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Alcohol is a risk factor for pancreatic cancer. However, the molecular mechanism by which chronic alcohol consumption influences pancreatic cancer development is not well understood. We have recently demonstrated that chronic ethanol exposure of pancreatic normal ductal epithelial cells (HPNE) induces cellular transformation by generating cancer stem cells (CSCs). Here, we examined whether chronic ethanol treatment induces epithelial-mesenchymal transition in HPNE cells and promotes pancreatic cancer development in KC (Pdx1-Cre, and LSL-KrasG12D ) mice. Our data demonstrate that chronic ethanol exposure of HPNE cells induces SATB2 gene and those cells became highly motile. Ethanol treatment of HPNE cells results in downregulation of E-Cadherin and upregulation of N-Cadherin, Snail, Slug, Zeb1, Nanog and BMI-1. Suppression of SATB2 expression in ethanol-transformed HPNE cells inhibits EMT phenotypes. KC mice fed with an ethanol-containing diet show enhanced pancreatic cancer growth and development than those fed with a control diet. Pancreas isolated from KC mice fed with an ethanol-containing diet show higher expression of stem cell markers (CD133, CD44, CD24), pluripotency-maintaining factors (cMyc, KLF4, SOX-2, and Oct-4), N-Cadherin, EMT-transcription factors (Snail, Slug, and Zeb1), and lower expression of E-cadherin than those isolated from mice fed with a control diet. Furthermore, pancreas isolated from KC mice fed with an ethanol-containing diet show higher expression of inflammatory cytokines (TNF-α, IL-6, and IL-8) and PTGS-2 (COX-2) gene than those isolated from mice fed with a control diet. These data suggest that chronic alcohol consumption may contribute to pancreatic cancer development by generating inflammatory signals and CSCs.

Special AT-rich sequence binding protein-2 (SATB2) is a nuclear matrix protein that binds to nuclear attachment regions and is involved in chromatin remodeling and transcription regulation. In stem cells, it regulates the expression of genes required for maintaining pluripotency and self-renewal and epithelial-mesenchymal transition (EMT). In this study, we examined the oncogenic role of SATB2 in prostate cancer, and assess whether overexpression of SATB2 in human normal prostate epithelial cells (PrECs) induces properties of cancer stem cells (CSCs). The results demonstrate that SATB2 is highly expressed in prostate cancer cell lines and CSCs, but not in PrECs. Overexpression of SATB2 in PrECs induces cellular transformation which was evident by the formation of colonies in soft agar and spheroids in suspension. Overexpression of SATB2 in PrECs also resulted in induction of stem cell markers (CD44 and CD133), pluripotency maintaining transcription factors (cMyc, Oct4, Sox2, KLF4, and Nanog), cadherin switch and EMT-related transcription factors. Chromatin immunoprecipitation assay demonstrated that SATB2 can directly bind to promoters of Bcl-2, Bsp, Nanog, XIAP, KLF4 and HOXA2, suggesting SATB2 is capable of directly regulating pluripotency/self-renewal, cell survival and proliferation. Since prostate CSCs play a crucial role in cancer initiation, progression, and metastasis, we also examined the effects of SATB2 knockdown on stemness. SATB2 knockdown in prostate CSCs inhibited spheroid formation, cell viability, colony formation, cell motility, migration, and invasion compared to their scrambled control groups. SATB2 knockdown in CSCs also upregulated the expression of E-Cadherin and inhibited the expression of N-Cadherin, Snail, Slug, and Zeb1. The expression of SATB2 was significantly higher in prostate adenocarcinoma compared to normal tissues. Overall, our data suggest that SATB2 acts as an oncogenic factor where it is capable of inducing malignant changes in PrECs by inducing CSC characteristics.

Several studies have confirmed the involvement of cancer stem cells (CSC) in tumour progression, metastasis, drug resistance and cancer relapse. SATB2 (special AT‐rich binding protein‐2) acts as a transcriptional co‐factor and modulates chromatin architecture to regulate gene expression. The purpose of this review was to discuss the pathophysiological roles of SATB2 and assess whether it could be used as a therapeutic target for cancer. SATB2 modulated the expression of those genes which regulated pluripotency and self‐renewal. Overexpression of SATB2 gene in normal epithelial cells was shown to induce transformation, as a result transformed cells gained CSC’s characteristics by expressing stem cell markers and pluripotency maintaining factors, suggesting its role as an oncogene. In addition, SATB2 induced epithelial‐mesenchymal transition (EMT) and metastasis. Interestingly, the expression of SATB2 was positively correlated with the activation of β‐catenin/TCF‐LEF pathway. Furthermore, SATB2 silencing inhibited EMT and their positive regulators, and tumour growth, and suppressed the expression of stem cell markers, pluripotency maintaining factors, cell cycle and cell survival genes, and TCF/LEF targets. Based on the cancer genome atlas (TCGA) expression data and published papers, SATB2 alone or in combination with other proteins could be used a diagnostic biomarker for cancer. Although there is no pharmacological inhibitor of SATB2, studies using genetic approaches suggest that SATB2 could be a potential target for cancer treatment and prevention.

Despite improved screening and surveillance guidelines, significant race/ethnicity‐specific disparities in hepatocellular carcinoma (HCC) continue to exist and disproportionately affect minority and disadvantaged populations. This trend indicates that social determinants, genetic, and environmental factors are driving the epidemic at the population level. Race and geography had independent associations with risk of mortality among patients with HCC. The present review discusses the risk factors and issues related to disparities in HCC. The underlying etiologies for these disparities are complex and multifactorial. Some of the risk factors for developing HCC include hepatitis B (HBV) and hepatitis C (HCV) viral infection, nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, smoking and alcohol consumption. In addition, population genetics; socioeconomic and health care access; treatment and prevention differences; and genetic, behavioral, and biological influences can contribute to HCC. Acculturation of ethnic minorities, insurance status, and access to health care may further contribute to the observed disparities in HCC. By increasing awareness, better modalities for screening and surveillance, improving access to health care, and adapting targeted preventive and therapeutic interventions, disparities in HCC outcomes can be reduced or eliminated., Population genetics, socioeconomic and health care access, treatment and prevention differences, dietary composition, and genetic, behavioral and biological influences can contribute to hepatocellular carcinoma (HCC). Acculturation of ethnic minorities, insurance status and access to healthcare may further contribute to the observed disparities in HCC. By increasing awareness and better modalities for screening and surveillance, targeted preventive and therapeutic interventions for reducing disparities can be successfully improved.

