Aamir Ahmad

Gemcitabine Sensitivity Can Be Induced in Pancreatic Cancer Cells through Modulation of miR-200 and miR-21 Expression by Curcumin or Its Analogue CDF

Curcumin induces cancer cell growth arrest and apoptosis in vitro, but its poor bioavailability in vivo limits its antitumor efficacy. We have previously evaluated the bioavailability of novel analogues of curcumin compared with curcumin, and we found that the analogue CDF exhibited greater systemic and pancreatic tissue bioavailability. In this study, we evaluated the effects of CDF or curcumin alone or in combination with gemcitabine on cell viability and apoptosis in gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer (PC) cell lines. Mechanistic investigations revealed a significant reduction in cell viability in CDF-treated cells compared with curcumin-treated cells, which were also associated with the induction of apoptosis, and these results were consistent with the downregulation of Akt, cyclooxygenase-2, prostaglandin E(2), vascular endothelial growth factor, and NF-kappaB DNA binding activity. We have also documented attenuated expression of miR-200 and increased expression of miR-21 (a signature of tumor aggressiveness) in gemcitabine-resistant cells relative to gemcitabine-sensitive cells. Interestingly, CDF treatment upregulated miR-200 expression and downregulated the expression of miR-21, and the downregulation of miR-21 resulted in the induction of PTEN. These results prompt further interest in CDF as a drug modality to improve treatment outcome of patients diagnosed with PC as a result of its greater bioavailability in pancreatic tissue.

miR-200 Regulates PDGF-D-Mediated Epithelial-Mesenchymal Transition, Adhesion, and Invasion of Prostate Cancer Cells

MicroRNAs have been implicated in tumor progression. Recent studies have shown that the miR‐200 family regulates epithelial–mesenchymal transition (EMT) by targeting zinc‐finger E‐box binding homeobox 1 (ZEB1) and ZEB2. Emerging evidence from our laboratory and others suggests that the processes of EMT can be triggered by various growth factors, such as transforming growth factor β and platelet‐derived growth factor‐D (PDGF‐D). Moreover, we recently reported that overexpression of PDGF‐D in prostate cancer cells (PC3 PDGF‐D cells) leads to the acquisition of the EMT phenotype, and this model offers an opportunity for investigating the molecular interplay between PDGF‐D signaling and EMT. Here, we report, for the first time, significant downregulation of the miR‐200 family in PC3 PDGF‐D cells as well as in PC3 cells exposed to purified active PDGF‐D protein, resulting in the upregulation of ZEB1, ZEB2, and Snail2 expression. Interestingly, re‐expression of miR‐200b in PC3 PDGF‐D cells led to reversal of the EMT phenotype, which was associated with the downregulation of ZEB1, ZEB2, and Snail2 expression, and these results were consistent with greater expression levels of epithelial markers. Moreover, transfection of PC3 PDGF‐D cells with miR‐200b inhibited cell migration and invasion, with concomitant repression of cell adhesion to the culture surface and cell detachment. From these results, we conclude that PDGF‐D‐induced acquisition of the EMT phenotype in PC3 cells is, in part, a result of repression of miR‐200 and that any novel strategy by which miR‐200 could be upregulated would become a promising approach for the treatment of invasive prostate cancer. STEM CELLS 2009;27:1712–1721

Targeting miRNAs involved in cancer stem cell and EMT regulation: An emerging concept in overcoming drug resistance

Although chemotherapy is an important therapeutic strategy for cancer treatment, it fails to eliminate all tumor cells due to intrinsic or acquired drug resistance, which is the most common cause of tumor recurrence. Emerging evidence suggests an intricate role of cancer stem cells (CSCs) and epithelial-mesenchymal transition (EMT)-type cells in anticancer drug resistance. Recent studies also demonstrated that microRNAs (miRNAs) play critical roles in the regulation of drug resistance. Here we will discuss current knowledge regarding CSCs, EMT and the role of regulation by miRNAs in the context of drug resistance, tumor recurrence and metastasis. A better understanding of the molecular intricacies of drug-resistant cells will help to design novel therapeutic strategies by selective targeting of CSCs and EMT-phenotypic cells through alterations in the expression of specific miRNAs towards eradicating tumor recurrence and metastasis. A particular promising lead is the potential synergistic combination of natural compounds that affect critical miRNAs, such as curcumin or epigallocatechin-3-gallate (EGCG) with chemotherapeutic agents.

