Todd Schuster

(−)−Epigallocatechin Gallate Overcomes Resistance to Etoposide-Induced Cell Death by Targeting the Molecular Chaperone Glucose-Regulated Protein 78

Many beneficial properties have been attributed to (-)-epigallocatechin gallate (EGCG), including chemopreventive, anticarcinogenic, and antioxidant actions. In this study, we investigated the effects of EGCG on the function of glucose-regulated protein 78 (GRP78), which is associated with the multidrug resistance phenotype of many types of cancer cells. Our investigation was directed at elucidating the mechanism of the EGCG and GRP78 interaction and providing evidence about whether EGCG modulates the activity of anticancer drugs through the inhibition of GRP78 function. We found that EGCG directly interacted with GRP78 at the ATP-binding site of protein and regulated its function by competing with ATP binding, resulting in the inhibition of ATPase activity. EGCG binding caused the conversion of GRP78 from its active monomer to the inactive dimer and oligomer forms. Further, we showed that EGCG interfered with the formation of the antiapoptotic GRP78-caspase-7 complex, which resulted in an increased etoposide-induced apoptosis in cancer cells. We also showed that EGCG significantly suppressed the transformed phenotype of breast cancer cells treated with etoposide. Overall, these results strongly suggested that EGCG could prevent the antiapoptotic effect of GRP78, which usually suppresses the caspase-mediated cell death pathways in drug-treated cancer cells, contributing to the development of drug resistance.

Differential involvement of reactive oxygen species in apoptosis induced by two classes of selenium compounds in human prostate cancer cells.

Selenium is a promising chemopreventive agent for prostate cancer, possibly via an induction of apoptosis. Earlier studies have shown that selenite induces DNA single strand breaks (SSBs), reactive oxygen species (ROS), p53 Ser‐15 phosphorylation and caspase‐dependent and ‐independent apoptosis, whereas a methylselenol precursor methylseleninic acid (MSeA) induces caspase‐mediated apoptosis regardless of p53 status. Here we address three main questions: What types of ROS are induced by selenite vs. MSeA in LNCaP (p53 wild type, androgen‐responsive) and DU145 (mutant p53, androgen‐independent) prostate cancer cells? Does ROS generation depend on androgen signaling? What are the relationships among ROS, DNA SSBs, p53 and caspases? We show that selenite (5 μM) induced superoxide and hydrogen peroxide in LNCaP cells much more than in DU145 cells and the ROS generation was not affected by physiological androgen stimulation. MSeA (10 μM) induced apoptosis without either type of ROS in both cell lines. In LNCaP cells, we established superoxide as a primary mediator for selenite‐induced DNA SSBs, p53 activation and caspase‐mediated apoptosis. Furthermore a p53‐dominant negative mutant attenuated selenite‐induced ROS, leading to a proportionate protection against apoptosis. The results support the p53‐mitochondria axis in a feedback loop for sustaining superoxide production to lead to efficient caspase‐mediated apoptosis by selenite. In contrast, caspase‐mediated apoptosis induced by MSeA does not involve ROS induction. Since p53 is frequently mutated or deleted in prostate cancer and many other cancers, our results suggest that genotoxic vs. nongenotoxic classes of selenium may exert differential apoptosis efficacy depending on the p53 status of the cancer cells.

Eleostearic Acid Inhibits Breast Cancer Proliferation by Means of an Oxidation-Dependent Mechanism

Eleostearic acid (α-ESA) is a conjugated linolenic acid that makes up ∼60% of Momordica charantia (bitter melon) seed oil. Prior work found that water extract from bitter melon was able to inhibit breast cancer. Here, we investigated effects of α-ESA on both estrogen receptor (ER)–negative MDA-MB-231 (MDA-wt) and ER-positive MDA-ERα7 human breast cancer cells. We found that α-ESA inhibited proliferation of both MDA-wt and MDA-ERα7 cells, whereas conjugated linoleic acid had comparatively weak antiproliferative activity at 20 to 80 μmol/L concentrations. We also found that α-ESA (40 μmol/L) treatment led to apoptosis in the range of 70% to 90% for both cell lines, whereas conjugated linoleic acid (40 μmol/L) resulted in only 5% to 10% apoptosis, similar to results for control untreated cells. Addition of α-ESA also caused loss of mitochondrial membrane potential and translocation of apoptosis-inducing factor as well as endonuclease G from the mitochondria to the nucleus. Additionally, α-ESA caused a G2-M block in the cell cycle. We also investigated the potential for lipid peroxidation to play a role in the inhibitory action of α-ESA. We found that when the breast cancer cells were treated with α-ESA in the presence of the antioxidant α-tocotrienol (20 μmol/L), the growth inhibition and apoptosis effects of α-ESA were lost. An AMP-activated protein kinase inhibitor (Dorsomorphin) was also able to partially abrogate the effects of α-ESA, whereas a caspase inhibitor (BOC-D-FMK) did not. These results illustrate that α-ESA can block breast cancer cell proliferation and induce apoptosis through a mechanism that may be oxidation dependent.