The incidence of obesity and type 2 diabetes (T2DM) in the Western world has increased dramatically during the recent decades. According to the American Cancer Society, pancreatic cancer (PC) is the fourth leading cause of cancer‐related death in the United States. The relationship among obesity, T2DM and PC is complex. Due to increase in obesity, diabetes, alcohol consumption and sedentary lifestyle, the mortality due to PC is expected to rise significantly by year 2040. The underlying mechanisms by which diabetes and obesity contribute to pancreatic tumorigenesis are not well understood. Furthermore, metabolism and microenvironment within the pancreas can also modulate pancreatic carcinogenesis. The risk of PC on a population level may be reduced by modifiable lifestyle risk factors. In this review, the interactions of diabetes and obesity to PC development were summarized, and novel strategies for the prevention and treatment of diabetes and PC were discussed.

Alcohol is a risk factor for hepatocellular carcinoma (HCC). However, the molecular mechanism by which chronic alcohol consumption contributes to HCC is not well understood. The purpose of the study was to demonstrate the effects of chronic ethanol exposure on the damage of human normal hepatocytes. Our data showed that chronic exposure of hepatocytes with ethanol induced changes similar to transformed hepatocytes that is, exhibited colonies and anchorage-independent growth. These damaged hepatocytes contained high levels of reactive oxygen species (ROS) and showed induction of the SATB2 gene. Furthermore, damaged hepatocytes gained the phenotypes of CSCs which expressed stem cell markers (CD133, CD44, CD90, EpCAM, AFP and LGR5), and pluripotency maintaining factors (Sox-2, POU5F1/Oct4 and KLF-4). Ethanol exposure also induced Nanog, a pluripotency maintaining transcription factor that functions in concert with Oct4 and SOX-2. Furthermore, ethanol induced expression of EMT-related transcription factors (Snail, Slug and Zeb1), N-Cadherin, and inhibited E-cadherin expression in damaged hepatocytes. Ethanol enhanced recruitment of SATB2 to promoters of Bcl-2, Nanog, c-Myc, Klf4 and Oct4. Ethanol also induced activation of the Wnt/TCF-LEF1 pathway and its targets (Bcl-2, Cyclin D1, AXIN2 and Myc). Finally, ethanol induced hepatocellular steatosis, SREBP1 transcription, and modulated the expression of SREBP1c, ACAC, ACLY, FASN, IL-1β, IL-6, TNF-α, GPC3, FLNB and p53. These data suggest that chronic alcohol consumption may contribute towards the development of HCC by damaging normal hepatocytes with the generation of inflammatory environment, induction of SATB2, stem cell-like characteristics, and cellular steatosis.

Alcohol is a risk factor for pancreatic cancer. However, the molecular mechanism by which chronic alcohol consumption influences pancreatic cancer development is not well understood. We have recently demonstrated that chronic ethanol exposure of pancreatic normal ductal epithelial cells (HPNE) induces cellular transformation by generating cancer stem cells (CSCs). Here, we examined whether chronic ethanol treatment induces epithelial–mesenchymal transition in HPNE cells and promotes pancreatic cancer development in KC (Pdx1‐Cre, and LSL‐KrasG12D) mice. Our data demonstrate that chronic ethanol exposure of HPNE cells induces SATB2 gene and those cells became highly motile. Ethanol treatment of HPNE cells results in downregulation of E‐Cadherin and upregulation of N‐Cadherin, Snail, Slug, Zeb1, Nanog and BMI‐1. Suppression of SATB2 expression in ethanol‐transformed HPNE cells inhibits EMT phenotypes. KC mice fed with an ethanol‐containing diet show enhanced pancreatic cancer growth and development than those fed with a control diet. Pancreas isolated from KC mice fed with an ethanol‐containing diet show higher expression of stem cell markers (CD133, CD44, CD24), pluripotency‐maintaining factors (cMyc, KLF4, SOX‐2, and Oct‐4), N‐Cadherin, EMT‐transcription factors (Snail, Slug, and Zeb1), and lower expression of E‐cadherin than those isolated from mice fed with a control diet. Furthermore, pancreas isolated from KC mice fed with an ethanol‐containing diet show higher expression of inflammatory cytokines (TNF‐α, IL‐6, and IL‐8) and PTGS‐2 (COX‐2) gene than those isolated from mice fed with a control diet. These data suggest that chronic alcohol consumption may contribute to pancreatic cancer development by generating inflammatory signals and CSCs.

In the United States, Hepatocellular Carcinoma (HCC) incidence has tripled over the past two decades. The disease has disproportionately affected minority and disadvantaged populations. The purpose of this study was to examine the expression of SATB2 gene in HCC cells derived from African Americans (AA) and Caucasian Americans (CA) and assess its oncogenic potential by measuring cell viability, spheroid formation, epithelial‐mesenchymal transition (EMT), stem cell markers and pluripotency maintaining factors in cancer stem cells (CSCs). We compared the expression of SATB2 in human primary hepatocytes, HCC cells derived from AA and CA, and HCC CSCs. Hepatocellular carcinoma cells derived from AA expressed the higher level of SATB2 than those from CA. By comparison, normal human hepatocytes did not express SATB2. Higher expression of SATB2 in HCC cells from AA was associated with greater growth rate, cell viability, colony formation and EMT characteristics than those from CA. Knockout of SATB2 in CSCs by Crispr/Cas9 technique significantly inhibited the expression of SATB2 gene, stem cell markers (CD24, CD44 and CD133), pluripotency maintaining factors (c‐Myc, KLF4, SOX2 and OCT4), and EMT compared with non‐targeting control group. The expression of SATB2 was negatively correlated with miR34a. SATB2 rescued the miR‐34a‐mediated inhibition of CSC's viability. These data suggest that SATB2 is an oncogenic factor, and its higher expression may explain the disparity in HCC outcomes among AA.

