Sharmila Shankar

Green tea catechin, epigallocatechin-3-gallate (EGCG): mechanisms, perspectives and clinical applications

An expanding body of preclinical evidence suggests EGCG, the major catechin found in green tea (Camellia sinensis), has the potential to impact a variety of human diseases. Apparently, EGCG functions as a powerful antioxidant, preventing oxidative damage in healthy cells, but also as an antiangiogenic and antitumor agent and as a modulator of tumor cell response to chemotherapy. Much of the cancer chemopreventive properties of green tea are mediated by EGCG that induces apoptosis and promotes cell growth arrest by altering the expression of cell cycle regulatory proteins, activating killer caspases, and suppressing oncogenic transcription factors and pluripotency maintain factors. In vitro studies have demonstrated that EGCG blocks carcinogenesis by affecting a wide array of signal transduction pathways including JAK/STAT, MAPK, PI3K/AKT, Wnt and Notch. EGCG stimulates telomere fragmentation through inhibiting telomerase activity. Various clinical studies have revealed that treatment by EGCG inhibits tumor incidence and multiplicity in different organ sites such as liver, stomach, skin, lung, mammary gland and colon. Recent work demonstrated that EGCG reduced DNMTs, proteases, and DHFR activities, which would affect transcription of TSGs and protein synthesis. EGCG has great potential in cancer prevention because of its safety, low cost and bioavailability. In this review, we discuss its cancer preventive properties and its mechanism of action at numerous points regulating cancer cell growth, survival, angiogenesis and metastasis. Therefore, non-toxic natural agent could be useful either alone or in combination with conventional therapeutics for the prevention of tumor progression and/or treatment of human malignancies.

Inhibition of PI3K/AKT and MAPK/ERK pathways causes activation of FOXO transcription factor, leading to cell cycle arrest and apoptosis in pancreatic cancer.

Background Mammalian forkhead members of the class O (FOXO) transcription factors, including FOXO1, FOXO3a, and FOXO4, are implicated in the regulation of several biological processes, including the stress resistance, metabolism, cell cycle, apoptosis and DNA repair. The objectives of this study were to examine the molecular mechanisms by which FOXO transcription factors induced cell cycle arrest and apoptosis and enhanced anti-proliferative effects of sulforaphane (SFN, an active compound in cruciferous vegetables) in pancreatic cancer cells. Results Our data demonstrated that SFN inhibited cell proliferation and colony formation, and induced apoptosis through caspase-3 activation in pancreatic cancer cells. The inhibition of PI3K/AKT and MEK/ERK pathways activated FOXO transcription factors. SFN inhibited phosphorylation of AKT and ERK, and activated FOXO transcription factors, leading to cell cycle arrest and apoptosis. Phosphorylation deficient mutants of FOXO proteins enhanced FOXO transcriptional activity, and further enhanced SFN-induced FOXO activity and apoptosis. SFN induced the expression of p21/CIP1 and p27/KIP1, and inhibited the expression of cyclin D1. Conclusion These data suggest that inhibition of PI3K/AKT and ERK pathways acts together to activate FOXO transcription factor and enhances SFN-induced FOXO transcriptional activity, leading to cell cycle arrest and apoptosis.

Resveratrol Inhibits Pancreatic Cancer Stem Cell Characteristics in Human and KrasG12D Transgenic Mice by Inhibiting Pluripotency Maintaining Factors and Epithelial-Mesenchymal Transition

Background Cancer stem cells (CSCs) can proliferate and self-renew extensively due to their ability to express anti-apoptotic and drug resistant proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which resveratrol inhibits stem cell characteristics of pancreatic CSCs derived from human primary tumors and KrasG12D transgenic mice. Methodology/Principal Findings Human pancreatic CSCs (CD133+CD44+CD24+ESA+) are highly tumorigenic and form subcutaneous tumors in NOD/SCID mice. Human pancreatic CSCs expressing high levels of CD133, CD24, CD44, ESA, and aldehyde dehydrogenase also express significantly more Nanog, Oct-4, Notch1, MDR1 and ABCG2 than normal pancreatic tissues and primary pancreatic cancer cells. Similarly, CSCs from KrasG12D mice express significantly higher levels of Nanog and Oct-4 than pancreatic tissues from Pdx-Cre mice. Resveratrol inhibits the growth (size and weight) and development (PanIN lesions) of pancreatic cancer in KrasG12D mice. Resveratrol inhibits the self-renewal capacity of pancreatic CSCs derived from human primary tumors and KrasG12D mice. Resveratrol induces apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2 and XIAP in human CSCs. Resveratrol inhibits pluripotency maintaining factors (Nanog, Sox-2, c-Myc and Oct-4) and drug resistance gene ABCG2 in CSCs. Inhibition of Nanog by shRNA enhances the inhibitory effects of resveratrol on self-renewal capacity of CSCs. Finally, resveratrol inhibits CSCs migration and invasion and markers of epithelial-mesenchymal transition (Zeb-1, Slug and Snail). Conclusions/Significance These data suggest that resveratrol inhibits pancreatic cancer stem cell characteristics in human and KrasG12D transgenic mice by inhibiting pluripotency maintaining factors and epithelial-mesenchymal transition. In conclusion, resveratrol can be used for the management of pancreatic cancer.