PKR regulates B56α-mediated BCL2 phosphatase activity in acute lymphoblastic leukemia-derived REH cells

Protein phosphatase 2A (PP2A) is a heterotrimer comprising catalytic, scaffold, and regulatory (B) subunits. There are at least 21 B subunit family members. Thus PP2A is actually a family of enzymes defined by which B subunit is used. The B56 family member B56α is a phosphoprotein that regulates dephosphorylation of BCL2. The stress kinase PKR has been shown to phosphorylate B56α at serine 28 in vitro, but it has been unclear how PKR might regulate the BCL2 phosphatase. In the present study, PKR regulation of B56α in REH cells was examined, because these cells exhibit robust BCL2 phosphatase activity. PKR was found to be basally active in REH cells as would be predicted if the kinase supports B56α-mediated dephosphorylation of BCL2. Suppression of PKR promoted BCL2 phosphorylation with concomitant loss of B56α phosphorylation at serine 28 and inhibition of mitochondrial PP2A activity. PKR supports stress signaling in REH cells, as suppression of PKR promoted chemoresistance to etoposide. Suppression of PKR promoted B56α proteolysis, which could be blocked by a proteasome inhibitor. However, the mechanism by which PKR supports B56α protein does not involve PKR-mediated phosphorylation of the B subunit at serine 28 but may involve eIF2α activation of AKT. Phosphorylation of serine 28 by PKR promotes mitochondrial localization of B56α, because wild-type but not mutant S28A B56α promoted mitochondrial PP2A activity. Cells expressing wild-type B56α but not S28A B56α were sensitized to etoposide. These results suggest that PKR regulates B56α-mediated PP2A signaling in REH cells.

Epicatechin-rich cocoa polyphenol inhibits Kras-activated pancreatic ductal carcinoma cell growth in vitro and in a mouse model

Activated Kras gene coupled with activation of Akt and nuclear factor‐kappa B (NF‐κB) triggers the development of pancreatic intraepithelial neoplasia, the precursor lesion for pancreatic ductal adenocarcinoma (PDAC) in humans. Therefore, intervention at premalignant stage of disease is considered as an ideal strategy to delay the tumor development. Pancreatic malignant tumor cell lines are widely used; however, there are not relevant cell‐based models representing premalignant stages of PDAC to test intervention agents. By employing a novel Kras‐driven cell‐based model representing premalignant and malignant stages of PDAC, we investigated the efficacy of ACTICOA‐grade cocoa polyphenol (CP) as a potent chemopreventive agent under in vitro and in vivo conditions. It is noteworthy that several human intervention/clinical trials have successfully established the pharmacological benefits of cocoa‐based foods. The liquid chromatography (LC)–mass spectrometry (MS)/MS data confirmed epicatechin as the major polyphenol of CP. Normal, nontumorigenic and tumorigenic pancreatic ductal epithelial (PDE) cells (exhibiting varying Kras activity) were treated with CP and epicatechin. CP and epicatechin treatments induced no effect on normal PDE cells, however, caused a decrease in the (i) proliferation, (ii) guanosine triphosphate (GTP)‐bound Ras protein, (iii) Akt phosphorylation and (iv) NF‐κB transcriptional activity of premalignant and malignant Kras‐activated PDE cells. Further, oral administration of CP (25 mg/kg) inhibited the growth of Kras‐PDE cell‐originated tumors in a xenograft mouse model. LC–MS/MS analysis of the blood showed epicatechin to be bioavailable to mice after CP consumption. We suggest that (i) Kras‐driven cell‐based model is an excellent model for testing intervention agents and (ii) CP is a promising chemopreventive agent for inhibiting PDAC development.

Role for PKC δ in Fenretinide-Mediated Apoptosis in Lymphoid Leukemia Cells

The synthetic Vitamin A analog fenretinide is a promising chemotherapeutic agent. In the current paper, the role of PKC δ was examined in fenretinide-induced apoptosis in lymphoid leukemia cells. Levels of proapoptotic cleaved PKC δ positively correlated with drug sensitivity. Fenretinide promoted reactive oxygen species (ROS) generation. The antioxidant Vitamin C prevented fenretinide-induced PKC δ cleavage and protected cells from fenretinide. Suppression of PKC δ expression by shRNA sensitized cells to fenretinide-induced apoptosis possibly by a mechanism involving ROS production. A previous study demonstrated that fenretinide promotes degradation of antiapoptotic MCL-1 in ALL cells via JNK. Now we have found that fenretinide-induced MCL-1 degradation may involve PKC δ as cleavage of the kinase correlated with loss of MCL-1 even in cells when JNK was not activated. These results suggest that PKC δ may play a complex role in fenretinide-induced apoptosis and may be targeted in antileukemia strategies that utilize fenretinide.