The current investigation was intended to elucidate the molecular mechanism of α‐Mangostin in the regulation of pancreatic cancer stem cell (CSC) characteristics. Here, we demonstrate that α‐Mangostin inhibited cell proliferation in pancreatic CSCs and cancer cell lines while it showed no effect on human pancreatic normal ductal epithelial cells. Also, α‐Mangostin inhibited colony formation and induced apoptosis in these cells. Further, α‐Mangostin inhibited the self‐renewal capacity of CSCs isolated from human primary tumours and KrasG12D mice. Furthermore, α‐Mangostin inhibited the invasive and metastatic ability of pancreatic CSCs by suppressing the epithelial‐to‐mesenchymal transition (EMT) via up‐regulation of E‐cadherin and down‐regulation of mesenchymal phenotype by inhibiting N‐cadherin, Snail and Slug expression. Interestingly, the pluripotency maintaining factors and CSC markers were inhibited by α‐Mangostin thus suggesting that α‐Mangostin can target CSCs to inhibit pancreatic cancer effectively. Gli signalling plays a crucial role in the self‐renewal and pluripotency of CSCs. α‐Mangostin inhibited the Gli transcription and the expression of Gli target genes (Nanog, Oct4, c‐Myc, Sox‐2 and KLF4) in CSCs. Using ChIP assay, we demonstrated that Nanog could directly bind to promoters of Cdk2, Cdk6, FGF4, c‐Myc and α‐Mangostin inhibited Nanog binding to these promoters. Conversely, the inhibitory effects of the α‐Mangostin on CSC proliferation and Gli or Nanog transcription and their targets were abrogated by either enforced activation of sonic hedgehog (Shh) or by the overexpression of Nanog. Taken together, our studies suggest that α‐Mangostin may act as Gli inhibitor and establishes the pre‐clinical significance of α‐Mangostin for the prevention and treatment of pancreatic cancer.

The incidence of pancreatic cancer is on the rise. Risk factors for pancreatic cancer include alcohol toxicity and metabolic conditions such as obesity, hypertension, dyslipidaemia, insulin resistance and type 2 diabetes. However, the molecular mechanism by which chronic alcohol consumption contributes to pancreatic cancer is not well understood. The purpose of the study was to demonstrate the effects of long‐term chronic ethanol exposure on the transformation of human pancreatic normal ductal epithelial (HPNE) cells. Our data showed that ethanol‐transformed HPNE cells were more progressively transformed exhibiting spheroids and colonies, and anchorage‐independent growth. These transformed cells contained high levels of reactive oxygen species and induced SATB2 expression. Furthermore, during ethanol‐induced cellular transformation, cells gained the phenotypes of cancer stem cells (CSCs) by expressing pluripotency maintaining factors (Oct4, Sox2, cMyc and KLF4) and stem cell markers (CD24, CD44 and CD133). Ethanol‐induced SATB2 can bind to the promoters of KLF4, Oct4, cMyc, Sox2, Bcl‐2 and XIAP genes. Suppression of SATB2 expression in ethanol‐transformed HPNE cells inhibited cell proliferation, colony formation and markers of CSCs and pluripotency. These data suggest that chronic alcohol consumption may contribute toward the development of pancreatic cancer by converting HPNE cells to cancer stem‐like cells.

Sonic hedgehog pathway is highly activated in pancreatic cancer stem cells (CSC) which play crucial roles in cancer initiation, progression and metastasis. However, the molecular mechanisms by which sanguinarine regulates pancreatic CSC characteristics is not well understood. The objectives of this study were to examine the molecular mechanisms by which sanguinarine regulates pancreatic CSC characteristics. Sanguinarine inhibited cell proliferation and colony formation and induced apoptosis through oxidative damage. Sanguinarine inhibited self-renewal capacity of pancreatic CSCs isolated from human and KrasG12D mice. Furthermore, sanguinarine suppressed epithelial-mesenchymal transition (EMT) by up-regulating E-cadherin and inhibiting N-cadherin. Significant decrease in expression level of Snail, Slug and Zeb1 corroborated the suppression of EMT in sanguinarine treated pancreatic CSCS. The ability of sanguinarine to inhibit pluripotency maintaining factors and CSC markers suggest that sanguinarine can be an effective agent for inhibiting pancreatic cancer growth and development by targeting CSCs. Furthermore, sanguinarine inhibited Shh-Gli pathway leading to modulation of Gli target genes in pancreatic CSCs. Chromatin immunoprecipitation assay demonstrated that Nanog directly binds to promoters of Cdk2, Cdk6, FGF4, c-Myc and Oct4, and sanguinarine inhibits the binding of Nanog with these genes, suggesting the direct involvement of Nanog in cell cycle, pluripotency and self-renewal. To further investigate the role of Shh-Gli-Nanog pathway, we regulated Shh signaling either by Shh protein or Nanog overexpression. Enforced activation of Shh or overexpression of Nanog counteracted the inhibitory effects of sanguinarine on pancreatic CSC proliferation, suggesting the actions of sanguinarine are mediated, at least in part, through Shh-Gli-Nanog pathway. Our studies suggest that sanguinarine can be used for the treatment and/or prevention of pancreatic cancer by targeting CSCs.

Recent studies have demonstrated the involvement of colorectal cancer (CRC) stem cells (CSC) in transformation, cancer progression and metastasis. The main goal of this paper was to examine the molecular mechanisms by which SATB2 induced malignant transformation of colorectal epithelial cells. SATB2 induced malignant transformation and these transformed cells gained the characteristics of CSCs by expressing stem cell markers (CD44, CD133, LGR5 and DCLK1) and transcription factors (c-Myc, Nanog and Sox2). Overexpression of SATB2 in normal colorectal epithelial cells increased cell motility, migration and invasion, which were associated with an increase in N-cadherin and Zeb1, and decrease in E-cadherin expression. SATB2 overexpression also upregulated XIAP and cyclin D1, suggesting its role in cell survival and cell cycle. Furthermore, the expression of SATB2 was positively correlated with β-catenin expression in CRC. In contrary, depletion of SATB2 inhibited cell proliferation, colony formation, cell motility and expression of β-catenin, Snail, Slug, Zeb1 and N-cadherin, and upregulated E-cadherin. Furthermore, SATB2 silencing inhibited the expression of stem cell markers, pluripotency maintaining transcription factors, cell cycle and cell proliferation/survival genes and TCF/LEF targets. Finally, β-catenin/TCF-LEF pathway mediated the biological effects of SATB2 in CSCs. These studies support the role of SATB2/β-catenin/TCF-LEF pathway in transformation and carcinogenesis.

Breast tumors are heterogeneous and carry a small population of progenitor cells that can produce various subtypes of breast cancer. SATB2 (special AT-rich binding protein-2) is a newly identified transcription factor and epigenetic regulator. It is highly expressed in embryonic stem cells, but not in adult tissues, and regulates pluripotency-maintaining factors. However, the molecular mechanisms by which SATB2 induces transformation of human mammary epithelial cells (HMECs) leading to malignant phenotype are unknown. The main goal of this paper is to examine the molecular mechanisms by which SATB2 induces cellular transformation of HMECs into cells that are capable of self-renewal. SATB2-transformed HMECs gain the phenotype of breast progenitor cells by expressing markers of stem cells, pluripotency-maintaining factor, and epithelial to mesenchymal transition. SATB2 is highly expressed in human breast cancer cell lines, primary mammary tissues and cancer stem cells (CSCs), but not in HMECs and normal breast tissues. Chromatin Immunoprecipitation assays demonstrate that SATB2 can directly bind to promoters of Bcl-2, c-Myc, Nanog, Klf4, and XIAP, suggesting a role of SATB2 in regulation of pluripotency, cell survival and proliferation. Furthermore, inhibition of SATB2 by shRNA in breast cancer cell lines and CSCs attenuates cell proliferation and EMT phenotype. Our results suggest that SATB2 induces dedifferentiation/transformation of mature HMECs into progenitor-like cells.