EGCG inhibits growth, invasion, angiogenesis and metastasis of pancreatic cancer

We have shown that epigallocatechin-3-gallate (EGCG), a polyphenolic compound from green tea, inhibits growth and induces apoptosis in human pancreatic cancer cells. However, the preclinical potential of EGCG in a suitable mouse model has not been examined. In this study, we examined the molecular mechanisms by which EGCG inhibited growth, invasion, metastasis and angiogenesis of human pancreatic cancer cells in a xenograft model system. EGCG inhibited viability, capillary tube formation and migration of HUVEC, and these effects were further enhanced in the presence of an ERK inhibitor. In vivo, AsPC-1 xenografted tumors treated with EGCG showed significant reduction in volume, proliferation (Ki-67 and PCNA staining), angiogenesis (vWF, VEGF and CD31) and metastasis (MMP-2, MMP-7, MMP-9 and MMP-12) and induction in apoptosis (TUNEL), caspase-3 activity and growth arrest (p21/WAF1). EGCG also inhibited circulating endothelial growth factor receptor 2 (VEGF-R2) positive endothelial cells derived from xenografted mice. Tumor samples from EGCG treated mice showed significantly reduced ERK activity, and enhanced p38 and JNK activities. Overall, our data suggest that EGCG inhibits pancreatic cancer growth, invasion, metastasis and angiogenesis, and thus could be used for the management of pancreatic cancer prevention and treatment.

Targeting Epigenetic Regulation of miR-34a for Treatment of Pancreatic Cancer by Inhibition of Pancreatic Cancer Stem Cells

Background MicroRNA-34a (miR-34a) is a transcriptional target of p53 and is down-regulated in pancreatic cancer. This study aimed to investigate the functional significance of miR-34a in pancreatic cancer progression through its epigenetic restoration with chromatin modulators, demethylating agent 5-Aza-2′-deoxycytidine (5-Aza-dC) and HDAC inhibitor Vorinostat (SAHA). Methodology/Principal Findings Re-expression of miR-34a in human pancreatic cancer stem cells (CSCs) and in human pancreatic cancer cell lines upon treatment with 5-Aza-dC and SAHA strongly inhibited the cell proliferation, cell cycle progression, self-renewal, epithelial to mesenchymal transition (EMT) and invasion. In pancreatic CSCs, modulation of miR-34a induced apoptosis by activating caspase-3/7. Treatment of pancreatic CSCs with the chromatin-modulating agents resulted in the inhibition of Bcl-2, CDK6 and SIRT1, which are the putative targets of miR-34a. MiR-34a upregulation by these agents also induced acetylated p53, p21WAF1, p27KIP1 and PUMA in pancreatic CSCs. Inhibition of miR-34a by antagomiR abrogates the effects of 5-Aza-dC and SAHA, suggesting that 5-Aza-dC and SAHA regulate stem cell characteristics through miR-34a. In CSCs, SAHA inhibited Notch pathway, suggesting its suppression may contribute to inhibition of the self-renewal capacity and induction of apoptosis. Interestingly, treatment of pancreatic CSCs with SAHA resulted in the inhibition of EMT with the transcriptional up-regulation of E-Cadherin and down-regulation of N-Cadherin. Expression of EMT inducers (Zeb-1, Snail and Slug) was inhibited in CSCs upon treatment with SAHA. 5-Aza-dC and SAHA also retard in vitro migration and invasion of CSCs. Conclusions The present study thus demonstrates the role of miR-34a as a critical regulator of pancreatic cancer progression by the regulating CSC characteristics. The restoration of its expression by 5-Aza-dC and SAHA in CSCs will not only provide mechanistic insight and therapeutic targets for pancreatic cancer but also promising reagents to boost patient response to existing chemotherapies or as a standalone cancer drug by eliminating the CSC characteristics.

