Arkene Levy

6-Shogaol reduced chronic inflammatory response in the knees of rats treated with complete Freund's adjuvant.

Background6-Shogaol is one of the major compounds in the ginger rhizome that may contribute to its anti-inflammatory properties. Confirmation of this contribution was sought in this study in Sprague- Dawley rats (200–250 g) treated with a single injection (0.5 ml of 1 mg/ml) of a commercial preparation of complete Freunds Adjuvant (CFA) to induce monoarthritis in the right knee over a period of 28 days. During this development of arthritis, each rat received a daily oral dose of either peanut oil (0.2 ml-control) or 6-shogaol (6.2 mg/Kg in 0.2 ml peanut oil).ResultsWithin 2 days of CFA injection, the control group produced maximum edematous swelling of the knee that was sustained up to the end of the investigation period. But, in the 6-shogaol treated group, significantly lower magnitudes of unsustained swelling of the knees (from 5.1 ± 0.2 mm to 1.0 ± 0.2 mm, p < 0.002, n = 6) were produced during the investigation period. Unsustained swelling of the knees (from 3.2 ± 0.6 mm to 0.8 ± 1.1 mm, p < 0.00008, n = 6) was also produced after 3 days of treatment with indomethacin (2 mg/Kg/day) as a standard anti-inflammatory drug, but during the first 2 days of drug treatment swelling of the knees was significantly larger (11.6 ± 2.0 mm, p < 0.0002, n = 6) than either the controls or the 6-shogaol treated group of rats. This exaggerated effect in the early stage of indomethacin treatment was inhibited by montelukast, a cysteinyl leukotriene receptor antagonist. Also, 6-shogaol and indomethacin were most effective in reducing swelling of the knees on day 28 when the controls still had maximum swelling. The effect of 6-shogaol compared to the controls was associated with significantly lower concentration of soluble vascular cell adhesion molecule-1 (VCAM-1) in the blood and infiltration of leukocytes, including lymphocytes and monocytes/macrophages, into the synovial cavity of the knee. There was also preservation of the morphological integrity of the cartilage lining the femur compared to damage to this tissue in the peanut oil treated control group of rats.ConclusionFrom these results, it is concluded that 6-shogaol reduced the inflammatory response and protected the femoral cartilage from damage produced in a CFA monoarthritic model of the knee joint of rats.

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 diagnos‐ tic 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.

Abstract 2090: Evaluation of the cytotoxic profile of Metformin and Y15 in platinum resistant ovarian cancer cells

Platinum resistance remains a major challenge in the chemotherapeutic management of ovarian cancer. The anti-diabetic drug metformin has been previously shown to induce cytotoxicity in platinum resistant ovarian cancer cells and overexpression and increased phosphorylation of a tyrosine kinase called Focal Adhesion Kinase (FAK) has been implicated in the development of this platinum resistance. Therefore, in the present study we evaluated the combined cytotoxic efficacy of Metformin and the focal adhesion kinase inhibitor 1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) in platinum resistant OVCAR3 ovarian cancer cells. Cells were initially treated with concentrations of Y15 ranging from 10-100 μM, and metformin from 10-100mM to determine 1C50 values. Subsequently, cells were treated with Y15 (80 μM) and metformin (26mM) alone and in combination. All treatments were triplicated with duration of 24hrs and control cells exposed to media only. The cytotoxic profile of each treatment was assessed using the automated trypan blue assay. DNA fragmentation and poly ADP ribose polymerase (PARP) cleavage assays were performed to evaluate the mechanism of cell death and we further evaluated the expression of phosphorylated FAK, p53 and p21 in response to treatments using western blot. Y15 alone produced 48% cell death. In combination, Y15 significantly increased the cytotoxic efficacy of metformin by 22%, when compared to the metformin only treatment. Cell death by apoptosis was confirmed by PARP cleavage and the presence of DNA fragments in Y15, metformin, and metformin +Y15 treatment groups. The Metformin +Y15 combination significantly downregulated the expression of phosphorylated FAK when compared to the individual treatments and control and this confirmed reduced FAK activity. Reduced FAK auto phosphorylation also correlated with increased expression of p53 AND p21 in metformin and Y15 treatment groups. Our findings show that Y15 significantly enhances the cytotoxic profile of metformin in platinum resistant OVCAR-3 cells. Furthermore, a FAK dependent apoptotic mechanism appears to underlie the cytotoxic effect of metformin as well as Y15 as both drugs significantly reduced the phosphorylation of FAK alone, and in combination. Reduced FAK activity also correlated with increased p53 and p21 expression. This study is the first to report a FAK dependent cytotoxic mechanism of metformin in ovarian cancer and in further work we will evaluate the mechanisms why which metformin cooperates with Y15 to inhibit FAK activity in platinum resistant ovarian cancer. Citation Format: Arkene S. Levy, Zara Khan, Samuel Batko, Keerthi Thallapureddy, Robert Smith, Thanigaivelan Kanagasabai, Julie Torruellas Garcia, Appu Rathinavelu. Evaluation of the cytotoxic profile of Metformin and Y15 in platinum resistant ovarian 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 2090.