// Wei Yu 1 , Yiming Ma 1 , Sharmila Shankar 1, 2 , Rakesh K. Srivastava 1, 3 1 Kansas City VA Medical Center, Kansas City, MO 66128, USA 2 Department of Pathology, University of Missouri-School of Medicine, Kansas City, MO 64108, USA 3 Department of Pharmaceutical Sciences, University of Missouri-School of Medicine, Kansas City, MO 64108, USA Correspondence to: Rakesh K. Srivastava, email: rsrivastava.lab@gmail.com Sharmila Shankar, email: sharmila.shankar@va.gov Keywords: pancreatic cancer, SATB2, cancer stem cell, stem cell, pluripotency Received: May 01, 2016 Accepted: July 10, 2016 Published: July 27, 2016 ABSTRACT SATB2 (special AT-rich binding protein-2), a transcription factor and chromatin modulator, regulates the expression of genes required for maintaining pluripotency and self-renewal. The molecular mechanisms by which human pancreatic normal ductal epithelial cells are transformed to cancer cells are not well understood. The main goal of the paper is to examine the molecular mechanisms by which SATB2 regulates transformation of human pancreatic normal ductal epithelial (HPNE) cells, and assess whether transformed HPNE cells gained the phenotypes of cancer stem cells (CSCs). The results demonstrate that SATB2 is highly expressed in pancreatic CSCs, primary tissues and cell lines, but not in HPNE cells. SATB2 induces cellular transformation, stemness and epithelial to mesenchymal transition in HPNE cells, and inhibition of its expression suppresses these activities. Overexpression of SATB2 in HPNE cells resulted in induction of stem cell markers (CD44, CD24 and CD133), and transcription factors (Oct4, Sox2 and Nanog). SATB2 can directly bind to promoters of Bcl-2, Bsp, Nanog, c-Myc, XIAP, Klf4 and Hoxa2, suggesting the role of SATB2 in pluripotency, cell survival and proliferation. SATB2-overexpressing HPNE cells (HPNE/SATB2) formed tumors in Balb C nude mice, whereas HPNE/Empty vector cells did not form any tumor. Since SATB2 is highly expressed in human pancreatic cancer tissues and cell lines, but not in HPNE cells and normal pancreatic tissue, it can drive pancreatic cancer growth and metastasis. Our findings suggest that SATB2 can induce dedifferentiation by inducing stemness and may have a role in pancreatic carcinogenesis, and can be used as a diagnostic biomarker.

Based on the quantitative structure-activity relationship (QSAR), some novel p-aminobenzoic acid derivatives as promising cholinesterase enzyme inhibitors were designed, synthesized, characterized and evaluated to enhance learning and memory. The in vitro enzyme kinetic study of the synthesized compounds revealed the type of inhibition on the respective acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) enzymes. The in vivo studies of the synthesized compounds exhibited significant reversal of cognitive deficits in the animal models of amnesia as compared to standard drug donepezil. Further, the ex vivo studies in the specific brain regions like the hippocampus, hypothalamus, and prefrontal cortex regions also exhibited AChE inhibition comparable to standard donepezil. The in silico molecular docking and dynamics simulations studies of the most potent compound 22 revealed the consensual interactions at the active site pocket of the AChE.

Trans‑3,4',5‑trihydroxystilbene (resveratrol) is a naturally occurring polyphenolic phytoalexin with marked anticancer activities, and is mainly found in grapes, berries and peanuts. However, due to a low bioavailability, it has not progressed to clinical practice for cancer treatment. Therefore, the aims of the present study were to examine the anticancer activities of the resveratrol derivative, triacetyl resveratrol (TCRV), in pancreatic cancer cells. Apoptosis was measured by fluorescence‑activated cell sorting and terminal deoxynucleotidyl transferase (TdT)‑mediated dUTP nick‑end labeling assays. Gene expression was measured by reverse transcription‑quantitative polymerase chain reaction. TCRV inhibited colony formation and induced apoptosis through caspase‑3 activation in human pancreatic cancer AsPC‑1 and PANC‑1 cells, whereas it exerted no effect on human pancreatic normal ductal epithelial cells (HPNE). TCRV inhibited epithelial‑mesenchymal transition (EMT) by upregulating the expression of E‑cadherin and suppressing the expression of N‑cadherin and the transcription factors, Snail, Slug and Zeb1. TCRV inhibited Zeb1 3'UTR‑luciferase activity through the upregulation of microRNA (miR)‑200 family members. The inhibitory effects of TCRV on pancreatic cancer cell migration and invasion were counteracted by anti‑miR‑200 family members. The inhibitory effects of TCRV on EMT and the induction of apoptosis were exerted through the suppression of the sonic hedgehog (Shh) pathway, and through the modulation of cyclin D1 and Bcl‑2 expression. The hyperactivation of the Shh pathway by either Shh protein or Gli1 overexpression abrogated the biological effects of TCRV. Taken together, the results of this study demonstrate that TCRV inhibits pancreatic cancer growth and EMT by targeting the Shh pathway and its downstream signaling mediators. TCRV inhibited EMT through the upregulation of miR‑200 family members. Since TCRV effectively inhibited the growth of human pancreatic cancer cells by modulating the Shh pathway, without affecting the growth of HPNE cells, our findings suggest the possible use of TCRV as a promising candidate for the treatment and/or prevention of pancreatic cancer.