HDAC inhibitors enhance the apoptosis-inducing potential of TRAIL in breast carcinoma

Histone deacetylase (HDAC) inhibitors induce differentiation and/or apoptosis in a variety of cell types by activating transcription of target genes. Activation of the death receptor (DR) pathway by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis preferentially in cancer cells. Here, we investigated the intracellular mechanisms by which HDAC inhibitors (suberoylanilide hydroxamic acid, m-carboxycinnamic acid bis-hydroxamide, MS-275 and trichostatin A) enhance the apoptosis-inducing potential of TRAIL in breast cancer cells in vitro. A synergism in apoptosis was observed in both TRAIL-sensitive and -resistant cells upon sequential treatments with HDAC inhibitors followed by TRAIL. HDAC inhibitors synergized with TRAIL by inducing DRs DR4/TRAIL-R1 and DR5/TRAIL-R2 through NFκB activation and some of the proapoptotic members of the Bcl-2 family, and engaging the mitochondrial pathway. The ability of HDAC inhibitors to sensitize TRAIL-resistant cells suggests that HDAC inhibitors may induce fundamental alterations in cell signaling pathways. Thus, the sequential treatments with HDAC inhibitors followed by TRAIL may be used as a new therapeutic approach for the treatment of human cancers.

Resveratrol Induces Growth Arrest and Apoptosis through Activation of FOXO Transcription Factors in Prostate Cancer Cells

Background Resveratrol, a naturally occurring phytopolyphenol compound, has attracted extensive interest in recent years because of its diverse pharmacological characteristics. Although resveratrol possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. The present study was carried out to examine whether PI3K/AKT/FOXO pathway mediates the biological effects of resveratrol. Methodology/Principal Findings Resveratrol inhibited the phosphorylation of PI3K, AKT and mTOR. Resveratrol, PI3K inhibitors (LY294002 and Wortmannin) and AKT inhibitor alone slightly induced apoptosis in LNCaP cells. These inhibitors further enhanced the apoptosis-inducing potential of resveratrol. Overexpression of wild-type PTEN slightly induced apoptosis. Wild type PTEN and PTEN-G129E enhanced resveratrol-induced apoptosis, whereas PTEN-G129R had no effect on proapoptotic effects of resveratrol. Furthermore, apoptosis-inducing potential of resveratrol was enhanced by dominant negative AKT, and inhibited by wild-type AKT and constitutively active AKT. Resveratrol has no effect on the expression of FKHR, FKHRL1 and AFX genes. The inhibition of FOXO phosphorylation by resveratrol resulted in its nuclear translocation, DNA binding and transcriptional activity. The inhibition of PI3K/AKT pathway induced FOXO transcriptional activity resulting in induction of Bim, TRAIL, p27/KIP1, DR4 and DR5, and inhibition of cyclin D1. Similarly, resveratrol-induced FOXO transcriptional activity was further enhanced when activation of PI3K/AKT pathway was blocked. Over-expression of phosphorylation deficient mutants of FOXO proteins (FOXO1-TM, FOXO3A-TM and FOXO4-TM) induced FOXO transcriptional activity, which was further enhanced by resveratrol. Inhibition of FOXO transcription factors by shRNA blocked resveratrol-induced upregulation of Bim, TRAIL, DR4, DR5, p27/KIP1 and apoptosis, and inhibition of cyclin D1 by resveratrol. Conclusion/Significance These data suggest that FOXO transcription factors mediate anti-proliferative and pro-apoptotic effects of resveratrol, in part due to activation of extrinsic apoptosis pathway.

The dietary bioflavonoid quercetin synergizes with epigallocathechin gallate (EGCG) to inhibit prostate cancer stem cell characteristics, invasion, migration and epithelial-mesenchymal transition

