tumor growth and metastasis in vivo. Gene expression analyses coupled with chromatin immunoprecipitation revealed that SUB1 binds to the promoter regions of several oncogenes such as PLK1 (Polo-like kinase 1), C-MYC, serine-threonine kinase BUB1B and regulates their expression. Additionally, we observed SUB1 downregulated CDKN1B expression. PLK1 knockdown or use of PLK1 inhibitor can mitigate oncogenic function of SUB1 in benign prostate cancer cells. Thus, our study suggests that miR-101 loss results in increased SUB1 expression and subsequent activation of known oncogenes driving prostate cancer progression and metastasis. This study therefore demonstrates functional role of SUB1 in prostate cancer, and identifies its regulation and potential downstream therapeutic targets of SUB1 in prostate cancer.