// Narinder Sharma 1 , Rajesh Nanta 1 , Jay Sharma 2 , Sumedha Gunewardena 3 , Karan P. Singh 4 , Sharmila Shankar 5, 6 , Rakesh K. Srivastava 1, 5 1 Department of Pharmacology, Toxicology and Therapeutics, and Medicine, University of Kansas Medical Center, Kansas City, KS, 66160, USA 2 Celprogen Inc. Torrance, CA 90503, USA 3 Department of Molecular and Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, 66160, USA 4 Division of Preventive Medicine, School of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35205, USA 5 Kansas City VA Medical Center, Kansas City, MO, 66128, USA 6 Department of Pathology, University of Missouri-School of Medicine, Kansas City, MO, 64108, USA Correspondence to: Rakesh K. Srivastava, e-mail: rakesh.srivastava@va.gov ; rsrivastava.lab@gmail.com Keywords: pancreatic cancer, PI3K/AKT/mTOR, sonic hedgehog, cancer stem cell, Gli Received: July 06, 2015 Accepted: September 22, 2015 Published: October 05, 2015 ABSTRACT Cancer stem cells (CSCs) play major roles in cancer initiation, progression, and metastasis. It is evident from growing reports that PI3K/Akt/mTOR and Sonic Hedgehog (Shh) signaling pathways are aberrantly reactivated in pancreatic CSCs. Here, we examined the efficacy of combining NVP-LDE-225 (PI3K/mTOR inhibitor) and NVP-BEZ-235 (Smoothened inhibitor) on pancreatic CSCs characteristics, microRNA regulatory network, and tumor growth. NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting pancreatic CSC's characteristics and tumor growth in mice by acting at the level of Gli. Combination of NVP-LDE-225 and NVP-BEZ-235 inhibited self-renewal capacity of CSCs by suppressing the expression of pluripotency maintaining factors Nanog, Oct-4, Sox-2 and c-Myc, and transcription of Gli. NVP-LDE-225 co-operated with NVP-BEZ-235 to inhibit Lin28/Let7a/Kras axis in pancreatic CSCs. Furthermore, a superior interaction of these drugs was observed on spheroid formation by pancreatic CSCs isolated from Pan kras/p53 mice. The combination of these drugs also showed superior effects on the expression of proteins involved in cell proliferation, survival and apoptosis. In addition, NVP-LDE-225 co-operated with NVP-BEZ-235 in inhibiting EMT through modulation of cadherin, vimentin and transcription factors Snail, Slug and Zeb1. In conclusion, these data suggest that the combined inhibition of PI3K/Akt/mTOR and Shh pathways may be beneficial for the treatment of pancreatic cancer.

Purpose: The purpose of this study was to examine the molecular mechanisms by which sulforaphane enhances the therapeutic potential of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in prostate cancer. Experimental Design: Cell viability and apoptosis assays were done by XTT and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, respectively. Tumor-bearing mice were treated with vehicle, sulforaphane, TRAIL, and sulforaphane plus TRAIL. Markers of apoptosis, angiogenesis, and metastasis were measured by immunohistochemistry. Results: Sulforaphane enhanced the therapeutic potential of TRAIL in PC-3 cells and sensitized TRAIL-resistant LNCaP cells. Sulforaphane-induced apoptosis in PC-3 cells correlated with the generation of intracellular reactive oxygen species (ROS), collapse of mitochondrial membrane potential, activation of caspase-3 and caspase-9, and up-regulation of DR4 and DR5. Sulforaphane induced the expression of Bax, Bak, Bim, and Noxa and inhibited the expression of Bcl-2, Bcl-X L , and Mcl-1. The quenching of ROS generation with antioxidant N -acetyl-l-cysteine conferred significant protection against sulforaphane-induced ROS generation, mitochondrial membrane potential disruption, caspase-3 activation, and apoptosis. Sulforaphane inhibited growth of orthotopically implanted PC-3 tumors by inducing apoptosis and inhibiting proliferation and also enhanced the antitumor activity of TRAIL. Sulforaphane up-regulated the expressions of TRAIL-R1/DR4, TRAIL-R2/DR5, Bax and Bak and inhibited the activation of nuclear factor-κB P13K/AKT and MEK/ERK pathways in tumor tissues. The combination of sulforaphane and TRAIL was more effective in inhibiting markers of angiogenesis and metastasis and activating FOXO3a transcription factor than single agent alone. Conclusions: The ability of sulforaphane to inhibit tumor growth, metastasis, and angiogenesis and to enhance the therapeutic potential of TRAIL suggests that sulforaphane alone or in combination with TRAIL can be used for the management of prostate cancer.

Background Glioblastoma multiforme is the most common form of primary brain tumor, often characterized by poor survival. Glioblastoma initiating cells (GICs) regulate self-renewal, differentiation, and tumor initiation properties and are involved in tumor growth, recurrence, and resistance to conventional treatments. The sonic hedgehog (SHH) signaling pathway is essential for normal development and embryonic morphogenesis. The objectives of this study were to examine the molecular mechanisms by which GIC characteristics are regulated by NPV-LDE-225 (Smoothened inhibitor; (2,2'-[[dihydro-2-(4-pyridinyl)-1,3(2H,4H)-pyrimidinediyl]bis(methylene)]bis[N,N-dimethylbenzenamine). Methods Cell viability and apoptosis were measured by XTT and annexin V-propidium iodide assay, respectively. Gli translocation and transcriptional activities were measured by immunofluorescence and luciferase assay, respectively. Gene and protein expressions were measured by quantitative real-time PCR and Western blot analyses, respectively. Results and conclusion NPV-LDE-225 inhibited cell viability, neurosphere formation, and Gli transcriptional activity and induced apoptosis by activation of caspase-3 and cleavage of poly(ADP-ribose) polymerase. NPV-LDE-225 increased the expression of tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-R1/DR4, TRAIL-R2/DR5, and Fas and decreased the expression of platelet derived growth factor receptor-α and Bcl2, and these effects were abrogated by Gli1 plus Gli2 short hairpin RNAs. NPV-LDE-225 enhanced the therapeutic potential of FasL and TRAIL by upregulating Fas and DR4/5, respectively. Interestingly, NPV-LDE-225 induced expression of programmed cell death 4 and apoptosis and inhibited cell viability by suppressing micro RNA (miR)-21. Furthermore, NPV-LDE-225 inhibited pluripotency-maintaining factors Nanog, Oct4, Sox2, and cMyc. The inhibition of Bmi1 by NPV-LDE-225 was regulated by induction of miR-128. Finally, NPV-LDE-225 suppressed epithelial-mesenchymal transition by upregulating E-cadherin and inhibiting N-cadherin, Snail, Slug, and Zeb1 through modulating the miR-200 family. Our data highlight the importance of the SHH pathway for self-renewal and early metastasis of GICs.