Abstract 1821: Identifying a novel mechanism underlying the enzalutamide and bicalutamide resistance in African-American prostate cancer patients

Recently, FDA approved the Enzalutamide as a drug for castration-resistant prostate cancer. Recent studies reported patient populations which are non-responsive to the Enzalutamide therapy. Notably, African-American patients do not fare well for the castration-therapies (Bicalutamide and Enzalutamide). To identify a new therapeutic strategy for such populations, it is very important to understand the mechanism underlying the resistance to Bicalutamide or Enzalutamide. This would involve the use of an appropriate Bicalutamide or Enzalutamide-resistant model. We hypothesized that heterogeneous sub-populations contribute to Enzalutamide and bicalutamide resistance. In this study, we generated cell models representing Bicalutamide- and Enzalutamide-resistant phenotypes of primary prostate tumor of African-Americans. We next isolated subpopulations of Enzalutamide RC-Enz; Eht-Enz) and bicalutamide-resistant (RCbc; Eht-bc) cells. The cell sub-populations were ranked into three major classes’ viz., (1) highly stem-cell like (expressing several stemness markers BMI1 & CD133), (2) less stem-cell like (express one stemness marker BM1), and (3) non-stem cell like (negative for BMI1 and CD133). Notably, African-American primary CaP cells exhibited higher number of stem cell-like populations than Caucasian primary (22Rν1) and metastatic cells (PC3). When compared, the African-American Enzalutamide/Bicalutamide-resistant stem cell-like (RC-EnzCD133+/BM1+, RC-bcCD133+/BM1+, Eht-bcCD133+/BM1+ & Eht-EnzCD133+/BM1+) cells exhibited increased rate of proliferation, invasiveness and migration than Enzalutamide-resistant Caucasian stem cell-like cells(LNCaP95EnzCD133+/BM1+). We show that RC-EnzCD133+/BM1+ and RC-bcCD133+/BM1+ cells resistant cells exhibit increased (i) promoter activity of BMI1 gene, (ii) localization of BMI1 protein on PTEN gene, and (iii) physical interaction of BMI1 tumor to E4F1 tumor suppressor protein. This was validated in prostatic tumors of African-Americans. We provide evidence that E4F1 under normal conditions negatively regulates the activity of Androgen receptor (AR)-associated signaling and inhibits growth of cells. We show that BMI1 protein sequesters E4F1 protein and inhibits its check-point type or negative regulation of AR-pathway in Enzalutamide and Bicalutamide-resistant cells. This leads to the growth of stem-cell like tumor cells during the Enzalutamide and Bicalutamide therapies. To conclude, we suggest that (1) E4F1-regulated AR-signaling plays an important role in prostate cancer, particularly in African-Americans, (2) ratio of E4F1 and BM1 expression has the potential as a biopsy biomarker for deciding the disease phenotype and (3) E4F1/BMI1 as a therapeutic target could be exploited to increase the sensitivity to Enzalutamide and Bicalutamide therapies in African-Americans. Citation Format: Arsheed Ganaie, Badrinath R. Konety, Todd Schuster, Mohammad Saleem. Identifying a novel mechanism underlying the enzalutamide and bicalutamide resistance in African-American prostate cancer patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1821.

Abstract 3847: Lupeol, a novel inhibitor of Wnt/β-catenin signaling: Implications in colon cancer therapy

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Wnt/β-catenin signaling pathway which regulates a variety of cellular processes has been reported to play a critical role in the pathogenesis of human colon cancer and found in almost 90% of the colon cancer patients. Recent studies from our laboratory and others showed that Lupeol a natural triterpene acts as a potent and selective inhibitor of Wnt signaling in prostate and melanoma tumor cells (Saleem et al, Carcinogenesis, 30: 801-17, 2009). Keeping in view these reports, we hypothesized that Lupeol will inhibit the tumorigenic growth of colon cancer cells (exhibiting aberrant Wnt/α signaling) under in vitro and in vivo conditions. Objectives: We investigated the effect of Lupeol on growth of human normal colon and carcinoma cells (exhibiting varied Wnt profile) and determined if Lupeol could enhances the efficacy of Sulindac, (Wnt signaling inhibitor and chemotherapeutic agent) that is used in clinics to treat human colon cancer. Methods: To test our hypothesis we employed in vitro and in vivo strategies. Firstly, normal human colon epithelial cells (FHC) and colon carcinoma cell lines (SW-480, HCT-116, HT-29) treated with either Lupeol (10-50 μM), or Sulindac (100-200 μM), or combination (Lupeol + Sulindac) were tested for growth, proliferation, clonogenicity and surrogate biomarkers of Wnt signaling by employing 3[H]thymidine uptake, MTT, Soft-agar colony, FACS, immunoblot and luciferase-reporter assays. Secondly, we investigated whether Lupeol is bio-available to mice after oral and intraperitoneal (i.p.) administration by using mass spectrometry. Next, we treated athymic mice bearing colon tumors-derived from HT-29 cells (highly chemoresistant among all colon cells) with either Lupeol (40 mg/kg) or Sulindac (20 mg/kg) or combination and measured tumor growth. In vivo proliferation and biochemical analysis of harvested tumors for surrogate biomarkers of apoptosis, proliferation and Wnt signaling were measured by employing BrdU uptake, immunoblot and immunhistochemical analysis. Results: Lupeol treatment was observed to (i) inhibit the proliferation of colon cancer cells (exhibiting activated Wnt) while sparing normal cells, (ii) induce G0/G1 arrest and apoptosis, (iii) decrease cytoslic as well as nuclear β-catenin levels (iv) increase degradation of cytosolic β-catenin, (iv) decrease TCF-transcriptional activity and (v) decrease the expression of Wnt target genes. Lupeol was observed to bioavailable in mice after oral and i.p. administration. Lupeol significantly inhibited the tumorigenicity of colon tumor cells in a xenograft mouse model. Most importantly, Lupeol treatment significantly sensitized highly chemoresistant colon cancer cells to Sulindac therapy, in vitro and in vivo. Conclusion: Lupeol alone or as an adjuvant to current therapies could be developed as an agent to treat subtype of human colon cancer exhibiting constitutive activation of Wnt signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3847. doi:1538-7445.AM2012-3847

Abstract A72: Punicic acid, a fatty acid from pomegranate seed oil, inhibits breast cancer cell proliferation

Introduction: Pomegranate (Punica granatum) is a well‐known fruit used for alternative medicine. Various pomegranate extracts are utilized as supplements with a wide variety of purported health benefits including anticancer actions. These supplements contain a number of different components that may either activate or inhibit estrogen9s action. Additionally factors associated with pomegranate fruit or extracts may lower oxidation of LDL cholesterol, reduced arterial plaques, lower oxidative stress, enhanced wound healing, inhibition of angiogenesis and tumor proliferation and increased tumor apoptosis. However, these studies have been conducted with fruit, seeds, pericarp or partially purified pomegranate extracts making it hard to determine what the active component(s) may be. Punicic acid is an omega‐5 long chain polyunsaturated fatty acid found in pomegranate seed oil. A number of long chain fatty acids have been reported to have cancer preventive actions. Here we investigated the potential ability of punicic acid to affect growth of both an estrogen insensitive breast cancer cell line (MDA‐MB‐231) and an estrogen sensitive cell line developed from the MDAMB‐231 cells (MDA‐ER 7). Methods: The two breast cancer cell lines were cultured in vitro in the presence of various amounts of punicic acid, conjugated linoleic acid, α‐linolenic acid and other substances to evaluate cell proliferation, apoptosis, mitochondrial membrane permeability and cell cycle. Results: We found that 40 µM punicic acid inhibited proliferation of MDA‐MB‐231 and MDA‐ER 7 cells 92% and 96% respectively compared to untreated cells. Furthermore, punicic acid induced apoptosis in the MDA‐MB‐231 and MDA‐ER 7 cells by 86% and 91% respectively compared to untreated control cells. Punicic acid also disrupted mitochondrial membrane potential of both cell lines. We next investigated whether lipid oxidation was required for the function of punicic acid by adding 20 µM of the antioxidant tocotrienol to the assays. This resulted in reversal of the effects of punicic acid on proliferation inhibition, apoptosis and disruption of the mitochondrial membrane potential in both the MDA‐MB‐231 and MDA‐ER 7 cells. Finally, we evaluated the role of PKC signaling in the anticancer effects of punicic acid by performing proliferation assays in the presence of the PKC inhibitor bisindolymaleimide I. Proliferation inhibition by punicic acid was partially blocked in both the MDA‐MB‐231 and MDA‐ER 7 cells. Conclusions: Our in vitro assays indicated that punicic acid inhibited both estrogen sensitive and estrogen insensitive cell lines equally. Additionally the results indicate that the antiproliferative effect of punicic acid on human breast cancer cells appears to be at least partially dependent on lipid peroxidation and the PKC pathway. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A72.