James Stokes

Targeting Hsp70: A possible therapy for cancer

In all organisms, heat-shock proteins (HSPs) provide an ancient defense system. These proteins act as molecular chaperones by assisting proper folding and refolding of misfolded proteins and aid in the elimination of old and damaged cells. HSPs include Hsp100, Hsp90, Hsp70, Hsp40, and small HSPs. Through its substrate-binding domains, Hsp70 interacts with wide spectrum of molecules, ranging from unfolded to natively folded and aggregated proteins, and provides cytoprotective role against various cellular stresses. Under pathophysiological conditions, the high expression of Hsp70 allows cells to survive with lethal injuries. Increased Hsp70, by interacting at several points on apoptotic signaling pathways, leads to inhibition of apoptosis. Elevated expression of Hsp70 in cancer cells may be responsible for tumorigenesis and for tumor progression by providing resistance to chemotherapy. In contrast, inhibition or knockdown of Hsp70 reduces the size of tumors and can cause their complete regression. Moreover, extracellular Hsp70 acts as an immunogen that participates in cross presentation of MHC-I molecules. The goals of this review are to examine the roles of Hsp70 in cancer and to present strategies targeting Hsp70 in the development of cancer therapeutics.

Resveratrol induces mitochondria-mediated, caspase-independent apoptosis in murine prostate cancer cells

Found in the skins of red fruits, including grapes, resveratrol (RES) is a polyphenolic compound with cancer chemopreventive activity. Because of this activity, it has gained interest for scientific investigations. RES inhibits tumor growth and progression by targeting mitochondria-dependent or -independent pathways. However, further investigations are needed to explore the underlying mechanisms. The present study is focused on examining the role of RES-induced, mitochondria-mediated, caspase-independent apoptosis of prostate cancer cells, namely transgenic adenocarcinoma of mouse prostate (TRAMP) cells. These cells were exposed to RES for various times, and cell killing, cell morphology, mitochondrial membrane potential (Δψm), expression of Bax and Bcl2 proteins, the role of caspase-3, and DNA fragmentation were analyzed. TRAMP cells exposed to RES showed decreased cell viability, altered cell morphology, and disrupted Δψm, which led to aberrant expression of Bax and Bcl2 proteins. Furthermore, since the caspase-3 inhibitor, z-VAD-fmk (benzyloxycarbonyl-valine-alanine-aspartic acid-fluoromethyl ketone), had no appreciable impact on RES-induced cell killing, the killing was evidently caspase-independent. In addition, RES treatment of TRAMP-C1, TRAMP-C2, and TRAMP-C3 cells caused an appreciable breakage of genomic DNA into low-molecular-weight fragments. These findings show that, in inhibition of proliferation of TRAMP cells, RES induces mitochondria-mediated, caspase-independent apoptosis. Therefore, RES may be utilized as a therapeutic agent to control the proliferation and growth of cancer cells.

Abstract 4328: Resveratrol inhibits NO-mediated oxidative stress in murine prostate cancer cells

Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA Background: Nitric oxide (NO) is an important signaling molecule in the immune, nervous and cardiovascular systems. NO involves in the regulation of cellular behavior as well as cytotoxic events. Studies demonstrated that resveratrol (RES) (a polyphenolic compound in the skin of red fruits) at high concentration (≥50μM) induces NO production in endothelial F-2 cells and in human umbilical vein endothelial cells. However, the impact of RES on the biological functions of NO are still poorly understood. Therefore, the goal of this study was to investigate the impact of RES on NO-mediated oxidative stress at mitochondria level in mouse prostate cancer (PCa) cells. Experimental Procedures: The transgenic adenocarcinoma of the mouse prostate (TRAMP) cells (mirrors the pathogenesis of human prostate cancer) were cultured in Dulbeccos Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin 100 IU/ml, streptomycin 100 μg/ml, 0.005mg/ml bovine insulin, and 10nM Dehydroisoandrosterone in 5% CO2 at 370C. Cells were harvested by trypsinization after 80% confluency (at 4-5 pass), counted (105/ml), and incubated with RES (50uM), RES (50uM) + Deta-NONOate (750uM), Deta-NONOate (750uM), RES (50uM) +L-NMMA (750uM), and L-NMMA (750uM). Deta-NONOate was used as nitric oxide donor while L-NMMA was used as nitric oxide inhibitor. Thereafter, cell viability, NO-estimation in culture supernatant, mitochondrial membrane potential (MMP), total ROS-production in mitochondria and cytoplasm, and apoptotic assay (Annexin-V and caspase-3/7) were carried out. Results and Conclusion: PCa cells incubated with RES showed decreased cell viability, decreased NO production in culture supernatant, decreased MMP, and decreased level of ROS in mitochondria. However, PS-externalization, and total ROS in cytoplasm were found high after normalization with control cells. Further, treatment with Deta-NONOate results in decreased cell viability, decreased total ROS in the cytoplasm, and decreased MMP, while mitochondrial ROS and NO production in culture supernatant were observed highly significant as compared control cells. Furthermore, cells were also exposed to the combination of RES and NO which suggest that RES reduced NO-mediated cell killing and reactive oxygen species generation in TRAMP cells. However, no significant changes were observed after L-NMMA exposure to TRAMP cells. The result demonstrates that RES protect cells from NO-mediated cell killing and induced NO-independent apoptosis in PCa cells. This suggests that role of RES as a potential therapeutic agent to control cancer. However, further investigation is needed to explore potential mechanism of action of RES in controlling cancer proliferation. Citation Format: Sanjay Kumar, James Stokes, Karyn Scissum Gunn, Selvarangan Ponnazhagan, Upender Manne, Manoj K. Mishra. Resveratrol inhibits NO-mediated oxidative stress in murine prostate cancer cells. [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 4328.