Multiple lines of evidence suggest that the Sonic Hedgehog (Shh) signaling pathway is aberrantly reactivated in pancreatic cancer stem cells (CSCs). The objectives of this study were to examine the molecular mechanisms by which GANT-61 (Gli transcription factor inhibitor) regulates stem cell characteristics and tumor growth. Effects of GANT-61 on CSC's viability, spheroid formation, apoptosis, DNA-binding and transcriptional activities, and epithelial-mesenchymal transition (EMT) were measured. Humanized NOD/SCID/IL2R gamma(null) mice were used to examine the effects of GANT-61 on CSC's tumor growth. GANT-61 inhibited cell viability, spheroid formation, and Gli-DNA binding and transcriptional activities, and induced apoptosis by activation of caspase-3 and cleavage of Poly-ADP ribose Polymerase (PARP). GANT-61 increased the expression of TRAIL-R1/DR4, TRAIL-R2/DR5 and Fas, and decreased expression of PDGFRα and Bcl-2. GANT-61 also suppressed EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Snail, Slug and Zeb1. In addition, GANT-61 inhibited pluripotency maintaining factors Nanog, Oct4, Sox-2 and cMyc. Suppression of both Gli1 plus Gli2 by shRNA mimicked the changes in cell viability, spheroid formation, apoptosis and gene expression observed in GANT-61-treated pancreatic CSCs. Furthermore, GANT-61 inhibited CSC tumor growth which was associated with up-regulation of DR4 and DR5 expression, and suppression of Gli1, Gli2, Bcl-2, CCND2 and Zeb1 expression in tumor tissues derived from NOD/SCID IL2Rγ null mice. Our data highlight the importance of Shh pathway for self-renewal and metastasis of pancreatic CSCs, and also suggest Gli as a therapeutic target for pancreatic cancer in eliminating CSCs.

Activation of sonic hedgehog (Shh) in cancer stem cell (CSC) has been demonstrated with aggressiveness of pancreatic cancer. In order to enhance the biological activity of α-mangostin, we formulated mangostin-encapsulated PLGA nanoparticles (Mang-NPs) and examined the molecular mechanisms by which they inhibit human and KC mice (PdxCre;LSL-KrasG12D) pancreatic CSC characteristics in vitro, and pancreatic carcinogenesis in KPC (PdxCre;LSLKrasG12D;LSL-Trp53R172H) mice. Mang-NPs inhibited human and KrasG12D mice pancreatic CSC characteristics in vitro. Mang-NPs also inhibited EMT by up-regulating E-cadherin and inhibiting N-cadherin and transcription factors Slug, and pluripotency maintaining factors Nanog, c-Myc, and Oct4. Furthermore, Mang-NPs inhibited the components of Shh pathway and Gli targets. In vivo, Mang-NPs inhibited the progression of pancreatic intraneoplasia to pancreatic ductal adenocarcinoma and liver metastasis in KPC mice. The inhibitory effects of Mang-NPs on carcinogenesis in KPC mice were associated with downregulation of pluripotency maintaining factors (c-Myc, Nanog and Oct4), stem cell markers (CD24 and CD133), components of Shh pathway (Gli1, Gli2, Patched1/2, and Smoothened), Gli targets (Bcl-2, XIAP and Cyclin D1), and EMT markers and transcription factors (N-cadherin, Slug, Snail and Zeb1), and upregulation of E-cadherin. Overall, our data suggest that Mang-NPs can inhibit pancreatic cancer growth, development and metastasis by targeting Shh pathway.

The objective of our study was to determine the groundwater quality in Mathadivagu basin, Adilabad district, Telanagana State, India. The study area lies between N. latitudes 19° 50' 48" and 20°13'30" and E. longitudes 78° 28' 25" and 78°58'00" with an area extent of 525 sq. kms. The current status of alluvial aquifer in the Mathadi vagu ba- sin, where the nitrate pollution of groundwater is being increasing along the last decades. A multi approach methodology using hydrogeology, nitrate concentrations. The analytical results revealed that the groundwater in the major part of the area is highly mineralized with high concentrations of Nitrate. Out of 37 ground water samples analysed, 31 samples were found to have nitrate concentration more than 45mg/1. The maximum concentration of nitrate in the area is 407.4 mg/l. The concentration of nitrate in excess of the permissible limit i.e., 45 mg/I in drinking water causes Methaemoglobinemia, particularly in infants. Agricultural practices in the study sites are the main cause of serious nitrate pollution given the su- perimposition of high nitrate concentrations with the distribution of irrigated perimeters. High nitrate levels are associated with high values, clearly indicating that significant quantities of evaporated irrigation waters infiltrate along with fertilizer nitrate to groundwater system.

According to the cancer stem cell hypothesis, the aggressive growth and early metastasis of cancer may arise through dysregulation of self-renewal of stem cells. The objectives of this study were to examine the molecular mechanisms by which sulforaphane (SFN, an active compound in cruciferous vegetables) inhibits self-renewal capacity of pancreatic cancer stem cells (CSCs), and synergizes with quercetin, a major polyphenol and flavonoid commonly detected in many fruits and vegetables. Our data demonstrated that SFN inhibited self-renewal capacity of pancreatic CSCs. Inhibition of Nanog by lentiviral-mediated shRNA expression enhanced the inhibitory effects of sulforaphane on self-renewal capacity of CSCs. SFN induced apoptosis by inhibiting the expression of Bcl-2 and XIAP, phosphorylation of FKHR, and activating caspase-3. Moreover, SFN inhibited expression of proteins involved in the epithelial-mesenchymal transition (beta-catenin, vimentin, twist-1, and ZEB1), suggesting the blockade of signaling involved in early metastasis. Furthermore, the combination of quercetin with SFN had synergistic effects on self-renewal capacity of pancreatic CSCs. These data suggest that SFN either alone or in combination with quercetin can eliminate cancer stem cell-characteristics.

Recent studies have demonstrated the involvement of colorectal cancer (CRC) stem cells (CSC) in transformation, cancer progression and metastasis. The main goal of this paper was to examine the molecular mechanisms by which SATB2 induced malignant transformation of colorectal epithelial cells. SATB2 induced malignant transformation and these transformed cells gained the characteristics of CSCs by expressing stem cell markers (CD44, CD133, LGR5 and DCLK1) and transcription factors (c-Myc, Nanog and Sox2). Overexpression of SATB2 in normal colorectal epithelial cells increased cell motility, migration and invasion, which were associated with an increase in N-cadherin and Zeb1, and decrease in E-cadherin expression. SATB2 overexpression also upregulated XIAP and cyclin D1, suggesting its role in cell survival and cell cycle. Furthermore, the expression of SATB2 was positively correlated with β-catenin expression in CRC. In contrary, depletion of SATB2 inhibited cell proliferation, colony formation, cell motility and expression of β-catenin, Snail, Slug, Zeb1 and N-cadherin, and upregulated E-cadherin. Furthermore, SATB2 silencing inhibited the expression of stem cell markers, pluripotency maintaining transcription factors, cell cycle and cell proliferation/survival genes and TCF/LEF targets. Finally, β-catenin/TCF-LEF pathway mediated the biological effects of SATB2 in CSCs. These studies support the role of SATB2/β-catenin/TCF-LEF pathway in transformation and carcinogenesis. Citation Format: Yiming Ma, Wei Yu, Sharmila Shankar, Rakesh K. Srivastava. SATB2/β-catenin/TCF-LEF pathway induces cellular transformation by generating cancer stem cells in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4509.

