Key Genes in Prostate Cancer Progression: Role of MDM2, PTEN, and TMPRSS2-ERG Fusions

In recent years, multiple genes or their protein products have been linked to initiation and progression of prostate cancer. Such genes include TMPRSS2, ERG, PTEN, and MDM2. This chapter discusses the pathological roles as well as the potential diagnostic and therapeutic applications of these genes that are highly expressed in prostate cancer when compared to other cancer types. The presence of these genes and related defects are linked to growth, progression, metastasis, invasiveness and resistance in prostate cancers. While knowledge related to TMPRSS2, ERG, and PTEN have been accumulating in the last two decades, the prometastatic role of MDM2 has been emerging in the last few years and revealing important functions related to prostate cancer progression

A Journey Towards Cancer Drug Discovery

Among the various drugs that are used for the treatment of human sufferings anticancer drugs may receive the distinction of being the most toxic to the human body. The side effects and toxicities of the anticancer drugs are primarily due to their inability to differentiate between the cancer cells and the normal cells in the body such as bone marrow cells and epithelial cells that are actively dividing. Some of the modern approaches are geared towards developing drugs or drug combination that are less toxic to the body and more effective towards cancer cells and tissues. In the process of effectively treating a cancer growth availability of sensitive methods for diagnosis also play a major role. The cancer research at the NSU (Nova Southeastern University) College of Pharmacy is focused on developing both therapeutics as well as sensitive diagnostic methods. One of the processes we have been targeting for the purpose of stopping the cancer growth and metastatic spread of cancer is known as angiogenesis, an intra tumoral process that helps the tumor to grow beyond 1 mm3 – 1 cm3 in size. Completion of angiogensis results in the formation of new blood vessels that traverse the tumor tissues and nourish them by providing oxygen and nutrients through establishment of blood circulation. Various growth factors, hormones and cytokines are known to be involved in the regulation of angiogenesis, however, the most important growth factor that controls angiogenesis is known as Vascular Endothelial Growth Factor (VEGF). Proangiogenic action of VEGF is mediated through VEGF receptors (VEGFR) and by the activation of associated kinases. For the purpose of inhibiting the angiogenic process several inhibitors of VEGF and VEGFR have been developed and one of them is a monoclonal antibody drug commercially available as Bevacizumab®. For the purpose of developing much stronger and highly specific angiogensis inhibitors we utilized molecular modeling approaches in collaboration with scientists from the Lombardi Comprehensive Cancer Center at Georgetown University. Through molecular modeling approach we were able to discover two drug molecules along with several others from a collection of molecules that exceeded 1 million in number. Two compounds, code named F16 and JFD, ranked at the top compared to several others that were identified through initial computer screening. Both these compounds showed strong cytotoxic as well as anti-angiogenic effects in our in vitro assays. The anti-tumor activity of these compounds was confirmed using the in vivo experiments also. Subsequently, patent applications were filed from NSU for the anti-tumor use of these compounds through their anti-angiogenic effect. The patent application for F16 was completed in 2006 and it is nearing approval at this time. The JFD patent application from NSU was completed in 2007 and is expected to be approved in 2009 Grants. This project was supported by the Center of Excellence for Marine Biology and Biotechnology grant from the state of Florida through FAU, also by the PFRD grant from NSU

Tumor Angiogenesis and Novel Vascular Endothelial Receptor (VEGFR)-Specific Small Molecule Inhibitors

The knowledge related to angiogenesis has grown exponentially over the past few decades with the recognition that angiogenesis is essential for numerous normal and pathological processes. Very importantly, angiogenesis is required for the growth and metastasis of solid tumors in human beyond the size of 1–2 mm³ (Arap et al. 1998; Folkman 1990, 1995). Angiogenesis is the process of developing buds and outgrowth of capillaries from existing blood vessels that are derived as extensions due to hypoxia or other forms of signaling that occurs in the microenvironment that surrounds a tissue or tumors. On the other hand neovascularization is defined as the formation of functional microvascular network with red blood cell perfusion. Angiogenesis is required for invasive tumor growth and metastasis and therefore constitutes an important step in the control of cancer progression. In general vascular tumors are severely restricted in their growth potential because of the lack of a blood supply. To achieve the new blood vessel formation, endothelial cells must first escape from their stable location by breaking through the basement membrane, and this degradation is associated with migration of endothelial cells out of the vascular channel toward the angiogenic stimulus. During this process, the subendothelial basement membrane, a dense meshwork of collagen, glycoproteins, and proteoglycans are proteolytically disrupted to allow formation of new capillaries. Though it is an integral component of normal processes such as reproduction and wound healing, angiogenesis is known to play an important role in other pathological processes ranging from tumor growth and metastasis to inflammation and ocular diseases. During angiogenesis tumor cells exploit their microenvironment by releasing cytokines and growth factors to activate normal, quiescent cells around them and initiate a cascade of events that quickly becomes dysregulated. For example, tumor cell-released vascular endothelial growth factor (VEGF) stimulates the sprouting and proliferation of endothelial cells and thereby play a crucial role in neovascularization of solid tumors (Leung et al. 1989). The expression of VEGF has been shown to correlate with the density of microvessels in various tumors and exhibit higher metastatic ability (Folkman 1995; Leung et al. 1989; Toi et al. 1994). Therefore, inhibition of angiogenesis triggered by VEGF or other factors is accepted as a valuable approach to cancer therapy.https://nsuworks.nova.edu/hpd_corx_facbooks/1004/thumbnail.jp