Notch-1 induces Epithelial-mesenchymal transition consistent with cancer stem cell phenotype in pancreatic cancer cells

Activation of Notch-1 is known to be associated with the development and progression of human malignancies including pancreatic cancer. Emerging evidence suggest that the acquisition of epithelial-mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem-like cell phenotype are interrelated and contributes to tumor recurrence and drug resistance. The molecular mechanism(s) by which Notch-1 contributes to the acquisition of EMT phenotype and CSC self-renewal capacity has not been fully elucidated. Here we show that forced over-expression of Notch-1 leads to increased cell growth, clonogenicity, migration and invasion of AsPC-1 cells. Moreover, over-expression of Notch-1 led to the induction of EMT phenotype by activation of mesenchymal cell markers such as ZEB1, CD44, EpCAM, and Hes-1. Here we also report, for the first time, that over-expression of Notch-1 leads to increased expression of miR-21, and decreased expression of miR-200b, miR-200c, let-7a, let-7b, and let-7c. Re-expression of miR-200b led to decreased expression of ZEB1, and vimentin, and increased expression of E-cadherin. Over-expression of Notch-1 also increased the formation of pancreatospheres consistent with expression of CSC surface markers CD44 and EpCAM. Finally, we found that genistein, a known natural anti-tumor agent inhibited cell growth, clonogenicity, migration, invasion, EMT phenotype, formation of pancreatospheres and expression of CD44 and EpCAM. These results suggest that the activation of Notch-1 signaling contributes to the acquisition of EMT phenotype, which is in part mediated through the regulation of miR-200b and CSC self-renewal capacity, and these processes could be attenuated by genistein treatment.

Metformin Inhibits Cell Proliferation, Migration and Invasion by Attenuating CSC Function Mediated by Deregulating miRNAs in Pancreatic Cancer Cells

Pancreatic cancer is the fourth leading cause of cancer-related deaths in the United States, which is, in part, due to intrinsic (de novo) and extrinsic (acquired) resistance to conventional therapeutics, suggesting that innovative treatment strategies are required for overcoming therapeutic resistance to improve overall survival of patients. Oral administration of metformin in patients with diabetes mellitus has been reported to be associated with reduced risk of pancreatic cancer and that metformin has been reported to kill cancer stem cells (CSC); however, the exact molecular mechanism(s) has not been fully elucidated. In the current study, we examined the effect of metformin on cell proliferation, cell migration and invasion, and self-renewal capacity of CSCs and further assessed the expression of CSC marker genes and microRNAs (miRNA) in human pancreatic cancer cells. We found that metformin significantly decreased cell survival, clonogenicity, wound-healing capacity, sphere-forming capacity (pancreatospheres), and increased disintegration of pancreatospheres in both gemcitabine-sensitive and gemcitabine-resistant pancreatic cancer cells. Metformin also decreased the expression of CSC markers,CD44, EpCAM,EZH2, Notch-1, Nanog and Oct4, and caused reexpression of miRNAs (let-7a,let-7b, miR-26a, miR-101, miR-200b, and miR-200c) that are typically lost in pancreatic cancer and especially in pancreatospheres. We also found that reexpression of miR-26a by transfection led to decreased expression of EZH2 and EpCAM in pancreatic cancer cells. These results clearly suggest that the biologic effects of metformin are mediated through reexpression of miRNAs and decreased expression of CSC-specific genes, suggesting that metformin could be useful for overcoming therapeutic resistance of pancreatic cancer cells. Cancer Prev Res; 5(3); 355–64. ©2011 AACR.

Pancreatic cancer: understanding and overcoming chemoresistance

Pancreatic cancer is a highly aggressive malignancy. This feature is believed to be partly attributable to the chemotherapy-resistant characteristics of specific subgroups of pancreatic cancer cells, namely those with an epithelial–mesenchymal transition (EMT) phenotype and cancer stem cells. Accumulating evidence suggests that several new and emerging concepts might be important in the drug-resistant phenotype of these cell types. An understanding of the molecular mechanisms underlying drug resistance in patients with pancreatic cancer might help researchers to devise novel strategies to overcome such resistance. In particular, microRNAs (miRNAs) seem to be critical regulators of drug resistance in pancreatic cancer cells. Selective and targeted elimination of cells with an EMT phenotype and cancer stem cells could be achieved by regulating the expression of specific miRNAs.

Curcumin Analogue CDF Inhibits Pancreatic Tumor Growth by Switching on Suppressor microRNAs and Attenuating EZH2 Expression

The histone methyltransferase EZH2 is a central epigenetic regulator of cell survival, proliferation, and cancer stem cell (CSC) function. EZH2 expression is increased in various human cancers, including highly aggressive pancreatic cancers, but the mechanisms underlying for its biologic effects are not yet well understood. In this study, we probed EZH2 function in pancreatic cancer using diflourinated-curcumin (CDF), a novel analogue of the turmeric spice component curcumin that has antioxidant properties. CDF decreased pancreatic cancer cell survival, clonogenicity, formation of pancreatospheres, invasive cell migration, and CSC function in human pancreatic cancer cells. These effects were associated with decreased expression of EZH2 and increased expression of a panel of tumor-suppressive microRNAs (miRNA), including let-7a, b, c, d, miR-26a, miR-101, miR-146a, andmiR-200b, c that are typically lost in pancreatic cancer. Mechanistic investigations revealed that reexpression of miR-101 was sufficient to limit the expression of EZH2 and the proinvasive cell surface adhesion molecule EpCAM. In an orthotopic xenograft model of human pancreatic cancer, administration of CDF inhibited tumor growth in a manner associated with reduced expression of EZH2, Notch-1, CD44, EpCAM, and Nanog and increased expression of let-7, miR-26a, and miR-101. Taken together, our results indicated that CDF inhibited pancreatic cancer tumor growth and aggressiveness by targeting an EZH2-miRNA regulatory circuit for epigenetically controlled gene expression.