Findings from epidemiologic studies and research with experimental animal models provide insights into alcohol toxicity-related disease pathogeneses. Epidemiologic data indicate that heavy drinking is associated with high risk of pancreatic cancer. However the molecular mechanisms by which chronic ethanol exposure changes pathophysiology of human pancreatic ductal epithelial (HPNE) cells is not well understood. Cancer stem cells (CSCs) play significant role in cellular transformation, and their emergence is responsible for cancer initiation, progression and metastasis. SATB2 is required for normal mammalian development; however, it is not expressed in normal adult cells. Inappropriate activation of SATB2 gene may cause malignant cellular transformation. SATB2 regulates transcription of pluripotency maintaining factors which are required for sustaining the stem cells capacity for self-renewal. The main goal of the paper is to examine the molecular mechanisms by which chronic alcohol consumption induces cellular transformation of HPNE cells. To investigate the role of SATB2 at an early step of cell transformation, we utilized HPNE cells to generate progenitor cells either by chronic ethanol exposure or overexpression with SATB2. Our data demonstrate that chronic exposure of HPNE cells to ethanol induces cellular transformation by upregulating SATB2. Furthermore, during ethanol-induced cell transformation, cells gain the phenotype of progenitor cells / CSCs and express pluripotency maintaining factors and epithelial-mesenchymal transition (EMT) markers. Suppression of SATB2 expression in ethanol-transformed HPNE cells inhibits stemness and EMT. SATB2 is highly expressed in pancreatic cancer tissues derived from human and KrasG12D mice. In conclusion, chronic ethanol exposure may contribute in pancreatic carcinogenesis by inducing the expression of SATB2 gene. Citation Format: We Yu, Yiming Ma, Sharmila Shankar, Rakesh K. Srivastava. Chronic alcohol exposure induces malignant transformation of pancreatic ductal epithelial cells and generates stem cell characteristics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3068.

Activation of sonic Hedgehog (Shh) in cancer stem cell (CSC) has been demonstrated with aggressiveness of pancreatic cancer. In order to enhance the biologic activity of α-mangostin, we formulated mangostin-encapsulated PLGA nanoparticles (Mang-NPs) and examined the molecular mechanisms by which they inhibit human and KC mice (PdxCre;LSL-KrasG12D) pancreatic CSC characteristics in vitro, and pancreatic carcinogenesis in KPC (PdxCre;LSLKrasG12D;LSL-Trp53R172H) mice. Mang-NPs inhibited human and KrasG12D mice pancreatic CSC characteristics in vitro. Mang-NPs also inhibited EMT by upregulating E-cadherin and inhibiting N-cadherin and transcription factors Slug, and pluripotency maintaining factors Nanog, c-Myc, and Oct4. Furthermore, Mang-NPs inhibited the components of Shh pathway and Gli targets. In vivo, Mang-NPs inhibited the progression of pancreatic intraneoplasia to pancreatic ductal adenocarcinoma and liver metastasis in KPC mice. The inhibitory effects of Mang-NPs on carcinogenesis in KPC mice were associated with downregulation of pluripotency-maintaining factors (c-Myc, Nanog and Oct4), stem cell markers (CD24 and CD133), components of Shh pathway (Gli1, Gli2, Patched1/2, and Smoothened), Gli targets (Bcl-2, XIAP and Cyclin D1), and EMT markers and transcription factors (N-cadherin, Slug, Snail and Zeb1), and upregulation of E-cadherin. Overall, our data suggest that Mang-NPs can inhibit pancreatic cancer growth, development and metastasis by targeting Shh pathway. Citation Format: Raj K. Verma, Wei Yu, Anju Shrivastava, Rakesh K. Srivastava, Sharmila Shankar. α-Mangostin-encapsulated PLGA nanoparticles inhibit pancreatic carcinogenesis by targeting cancer stem cells in KC and KPC mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3728.

Resveratrol (trans-3,5,4′-trihydroxystilbene), a compound found largely in the skins of red grapes and wines, possesses anti-cancer and anti-angiogenic properties and protects the cardiovascular system. However, the molecular mechanisms by which resveratrol inhibits angiogenesis are currently subjects of intense investigation. The purpose of this study was to examine whether FOXO transcription factors mediate anti-angiogenic effects of resveratrol, and whether vascular endothelial growth factor (VEGF) neutralizing antibody can enhance these effects of resveratrol. Inhibition of PI3 kinase (PI3K)/AKT and MEK/ERK pathways synergistically inhibited migration and capillary tube formation of Human Umbilical Vein Endothelial Cells (HUVECs) and further enhanced the anti-angiogenic effects of resveratrol. Inhibitors of AKT and MEK kinase synergistically inhibited cytoplasmic FOXO3a phosphorylation, which was accompanied by its nuclear translocation in HUVECs. Interestingly, inhibition of PI3K/AKT and MEK/ERK pathways synergistically induced FOXO transcriptional activity and inhibited cell migration and capillary tube formation. Antiangiogenic effects of resveratrol were enhanced by inhibitors of AKT and MEK. Phosphorylation-deficient mutants of FOXOs induced FOXO transcriptional activity, inhibited HUVEC cell migration, and capillary tube formation, and also enhanced antiangiogenic effects of resveratrol. Finally, VEGF neutralizing antibody enhanced the anti-proliferative and anti-angiogenic effects of resveratrol. In conclusion, regulation of FOXO transcription factors by resveratrol may play an important role in angiogenesis which is critical for cancer, diabetic retinopathy, rheumatoid arthritis, psoriasis, and cardiovascular disorders.