Abstract 1751: Y15 and Pyridoxal 5′ Phosphate enhances the cytotoxic profile of Cisplatin and Paclitaxel in platinum resistant ovarian cancer cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Overexpression of the Tyrosine Kinase, Focal Adhesion Kinase (FAK) has been implicated in the development of resistance to front line agents, cisplatin and paclitaxel, in the treatment of ovarian cancer. Moreover vitamins such as Pyridoxal 5′ phosphate (PLP) has been shown to induce cytotoxicity in cancers. Therefore, the aim of this study was to assess the cytotoxic efficacy of FAK inhibition using 1,2,4,5-Benzenetetraamine tetrahydrochloride (Y15) and PLP combined with cisplatin and paclitaxel. Platinum resistant OVCAR-3 cells were treated with varying concentrations of Y15 (10μM, 15 μM, 30 μM, 100 μM), and PLP (0.25mM, 1mM, 2Mm). Cells were also treated with Y15 + PLP, Y15 + Cisplatin + Paclitaxel, PLP + cisplatin + paclitaxel and Cisplatin + paclitaxel for 24 hrs in triplicate and control cells with 2N HCl or media only. The cytotoxic profile of each treatment was assessed using the automated trypan blue assay and DNA fragmentation assay was performed to evaluate the mechanism of cell death. Dose dependent and statistically significant cytotoxicities were seen with Y15 and PLP compared to the controls with IC 50 values 80 μM (p <0.05) and 2 mM (p<0.05) respectively. The use of Y15 in combination with PLP showed enhanced cytotoxic efficacy in OVCAR-3 cell (p<0.0001). Similarly, Y15 significantly increased percentage cell death when combined with cisplatin + paclitaxel (62%, p<0.0001) in comparison to cisplatin + paclitaxel (35%). The use of PLP with cisplatin + paclitaxel also showed a greater cytotoxic profile of 55% (p<0.002). Cell death by apoptosis was not confirmed by DNA fragments in Y15 and PLP positive treatment groups. Y15 and PLP significantly enhances the cytotoxic profile of cisplatin, paclitaxel in resistant OVCAR-3 cells suggesting that targeting FAK activity through the inhibition of Y397 site and the use of PLP augments resistance to paclitaxel and cisplatin. However further studies are needed to determine the mechanistic pathway of Y15 and Pyridoxal 5′Phosphate induce cytotoxicity. Citation Format: Monique P. Reboe, Terry-ann Waite, Arkene S. Levy, Sivanesan Dhandayuthapani, Appu Rathinavelu. Y15 and Pyridoxal 5′ Phosphate enhances the cytotoxic profile of Cisplatin and Paclitaxel in platinum resistant ovarian cancer cells. [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 1751. doi:10.1158/1538-7445.AM2015-1751

Abstract 4549: A comparative efficacy study of Saquinavir in combination with cisplatin or paclitaxel in platinum-resistant ovarian cancer cells