Prolonged exposure of resveratrol induces reactive superoxide species–independent apoptosis in murine prostate cells

Nitric oxide, a signaling molecule, inhibits mitochondrial respiration by binding with cytochrome c oxidase, resulting in elevated production of reactive superoxide species (reactive oxygen and nitrogen) in the mitochondria and increased susceptibility to cell death. Generation of mitochondrial superoxide species can be suppressed by natural compounds such as resveratrol, a dietary polyphenol found in the skin of red fruits. In various cancer cells, resveratrol shows anti-oxidant and cancer preventive properties. Since, the effect of resveratrol on reactive superoxide species–independent apoptosis in prostate cancer cells is not well illustrated; therefore, we investigated this phenomenon in TRAMP murine prostate cancer cells. To accomplish this, TRAMP cells were incubated with resveratrol, resveratrol + DETA-NONOate, DETA-NONOate (nitric oxide donor), resveratrol + L-NMMA, or L-NMMA (nitric oxide inhibitor) for 48 h, and reactive superoxide species in the mitochondria and culture supernatant were measured. In addition, the mitochondrial membrane potential, cell viability, expression of apoptotic markers (Bax and Bcl2), γ-H2A.x, p53, and caspase-3 was determined. We found that resveratrol suppressed reactive superoxide species such as reactive oxygen species in the mitochondria and nitric oxide in culture supernatant when compared to the DETA-NONOate treatment and disrupted the mitochondrial membrane potential. Resveratrol also reduced cell viability, altered the expression of apoptotic markers (Bax and Bcl2), and increased expression of γ-H2A.x (indicative marker of DNA fragmentation) and p53 (a critical DNA damage response protein). However, there was no appreciable modulation of the caspase-3. Therefore, our data suggest that resveratrol induces superoxide species–independent apoptosis and may act as a therapeutic agent against prostate cancer.

Enhancer of Zeste Homology 2 (Ezh2), an Epigenetic Regulator: A Possibility for Prostate Cancer Treatment

Mounting evidence of epigenetic dysregulation in cancer development has received lots of attention from cancer researchers and physicians. Enhancer of zeste homology 2 (Ezh2), an epigenetic regulator, plays an important role in cellular growth and survival. Ezh2 is a catalytic core protein, which belongs to Poly comb Repressor Complex 2 (PRC2). Ezh2 encodes histone methyltransferase, which induces the trimethylation of histone3 lysine27 (H3K27) and is involved in gene repression. The expression of Ezh2 is increased in breast, colon, lung, pancreatic and prostate cancer. Increased expression of Ezh2 is associated with the development and progression of malignant cancers, especially with the prostate cancer. Therefore, studies on Ezh2 inhibition have added more focus towards drug discovery and development to combat cancers. Thus, this offers great potential for the development of second-generation cancer therapies. Therefore, in this chapter we provide an analysis of novel cancer treatment methods that target epigenetic factors such as Ezh2. We will examine key examples of Ezh2 regulation in prostate cancer as well as novel drug concepts that employ epigenetic targets. This chapter will also provide a glimpse of recent successes in cancer epigenetic drug discovery and consider important epigenetic factors for scientific success in combating cancer.

Epigenetic and Cancer: An Evaluation of the Impact of Dietary Components

Epigenetics is an area of research that has recently gained much attention from scientists. Epigenetic processes can induce changes within an organism without altering its genetic makeup. More interestingly, epigenetic mechanisms have the strong ability to modulate gene expression without directly altering the sequences of DNA bases. Dietary compounds consist of several bioactive constituents, which actively regulate different molecular targets involved in tumorigenesis. Keeping these facts in view, we provide evidence that these dietary components (e.g. resveratrol (RES), curcumin, genistein, polyphenols and sulforaphane) might interact with various epigenetic targets in cancer therapeutics. These bioactive compounds can modulate normal DNA methylation and histone acetylation patterns, which are essential for the activation of cancer fighting genes. Compounds, such as the ones listed above, induce epigenetic changes associated with the expression of tumor suppressor genes, such as p53, and inhibition of tumor promoting genes such as telomerase reverse transcriptase during tumor progression. Therefore, in this chapter, we present considerable evidence that bioactive compounds and their epigenetic targets are linked with cancer therapeutics, which may open the door to novel drug discovery and development. Remarkable improvements in our understanding of basic epigenetic mechanisms coupled with the rapid progress in the development of powerful new technologies hold great promise for the advancement of cancer treatment.

Abstract 908: Impact of microbial metabolites and resveratrol in prostate cancer progression and clearance

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Short-chain fatty acids (SCFAs) are the chemical by-products of microbial fermentation. Resistant carbohydrates, some starches, and foods generally rich in fiber are used as substrates for this process. SCFAs are known to activate key cellular receptors such as GPR43/FFA2, which mediate programmed cell death in different cancer cells. Resveratrol (RES) is a polyphenolic compound found in select vegetables and fruits and has been known for its versatility in preventing and reversing a number of diseases including some cancers. Much like SCFAs, RES has the ability to induce programmed cell death in many clinical cancer models. To find out if the SCFAs and RES modulates the different pathways that controls the cancer progression, we utilized prostate cancer (PCa) cell lines to establish a) if, there is any homology between SCFAs produced by normal host epithelial cells and SCFAs produced by host intestinal microflora, and b) assess the in vitro/vivo effectiveness of derived SCFAs utilizing transgenic adenocarcinoma mouse prostate (TRAMP C1, C2, and C3) cell line(s), human prostate cancer cell line (PC-3) and live murine specimens, respectively. Preliminary data indicates that SCFAs and RES together regulate cell death pathways of PCa cells. Additionally, the SCFAs have the capability to manipulate different molecular signatures that directly govern the progression of PCa cells. Therefore, our data suggests that these two compounds (SCFAs and RES) share related biological pathways facilitated by intestinal microflora that can be used to regulate prostate cancer progression and clearance. Citation Format: James Stokes, Manoj K. Mishra. Impact of microbial metabolites and resveratrol in prostate cancer progression and clearance. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 908. doi:10.1158/1538-7445.AM2015-908

Abstract 4107: Efficient delivery of dietary compound modulates mcp-1 in murine prostate cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Monocyte chemo-attractant protein (MCP-1) is a chemokine, which recruits monocytes, neutrophils, and lymphocytes in response to trauma and proinflammatory cytokines such as TNF-α. MCP-1 generally is not expressed in healthy individuals, but in response to various stressful stimuli it gets up-regulated, which in turn contributes to increased inflammation. MCP-1 has been implicated in mediating apoptosis by producing a proapoptotic protein designated MCP-induced protein (MCPIP). Resveratrol (RES), a polyphenolic compound found in the skin of red fruits, exhibits anti-inflammatory, anti-oxidative, anti-cancer, and anti-proliferative characteristics. In this study, we hypothesized that the functionalization of RES with detonated nano-diamond (DND, hereafter called fDND) will enhance the effective delivery of RES to tumor cell and impact the modulation of molecular mechanisms in murine prostate cancer (PCa) cells. This hypothesis was tested on mouse PCa cell lines, derived from transgenic adenocarcinoma of mouse prostate (TRAMP), in C57/B6 mice. Interestingly, TRAMP-C1 and TRAMP-C2 form tumor while TRAMP-C3 fails to develop tumor. TRAMP cells were treated with different concentrations of fDND at different time points and analyzed for underlying cellular and molecular mechanisms such as cell viability/proliferation, mitochondrial membrane potential, cytokines and growth factors production. The initial results indicate that the fDND significantly induce molecular signatures in TRAMP-C3 cells in comparison with TRAMPC1 and C2. Interestingly, the level of MCP-1 was significant elevated in TRAMP C1 and C2 cells. Thus, suggesting a major role of MCP-1 in the regulation of cancer progression and clearance. This data also suggest that the functionality of dietary compounds can be enhanced by conjugating or functionalizing them with DND, which can be used as a therapeutic carrier. Citation Format: Manoj K. Mishra, James A. Stokes, Amanee Salaam, Elijah Nyairo, Udai Singh, Derrick Dean. Efficient delivery of dietary compound modulates mcp-1 in murine prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4107. doi:10.1158/1538-7445.AM2014-4107