Background Much attention has been recently focused on the role of cancer stem cells (CSCs) in the initiation and progression of solid malignancies. Since CSCs are able to proliferate and self-renew extensively due to their ability to express anti-apoptotic and drug resistant proteins, thus sustaining tumor growth. Therefore, the strategy to eradicate CSCs might have significant clinical implications. The objectives of this study were to examine the molecular mechanisms by which epigallocathechin gallate (EGCG) inhibits stem cell characteristics of prostate CSCs, and synergizes with quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables. Results Our data indicate that human prostate cancer cell lines contain a small population of CD44+CD133+ cancer stem cells and their self-renewal capacity is inhibited by EGCG. Furthermore, EGCG inhibits the self-renewal capacity of CD44+α2β1+CD133+ CSCs isolated from human primary prostate tumors, as measured by spheroid formation in suspension. EGCG induces apoptosis by activating capase-3/7 and inhibiting the expression of Bcl-2, survivin and XIAP in CSCs. Furthermore, EGCG inhibits epithelial-mesenchymal transition by inhibiting the expression of vimentin, slug, snail and nuclear β-catenin, and the activity of LEF-1/TCF responsive reporter, and also retards CSCs migration and invasion, suggesting the blockade of signaling involved in early metastasis. Interestingly, quercetin synergizes with EGCG in inhibiting the self-renewal properties of prostate CSCs, inducing apoptosis, and blocking CSCs migration and invasion. These data suggest that EGCG either alone or in combination with quercetin can eliminate cancer stem cell-characteristics. Conclusion Since carcinogenesis is a complex process, combination of bioactive dietary agents with complementary activities will be beneficial for prostate cancer prevention and/ortreatment.

Hedgehog Signaling Antagonist GDC-0449 (Vismodegib) Inhibits Pancreatic Cancer Stem Cell Characteristics: Molecular Mechanisms

Background Recent evidence from in vitro and in vivo studies has demonstrated that aberrant reactivation of the Sonic Hedgehog (SHH) signaling pathway regulates genes that promote cellular proliferation in various human cancer stem cells (CSCs). Therefore, the chemotherapeutic agents that inhibit activation of Gli transcription factors have emerged as promising novel therapeutic drugs for pancreatic cancer. GDC-0449 (Vismodegib), orally administrable molecule belonging to the 2-arylpyridine class, inhibits SHH signaling pathway by blocking the activities of Smoothened. The objectives of this study were to examine the molecular mechanisms by which GDC-0449 regulates human pancreatic CSC characteristics in vitro. Methodology/Principal Findings GDC-0499 inhibited cell viability and induced apoptosis in three pancreatic cancer cell lines and pancreatic CSCs. This inhibitor also suppressed cell viability, Gli-DNA binding and transcriptional activities, and induced apoptosis through caspase-3 activation and PARP cleavage in pancreatic CSCs. GDC-0449-induced apoptosis in CSCs showed increased Fas expression and decreased expression of PDGFRα. Furthermore, Bcl-2 was down-regulated whereas TRAIL-R1/DR4 and TRAIL-R2/DR5 expression was increased following the treatment of CSCs with GDC-0449. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GDC-0449-treated pancreatic CSCs. Thus, activated Gli genes repress DRs and Fas expressions, up-regulate the expressions of Bcl-2 and PDGFRα and facilitate cell survival. Conclusions/Significance These data suggest that GDC-0499 can be used for the management of pancreatic cancer by targeting pancreatic CSCs.

Inhibition of sonic hedgehog pathway and pluripotency maintaining factors regulate human pancreatic cancer stem cell characteristics.

Activation of the sonic hedgehog (SHh) pathway is required for the growth of numerous tissues and organs and recent evidence indicates that this pathway is often recruited to stimulate growth of cancer stem cells (CSCs) and to orchestrate the reprogramming of cancer cells via epithelial mesenchymal transition (EMT). The objectives of this study were to examine the molecular mechanisms by which (‐)‐epigallocatechin‐3‐gallate (EGCG), an active compound in green tea, inhibits self‐renewal capacity of pancreatic CSCs and synergizes with quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables. Our data demonstrated that EGCG inhibited the expression of pluripotency maintaining transcription factors (Nanog, c‐Myc and Oct‐4) and self‐renewal capacity of pancreatic CSCs. Inhibition of Nanog by shRNA enhanced the inhibitory effects of EGCG on self‐renewal capacity of CSCs. EGCG inhibited cell proliferation and induced apoptosis by inhibiting the expression of Bcl‐2 and XIAP and activating caspase‐3. Interestingly, EGCG also inhibited the components of SHh pathway (smoothened, patched, Gli1 and Gli2) and Gli transcriptional activity. Furthermore, EGCG inhibited EMT by inhibiting the expression of Snail, Slug and ZEB1, and TCF/LEF transcriptional activity, which correlated with significantly reduced CSCs migration and invasion, suggesting the blockade of signaling involved in early metastasis. Furthermore, combination of quercetin with EGCG had synergistic inhibitory effects on self‐renewal capacity of CSCs through attenuation of TCF/LEF and Gli activities. Since aberrant SHh signaling occurs in pancreatic tumorigenesis, therapeutics that target SHh pathway may improve the outcomes of patients with pancreatic cancer by targeting CSCs.