Background and Objective: Ovarian cancer is the 7th most common cancer affecting women Worldwide which has a 5-year survival rate of 45%. First line therapy, platinum and taxol compounds, produces 60% to 80% response rates in platinum sensitive ovarian cancer. However, incidence of resistance to this therapy is high resulting in poor prognosis. The present study evaluated the comparative efficacy of Saquinavir (SAQ) in combination with Cisplatin (CIS) or Paclitaxel (TAX) versus first line therapy, CIS+TAX, in platinum-resistant OVCAR-3 cells. Method: OVCAR-3cells were treated with varying concentrations of SAQ (20μM, 40μM, 60μM and 100μM). OVCAR-3 cells were treated with SAQ (100μM), SAQ+CIS(26μM), SAQ+TAX(1.5nM) and CIS+TAX. Cells were treated over a 24hr period in triplicate. Media only and DMSO only treated cells were used as controls. The cytotoxic effect of each treatment was determined using the automated Trypan Blue exclusion protocol and DNA fragmentation assay was performed to evaluate the mechanisms of induced cell death. Results: Dose dependent and statistically significant (p Conclusion: SAQ+CIS produced more significant cytotoxic effect than first line therapy, CIS+TAX, in platinum-resistant ovarian cancer cells. This data supports further evaluation of SAQ for its potential utilization in the treatment of platinum-resistant ovarian cancer. Note: This abstract was not presented at the meeting. Citation Format: Terry-Ann E. Waite, Monique Reboe, Arkene Levy, Sivanesan Dhandayuthapani, Appu Rathinavelu. A comparative efficacy study of Saquinavir in combination with cisplatin or paclitaxel in platinum-resistant ovarian cancer cells. [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 4549. doi:10.1158/1538-7445.AM2015-4549

Abstract 259: Analysis of Q356R polymorphism in BRCA1 gene of Jamaican ovarian cancer patients

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Studies in different populations worldwide have demonstrated that germ line mutations in BRCA1 and BRCA2 account for the majority of hereditary breast and ovarian cancers. In addition, polymorphic variants in these genes have been implicated in an increased breast and ovarian cancer risks. Indeed, it has been known that the germ line mutations in the BRCA1 gene can strikingly increase the life time risk of ovarian cancer by 15-30%. Among BRCA mutations the Q356R polymorphism is a variant causing the amino acid switch from glutamine (Q) to arginine (R) in the putative functional domain of the protein. The present study was carried out to assess the frequency of Q356R polymorphism in BRCA1 gene among Jamaican ovarian cancer patients, since there is lack of data for this population pertaining to this polymorphism. For this analysis the ovarian cancer samples were collected from 23 Jamaican women with 21 benign and 2 malignant tumors. The genomic DNA of cancer samples was extracted and the purity of the DNA was analyzed prior to polymorphism determination. Polymerase chain reaction was performed using the DNA of cancer patients as a template with the primers specific for BRCA1 gene. The Q356R polymorphism status within BRCA1 gene was analyzed through restriction enzyme digestion. The undigested or partially digested arginine (R) coding allele was seen as a band at 211 bp of length, whereas the glutamine (Q) alleles represented by the digestion products were seen at 134 and 77 bp length. The incomplete digestion of R allele indicates a high risk and the digested Q alleles signify the medium or low risk category. Genotyping analyses were performed in all 23 cases using the restriction fragment length analysis. Out of 23 samples, 22 showed digestion fragments of 134 bp and 77 bp, remaining one was partially digested and showed a 211 bp band. Among the samples analyzed the percentage of R allele was (4.34%) and the Q alleles were (95.65%). Our current analysis indicates that the QR genotype frequency for Jamaican women with non-malignant ovarian tumors is only 0.04. However, the QR genotype frequency for Jamaican women with malignant ovarian cancer appears to be 0.5. Thus, functional polymorphisms in BRCA1 might be postulated to be more likely affecting ovarian cancer risk than those in BRCA2. Though the frequency of R allele is only 4.3% in Jamaican population, this polymorphism seems to correlate very well with the malignant status of the tumor. Therefore, patients with this polymorphism can be selected for more aggressive surveillance and therapeutic options. The results presented here are from a small population of Jamaican women with ovarian tumors but further validation of this conclusion can be obtained by conducting a study with larger population of patients (The authors would like to thank the Royal Dames of Cancer Research Inc., Ft. Lauderdale, FL, for their generous support). Citation Format: Aiyavu Chinnaiyan, Colleen Salmon, Thiagarajan Venkatesan, Arkene Levy, Sivanesan Dhandayuthapani, Appu Rathinavelu. Analysis of Q356R polymorphism in BRCA1 gene of Jamaican ovarian cancer patients. [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 259. doi:10.1158/1538-7445.AM2014-259