Effect of the HDAC Inhibitor on Histone Acetylation and Methyltransferases in A2780 Ovarian Cancer Cells

Background andObjective: Epigenetic modifications are believed to play a significant role in the development of cancer progression, growth, differentiation, and cell death. One of the most popular histone deacetylases inhibitors (HDACIs), suberoylanilide hydroxamic acid (SAHA), also known as Vorinostat, can directly activate p21WAF1/CIP1 gene transcription through hyperacetylation of histones by a p53 independent mechanism. In the present investigation, we evaluated the correlation between histone modifications and DNA methyltransferase enzyme levels following SAHA treatments in A2780 ovarian cancer cells. Materials and Methods: Acetylation of histones and methyltransferases levels were analyzed using RT2 profiler PCR array, immunoblotting, and immunofluorescence methods in 2D and 3D cell culture systems. Results: The inhibition of histone deacetylases (HDAC) activities by SAHA can reduce DNA methyl transferases / histone methyl transferases (DNMTs/HMTs) levels through induction of hyperacetylation of histones. Immunofluorescence analysis of cells growing in monolayers and spheroids revealed significant up-regulation of histone acetylation preceding the above-described changes. Conclusions: Our results depict an interesting interplay between histone hyperacetylation and a decrease in methyltransferase levels in ovarian cancer cells, which may have a positive impact on the overall outcomes of cancer treatment

Apoptosis Induction by Ocimum sanctum Extract in LNCaP Prostate Cancer Cells.

Tulsi (Ocimum sanctum Linn), commonly known as holy basil, has been used for the treatment of a wide range of ailments in many parts of the world. This study focuses on apoptosis-inducing ability of tulsi extract on prostate cancer cells. For this purpose LNCaP prostate cancer cells were treated with different concentrations of 70% ethanolic extract of tulsi (EET) and then the cytotoxicity was determined after 24 and 48 h. After treatment with EET externalization of phosphatidyl serine (PS) from the inner membrane to outer leaflet of the plasma membrane was clearly evidenced by the results obtained from both flow cytometry analysis with Annexin V-FITC and pSIVA-IANBD binding fluorescence microscopy assay. Depolarization of the mitochondrial membrane potential was also evidenced by the presence of 5,5\u27,6,6\u27-tetrachlolo-1,1\u27,3,3\u27-tetraethyl benzimedazolyl carbocyanine iodide (JC-1) monomeric form in the EET-treated cells that emitted the green fluorescence when compared with the control cells that emitted the red fluorescence due to aggregation of JC-1. Furthermore, the level of poly (ADP-ribose) polymerase (PARP) cleavage and Bcl-2 were determined using western blot analysis. When compared to the control cells the level of cleaved PARP was found to be higher with a concomitant decrease in the Bcl-2 level after 24 h of treatment of cells with EET. In addition, treatment with EET significantly elevated the activities of caspase-9 and caspase-3 in LNCaP cells compared with the control. Also, after 48 h of treatment all doses used in this study showed clear fragments of DNA, which is one of the hallmarks of apoptosis. Taken together, our findings suggest that, EET can effectively induce apoptosis in LNCaP cells via activation of caspase-9 and caspase-3 that can eventually lead to DNA fragmentation and cell death

The Effects of the Herbal Enzyme Bromelain Against Breast Cancer Cell Line GI-101A

Bromelain is a proteinase derived from the stem of pineapple and has been studied for its anti-inflammatory, antithrombotic, and antimetastatic properties. Bromelain has also been known to significantly reduce local tumor growth and to raise the impaired cytotoxicity of monocytes in the immune system against tumor cells. The goal of this project is to advance the mechanistic knowledge of herbal remedies and to confirm the already known antimetastatic properties of Bromelain. The MTS assay method was used 24 hours after Bromelain treatment to detect the cell death. The data show that after 1 µM of Bromelain treatment, the population of GI101A cells is significantly reduced by up to 70%. Using the M30 Apoptosense assay, levels of the protein cytokeratin 18 (CK18) were measured to detect any apoptotic activity. After 10 µL of Bromelain treatment, CK18 levels increased and a large number of apoptotic cell bodies were observed. The antitumor effects of Bromelain are mainly involved in cancer cell division by the induction of apoptosis

Effect of HDAC Inhibitor on DNA Methylation and Cell Cycle Regulation in Prostate Cancer