Purpose: The purpose of this study was to examine the molecular mechanisms by which sulforaphane enhances the therapeutic potential of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in prostate cancer. Experimental Design: Cell viability and apoptosis assays were done by XTT and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, respectively. Tumor-bearing mice were treated with vehicle, sulforaphane, TRAIL, and sulforaphane plus TRAIL. Markers of apoptosis, angiogenesis, and metastasis were measured by immunohistochemistry. Results: Sulforaphane enhanced the therapeutic potential of TRAIL in PC-3 cells and sensitized TRAIL-resistant LNCaP cells. Sulforaphane-induced apoptosis in PC-3 cells correlated with the generation of intracellular reactive oxygen species (ROS), collapse of mitochondrial membrane potential, activation of caspase-3 and caspase-9, and up-regulation of DR4 and DR5. Sulforaphane induced the expression of Bax, Bak, Bim, and Noxa and inhibited the expression of Bcl-2, Bcl-XL, and Mcl-1. The quenching of ROS generation with antioxidant N-acetyl-l-cysteine conferred significant protection against sulforaphane-induced ROS generation, mitochondrial membrane potential disruption, caspase-3 activation, and apoptosis. Sulforaphane inhibited growth of orthotopically implanted PC-3 tumors by inducing apoptosis and inhibiting proliferation and also enhanced the antitumor activity of TRAIL. Sulforaphane up-regulated the expressions of TRAIL-R1/DR4, TRAIL-R2/DR5, Bax and Bak and inhibited the activation of nuclear factor-κB P13K/AKT and MEK/ERK pathways in tumor tissues. The combination of sulforaphane and TRAIL was more effective in inhibiting markers of angiogenesis and metastasis and activating FOXO3a transcription factor than single agent alone. Conclusions: The ability of sulforaphane to inhibit tumor growth, metastasis, and angiogenesis and to enhance the therapeutic potential of TRAIL suggests that sulforaphane alone or in combination with TRAIL can be used for the management of prostate cancer.

Curcumin, an active ingredient of turmeric (Curcuma longa), inhibits proliferation and induces apoptosis in cancer cells, but the sequence of events leading to cell death is poorly defined. The objective of this study was to examine the molecular mechanisms by which multidomain pro-apoptotic Bcl-2 family members Bax and Bak regulate curcumin-induced apoptosis using mouse embryonic fibroblasts (MEFs) deficient in Bax, Bak or both genes. Curcumin treatment resulted an increase in the protein levels of both Bax and Bak, and mitochondrial translocation and activation of Bax in MEFs to trigger drop in mitochondrial membrane potential, cytosolic release of apoptogenic molecules [cytochrome c and second mitochondria-derived activator of caspases (Smac)/direct inhibitor of apoptosis protein-binding protein with low isoelectric point], activation of caspase-9 and caspase-3 and ultimately apoptosis. Furthermore, MEFs derived from Bax and Bak double-knockout (DKO) mice exhibited even greater protection against curcumin-induced release of cytochrome c and Smac, activation of caspase-3 and caspase-9 and induction of apoptosis compared with wild-type MEFs or single-knockout Bax(-/-) or Bak(-/-) MEFs. Interestingly, curcumin treatment also caused an increase in the protein level of apoptosis protease-activating factor-1 in wild-type MEFs. Smac N7 peptide enhanced curcumin-induced apoptosis, whereas Smac siRNA inhibited the effects of curcumin on apoptosis. Mature form of Smac sensitized Bax and Bak DKO MEFs to undergo apoptosis by acting downstream of mitochondria. The present study demonstrates the role of Bax and Bak as a critical regulator of curcumin-induced apoptosis and over-expression of Smac as interventional approaches to deal with Bax- and/or Bak-deficient chemoresistant cancers for curcumin-based therapy.

Multiple myeloma is an incurable disease for the majority of patients, therefore requiring new biological targeted therapies. In primary myeloma cells, IMP dehydrogenase (IMPDH) was shown to be consistently overexpressed. We therefore tested the IMPDH inhibitor mycophenolate mofetil (MMF) currently available as a clinical therapeutic agent for its antimyeloma activity in vitro. MMF depleted intracellular guanosine 5′-triphosphate (GTP) levels in myeloma cells. We showed apoptosis induction in myeloma cell lines and primary myeloma cells between 1 and 5 μmol/L MMF. MMF was also cytotoxic at this concentration in dexamethasone-resistant and Mcl-1-overexpressed myeloma cell lines shown by the tetrazolium salt XTT assay along with cell survival measured by a modified flow cytometric assay. Apoptosis was not inhibited by the presence of an antioxidant, suggesting that MMF-induced apoptosis is less likely to be associated with reactive oxygen species. However, apoptosis was abrogated by exogenously added guanosine, which activates an alternative pathway for GTP formation, implicating that this effect is directly mediated by IMPDH inhibition. MMF-induced G1-S phase cell cycle arrest and its apoptosis induction mechanism were associated with a caspase-dependent pathway as shown by alteration of mitochondrial membrane potential and cytochrome c release followed by activation of the caspases. MMF-induced apoptosis was also inhibited by a pan-caspase inhibitor Z-VAD-fmk. MMF-treated myeloma cells showed an up-regulation of Bak, which most likely together with Bax resulted in the release of cytochrome c. In summary, MMF attenuates G1-S phase cell cycle progression and activates the pathway of mitochondrial dysfunction, leading to cytochrome c release followed by activation of caspases. [Mol Cancer Ther 2006;5(2):457–66]

In this study, we have evaluated the cytotoxic effect of combining two HDAC inhibitors, SAHA and TSA, with TRAIL in human multiple myeloma cell lines. Low doses of SAHA or TSA enhanced the cytotoxic and apoptotic effects of TRAIL and upregulated the surface expression of TRAIL death receptors (DR4 and/or DR5). SAHA and TSA induced G1 phase cell cycle growth arrest by upregulating p21(WAF1) and p27(Kip1) expression and by inhibiting E2F transcriptional activity. The enhanced TRAIL effect after pretreatment with HDAC inhibitors was consistent with the upregulation of the proapoptotic Bcl-2 family members (Bim, Bak, Bax, Noxa, and PUMA), the downregulation of the anti-apoptotic members of the Bcl-2 family (Bcl-2 and Bcl-X(L)), and IAPs. SAHA and TSA dissipated the mitochondrial membrane potential and enhanced the release of Omi/HtrA2 and AIF from the mitochondria to the cytosol. The cytotoxic effect of both SAHA and TSA was caspase- and calpain-independent. Inhibition of NF(kappa)B activation by the proteasome inhibitor, MG132, enhanced the apoptotic effect of TSA. Our study demonstrated the enhancing effects of HDAC inhibitors on apoptosis when combined with TRAIL and, for the first time, emphasized the role of AIF in mediating the cytotoxic effects of HDAC inhibitors.