Putative Mechanism for Anticancer and Apoptosis-Inducing Properties of Plant-Derived Polyphenolic Compounds

Several plant‐derived polyphenolic compounds are considered to possess anticancer and apoptosis‐inducing properties in cancer cells. Such compounds are recognized as naturally occurring antioxidants but also exhibit prooxidant properties under appropriate conditions. Evidence in the literature suggests that the antioxidant properties of polyphenolics such as gallotannins, curcumin, and resveratrol may not fully account for their chemopreventive effects. We propose a mechanism for the cytotoxic action of these compounds against cancer cells that involves mobilization of endogenous copper and the consequent prooxidant action.

Over‐expression of FoxM1 leads to epithelial–mesenchymal transition and cancer stem cell phenotype in pancreatic cancer cells

FoxM1 is known to play important role in the development and progression of many malignancies including pancreatic cancer. Studies have shown that the acquisition of epithelial‐to‐mesenchymal transition (EMT) phenotype and induction of cancer stem cell (CSC) or cancer stem‐like cell phenotypes are highly inter‐related, and contributes to drug resistance, tumor recurrence, and metastasis. The molecular mechanism(s) by which FoxM1 contributes to the acquisition of EMT phenotype and induction of CSC self‐renewal capacity is poorly understood. Therefore, we established FoxM1 over‐expressing pancreatic cancer (AsPC‐1) cells, which showed increased cell growth, clonogenicity, and cell migration. Moreover, over‐expression of FoxM1 led to the acquisition of EMT phenotype by activation of mesenchymal cell markers, ZEB1, ZEB2, Snail2, E‐cadherin, and vimentin, which is consistent with increased sphere‐forming (pancreatospheres) capacity and expression of CSC surface markers (CD44 and EpCAM). We also found that over‐expression of FoxM1 led to decreased expression of miRNAs (let‐7a, let‐7b, let‐7c, miR‐200b, and miR‐200c); however, re‐expression of miR‐200b inhibited the expression of ZEB1, ZEB2, vimentin as well as FoxM1, and induced the expression of E‐cadherin, leading to the reversal of EMT phenotype. Finally, we found that genistein, a natural chemo‐preventive agent, inhibited cell growth, clonogenicity, cell migration and invasion, EMT phenotype, and formation of pancreatospheres consistent with reduced expression of CD44 and EpCAM. These results suggest, for the first time, that FoxM1 over‐expression is responsible for the acquisition of EMT and CSC phenotype, which is in part mediated through the regulation of miR‐200b and these processes, could be easily attenuated by genistein. J. Cell. Biochem. 112: 2296–2306, 2011.

Down-regulation of Notch-1 and Jagged-1 inhibits prostate cancer cell growth, migration and invasion, and induces apoptosis via inactivation of Akt, mTOR, and NF-κB signaling pathways

Notch signaling is involved in a variety of cellular processes, such as cell fate specification, differentiation, proliferation, and survival. Notch‐1 over‐expression has been reported in prostate cancer metastases. Likewise, Notch ligand Jagged‐1 was found to be over‐expressed in metastatic prostate cancer compared to localized prostate cancer or benign prostatic tissues, suggesting the biological significance of Notch signaling in prostate cancer progression. However, the mechanistic role of Notch signaling and the consequence of its down‐regulation in prostate cancer have not been fully elucidated. Using multiple cellular and molecular approaches such as MTT assay, apoptosis assay, gene transfection, real‐time RT‐PCR, Western blotting, migration, invasion assay and ELISA, we found that down‐regulation of Notch‐1 or Jagged‐1 was mechanistically associated with inhibition of cell growth, migration, invasion and induction of apoptosis in prostate cancer cells, which was mediated via inactivation of Akt, mTOR, and NF‐κB signaling. Consistent with these results, we found that the down‐regulation of Notch‐1 or Jagged‐1 led to decreased expression and the activity of NF‐κB downstream genes such as MMP‐9, VEGF, and uPA, contributing to the inhibition of cell migration and invasion. Taken together, we conclude that the down‐regulation of Notch‐1 or Jagged‐1 mediated inhibition of cell growth, migration and invasion, and the induction of apoptosis was in part due to inactivation of Akt, mTOR, and NF‐κB signaling pathways. Our results further suggest that inactivation of Notch signaling pathways by innovative strategies could be a potential targeted approach for the treatment of metastatic prostate cancer. J. Cell. Biochem. 109: 726–736, 2010.