Objective: Our study was aimed to analyse the expression of methyltransferase levels in LNCaP (prostate Cancer) cells during SAHA treatment. Background: Prostate cancer is the second leading cause of death in men after lung cancer in the US. Nearly 1 in 8 men will be diagnosed of prostate cancer in their lifetime, and the risk increases significantly once the men cross the age of 70. Recognizing ways to reduce the death of prostate cancer is therefore a top research priority. Epigenetic regulation of gene plays an important role in the controlling cell cycle and tumor growth in various cancers. Epigenetic changes generally occur through alterations in DNA and Histone modification such as acetylation, methylation, phosphorylation, and ubiquitination. SAHA (Suberoylanilide Hydroxamic Acid) is a broad spectram inhibitor of histone deacetylase (HDAC), which is used to modify the status of Histone Acetylation during cancer treatments. However, the impact of SAHA on methyltransferase levels or methylation status of DNA has not been studied in detail. Methods: LNCaP cells were cultured in complete RPMI-1640 growth medium and treated with SAHA (7.5 uM) for 24 hours. Western blot technique was used to analyze the expression levels of DNMT3A, SUV39H1, PRMT1, and p21. Results: Our experimental results have shown that SAHA treatment reduce the levels of the methyltransferase enzymes listed above. Furthermore, SAHA treatment increased the protein levels of p21, which is a CDKI (cyclin-dependent kinase inhibitor). Conclusion: Methylation is an important modification of DNA that can regulate gene expressions. Our results indicate that SAHA treatment, which is known to regulate histone acetylation, can impact the methylation status also through an indirect mechanism and allow for the control of transcription of tumor suppressor genes. Acknowledgement: This Research was supported by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida

Effect of SAHA on the Expression of Chromatin Modifying Enzymes in Prostate and Breast Cancer Cells

Objective. Our study analyzed the effects of HDAC inhibitor SAHA treatment on gene expressions of Chromatin Modifying Enzymes. Background. Histone deacetylase (HDAC) inhibitors are one of the important epigenetic regulators that have enormous therapeutic potential in various diseases, including cancers. For example, SAHA (Suberoylanilidehydroxamic acid) has been known as a potent inhibitor of histone deacetylases that eventually lead to differentiation, growth arrest, and apoptosis of various cancer cells. Methods. In our study, we utilized the RT2 Profiler PCR Array that was specific for the Human Epigenetic Chromatin Modifying Enzymes. We examined the impact of SAHA (7.5 µM) treatment on gene expression patterns of LNCaP (prostate cancer cells) and MCF-7 (breast cancer) cells. Results. As a result of SAHA treatment, the expression levels of AURKB (0.11), SUV39H1 (0.23), AURKA (0.4), and SETD7 (0.49) were found to be significantly down-regulated compared to the control in the LNCaP cells. In addition, the mRNA level of KDM6B was also up-regulated (by 2.4 folds) after SAHA treatment. On the other hand, in the MCF-7 cells PAK1 (0.06), NSD1 (0.19), SETD7 (0.24), DNMT3A (0.31), NEK6 (0.34), SETD6 (0.38), PRMT1 (0.4), AURKB (0.4) and SUV39H1 (0.45) were found to be significantly down-regulated after 24 hr of SAHA treatment. Conclusion. Our results offer evidence that SAHA can impact the gene expression profile of epigenetic chromatin modification enzymes and exert its anti-cancer effect in both prostate and breast cancer cells. Grants. The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged

HDAC Inhibitor SAHA Sensitizes Metformin-Induced Cell Death in A2780 Ovarian Cancer Cells

HDAC inhibitor SAHA sensitizes Metformin-induced cell death in A2780 ovarian cancer cells Amal Alzahrani1, Ph.D student Theodore Lemuel Mathuram2, Ph.D., Research Associate Appu Rathinavelu1,2, Ph.D., Professor, Executive Director 1College of Pharmacy, 2Rumbaugh-Goodwin Institute for Cancer Research Objective. The study was conducted to assess the efficacy of metformin in combination with SAHA in ovarian cancer cell line (A2780). Background. Ovarian cancer is the seventh most common cancer among women with the highest mortality rate. The high mortality rate of ovarian cancer is attributed to the fact that most women are diagnosed at an advanced stage with poor survival rate. Recently, many studies have confirmed a profound effect of the known anti-diabetic drug, metformin, on various types of cancer including the ovarian cancer. Our study aims to repurpose metformin, by sensitizing A2780 cells with SAHA (pan-HDAC inhibitor) at lower doses. Methods. For this study, MTT assay was conducted with A2780 cells treated with metformin or SAHA, combination of metformin and SAHA at different doses for 24, 48 h. Western Blotting analysis was conducted to assess the protein expression levels of cytochrome C and p21. Results. Combination of metformin 0.1mM and SAHA 10 µM was able to significantly reduce cell viability after 48 h compared to metformin and SAHA alone. Moreover, the combination of metformin 0.1mM and SAHA 10 µM demonstrated significant upregulation of cytochrome C and p21 levels comparing to metformin and SAHA alone. Conclusion. Our results indicate that the reduction in cell viability, and upregulation of cytochrome C and p21 levels may be due to the sensitizing effect of SAHA to metformin. Grants. This study was funded by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida