Thanigaivelan Kanagasabai

MDM2 Overexpression Modulates the Angiogenesis-Related Gene Expression Profile of Prostate Cancer Cells

The Murine Double Minute 2 (MDM2) amplification or overexpression has been found in many tumors with high metastatic and angiogenic ability. Our experiments were designed to explore the impact of MDM2 overexpression, specifically on the levels of angiogenesis-related genes, which can also play a major role in tumor propagation and increase its metastatic potential. In the present study, we have used the human angiogenesis RT2 profiler PCR array to compare the gene expression profile between LNCaP and LNCaP-MST (MDM2 transfected) prostate cancer cells, along with LNCaP-MST cells treated with Nutlin-3, an MDM2 specific inhibitor. As a result of the overexpression of MDM2 gene in LNCaP-MST (10.3-fold), Thrombospondin 1 (THBS1), Tumor necrosis factor alpha (TNF-α) and Matrix metallopeptidase 9 (MMP9) were also found to be significantly up-regulated while genes such as Epiregulin (EREG), Tissue inhibitor of metalloproteinases 1 (TIMP1) were down-regulated. Also, we determined the total MMP activity and MMP9 expression in LNCaP, LNCaP-MST and SJSA-1 cells. Our results indicated that MDM2 level is positively correlated with MMP activity and MMP9 secretion. Our findings offer strong supporting evidence that MDM2 can impact growth and metastatic potential of cancer cells through tilting the balance towards pro-angiogenic mechanisms.

Anti-cancer effects of F16: A novel vascular endothelial growth factor receptor–specific inhibitor

Vascular endothelial growth factor receptor-2 is a dynamic target for therapeutic intervention in various types of cancers. This study was aimed to explore the anti-angiogenic activity of a novel vascular endothelial growth factor receptor–specific inhibitor named F16 in both in vitro and in vivo experimental models. This compound effectively reduced cell proliferation, tube formation, and migration of human umbilical vein endothelial cells in a concentration-dependent manner by directly inhibiting vascular endothelial growth factor binding and subsequent vascular endothelial growth factor receptor-2 phosphorylation. The F16 was also able to inhibit the phosphoinositide 3-kinase/protein kinase B–mediated survival and migration pathways in cancer in addition to inhibiting the focal adhesion kinase and mitogen-activated protein kinases–mediated signaling in GI-101A cancer cells. The chorioallantoic membrane assay followed by tumor growth inhibition measurements with GI-101A breast cancer xenograft implanted athymic nude mice confirmed the in vivo tumor reductive effects of F16. It was interesting to observe a decrease in tumor burden after F16 treatment which correlated very well with the decrease in the plasma levels of mucin-1 (MUC-1). Our studies so far have confirmed that F16 is a specific inhibitor of angiogenesis in both in vitro and in vivo models. The F16 also works very efficiently with Taxol in combination by limiting the tumor growth that is better than the monotherapy with any one of the drugs that were tested individually. Thus, F16 offers a promising anti-proliferative and anti-angiogenic effects with better specificity than some of the existing multi-kinase inhibitors.

Abstract 309: Effect of histone deacetylase (HDAC) inhibitor on gene expression in MDM2 transfected prostate cancer cells

Deacetylation of histone gives a tag for epigenetic repression and plays an important role in transcriptional regulation, cell cycle progression, and developmental events. HDACs catalyse the removal of the acetyl moiety from the lysine residues of proteins including the core nucleosomal histones. Through removal of critical acetyl groups from histones, HDACs can create a chromatin conformation that can prevent the transcription of genes that encode for proteins involved in cell cycle regulation. Thus together with histone acetyltransferases (HATs), HDACs regulate the level of acetylation and alter multitude of cellular functions and their characteristics. Several alterations of HDAC and HAT levels and activities have been found to be enacted by translocation, amplification, overexpression, or mutation of the relevant genes in a variety of cancers. In many cancer cell lines, overexpression or activation of the HDAC enzymes result in histone hypo-acetylation and consequent promotion of pro-cancerous mechanisms. Therefore, HDAC inhibitors represent a potential new class of antitumor agents with cytotoxic activity and the ability to regulate gene expression in tumor cells. In this study we evaluated the effects of Vorinostat (suberoylanilide hydroxamic acid), which is a potent inhibitor of HDAC activity, on cell cycle regulation in MDM2 (mouse double minute 2 homolog) overexpressing cells. MDM2 amplification or overexpression is found in many tumors that eventually lead to the inactivation of the cell cycle control and loss of pro-apoptotic functions through both p53 dependent and independent mechanisms. The PCR array, qRT-PCR, and western blot analysis of MDM2 overexpressing prostate cancer cells (LNCaP-MST), after treating with Nutlin-3 (20 µm) and 17-AAG (10 µm), was able to trigger p21 expression and down-regulation of BIRC5 (Baculoviral IAP Repeat Containing 5). Similarly, when we treated the MDM2 transfected LNCaP-MST cells with vorinostat (7.5 µm for 24 hrs), some of the above mentioned changes, similar to Nutlin-3 treatment, were observed. As a result of HDAC inhibition the mRNA levels of p21, p53 and TIMP-1 were significantly elevated, while the levels of BIRC5 was significantly down-regulated. Thus, treatment of MDM2 overexpressing cell lines with HDAC inhibitor resulted in activation of p21 and consequent decrease in cell proliferation due to resumption of cell cycle arrest. Our results with LNCaP-MST cells offer convincing evidence to suggest that the inhibition of HDAC can control cell proliferative signals in MDM2 overexpressing prostate cancer cells. (The generous support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged). Citation Format: Thiagarajan Venkatesan, Ali Alaseem, Khalid Alhazzani, Thanigaivelan Kanagasabai, Appu Rathinavelu. Effect of histone deacetylase (HDAC) inhibitor on gene expression in MDM2 transfected prostate cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 309. doi:10.1158/1538-7445.AM2017-309

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 A05: The anti-angiogenic activity and pharmacokinetic evaluation of a small molecule JFD-WS in preclinical testing

VEGF signaling through activation of Vascular Endothelial Growth Factor Receptor 2 (VEGFR2), an endothelial cell-specific receptor tyrosine kinase is indispensable for developmental angiogenesis and cancer progression. Since VEGFR2 is the dominant angiogenic signaling receptor, it has become an important target in the development of anti-angiogenic therapies. As part of this strategy inducing apoptosis in cancer cells could make the therapeutic agents very effective. The in vitro anti-angiogenic activity of water soluble form of JFD named as JFD-WS was examined by ECMatrixTM gel assay with human umbilical vein endothelial cells (HUVEC) using 0.01-10µM concentrations at different time intervals (0, 4 and 8 hrs). A complete inhibition of in vitro angiogenesis was observed at 8 hrs after treating with 10µM concentration of JFD-WS. Furthermore, the VEGFR2 phosphorylation was inhibited by 38.65% in JFD-WS (1.0µM) treated HUVEC cells. Besides the in vitro anti-angiogenic activity, JFD-WS is also proven as a potent anti-cancer drug in this study by using GI-101A (human breast adenocarcinoma) xenograft implanted athymic nude mice. In our in vivo experiments, the intraperitoneal (i.p.) injection of JFD-WS (at a dose of 100 mg/kg body weight) was able to significantly inhibit the tumor growth compared to the control group. At the end of the treatment period nearly 35.19% inhibition of tumor growth was observed in JFD-WS treated animals along with prolongation of survival. The tumor inhibition was lot more effective when JFD-WS was combined with Taxol (10 mg/kg). Eventually, the serum levels of MUC1 proved a significant regression of the tumor burden in the experimental animals. In addition to the anti-angiogenic ability, the anti-tumor activity of JFD-WS seems to be further enhanced due to induction of apoptosis signals in the xenograft tumor implanted animals. The protein expression levels of key apoptotic signaling molecules such as Bax, Bcl2, cytochrome c, Apaf-1 and cleaved caspase-3 and p53 were analyzed in the tumor samples that were extracted from experimental animals. Interestingly, a significant increase in the expression of pro-apoptotic proteins p53 (3 fold), Bax (10.6 fold) and Apaf-1 (2.2 fold) were observed in JFD-WS treated animals. In support of the induction of apoptotic signals, the expression of anti-apoptotic protein Bcl2 was decreased by 1.8 fold. Consequently, the release of cytochrome c and the cleavage of caspase 3 found in the cytosolic fraction were significantly higher in JFD-WS treated group as compared with the untreated controls. Finally, the pharmacokinetics (PK) property of JFD-WS was analyzed by measuring the concentration in plasma and urine samples of Balb/c mice at different time intervals (Plasma: 2.5, 5, 10, 15, 30, 60, 120, 1440 min; and Urine: 15, 30, 60, 120, 1440 min), using a HPLC method. Our experiments showed that JFD-WS can reach peak plasma concentration after 15 mins and a maximum elimination in urine was observed after 30 mins of i.p. injection. The compound was undetectable in both plasma and urine after 24 hrs. Moreover, a two compartment model of absorption, distribution and elimination for JFD-WS, with the half-life (t1/2) of around 33 mins is suspected. Our results from both in vivo and in vitro experiments confirm the anti-angiogenic and pro-apoptotic effects of JFD-WS in xenograft tumor implanted athymic nude mice. Furthermore, our data clearly indicate that, JFD-WS is highly effective in causing tumor inhibition with minimal toxicity. (This project was supported by The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida). Citation Format: Thanigaivelan Kanagasabai, Sivanesan Dhandayuthapani, Manasa Subbarao, Janelle Alvarez, Meera Bhalani, Appu Rathinavelu. The anti-angiogenic activity and pharmacokinetic evaluation of a small molecule JFD-WS in preclinical testing. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr A05.

Abstract 1380: The in vivo activity of a novel anti-angiogenic compound, JFD-WS, in human breast adenocarcinoma xenograft implanted athymic nude mice

The tumor growth and metastasis of several cancers depend on the extent of both angiogenesis and lymphangiogenesis triggered by chemical signals that are originating from cancer cells with aggressive growth ability. The discovery of angiogenic inhibitors have been helping to reduce both morbidity and mortality resulting from multiple types of cancers. As part of the comprehensive treatment strategy combining anti-angiogenic agents with conventional cytoreductive treatments is considered to be the most effective approach. As part of this strategy an added ability for inducing apoptosis in cancer cells could make the therapeutic agents very effective. The in vitro anti-angiogenic activity of water soluble form of JFD (JFD-WS) was examined by ECMatrixTM gel assay with human umbilical vein endothelial cells (HUVEC) using 0.01-10 μM concentrations at different time intervals (0, 4 and 8 hrs). A complete inhibition of in vitro angiogenesis was observed in 8 hrs at 10 μM concentration of JFD-WS. Our newly discovered JFD-WS is also proven as a potent anti-cancer drug in this study by using GI-101A (human breast adenocarcinoma) xenograft implanted athymic nude mice. In addition to the anti-angiogenic ability, the anti-tumor activity of JFD-WS seems to be further enhanced due to induction of apoptosis in the xenograft tumor implanted animals. In addition, the intraperitoneal (i.p.) injection of JFD-WS (at a dose of 100 mg/kg body weight) was able to significantly inhibit the tumor growth compared to the control group. At the end of the treatment period 35.19% inhibition of tumor growth was observed in JFD-WS treated animals along with prolongation of survival. The tumor inhibition was lot more effective when JFD-WS was combined with Taxol (10 mg/kg). The analysis of MUC1 levels in the serum proved a significant regression of the tumor burden in the experimental animals. Furthermore, when the key apoptotic signaling molecules such as Bax, Bcl2, Cytochrome c, Apaf-1, cleaved caspase-3 and p53 were analyzed in the tumor samples, that were extracted from JFD-WS treated animals. A significant increase in the expression of Bax (10.6 fold), Apaf-1 (2.2 fold), and p53 (3 fold) were observed. In support of the induction of apoptotic signals, the expression of anti-apoptotic protein Bcl2 was decreased by 1.8 fold. Consequently, the release of Cytochrome c found in the cytosolic fraction was significantly higher in JFD-WS treated animals as compared with the untreated controls. Finally, the induction of apoptosis in JFD-WS treated group was confirmed by the presence of cleaved caspase-3. Our results from both in vivo and in vitro experiments confirm the anti-angiogenic and pro-apoptotic effects of JFD-WS in xenograft tumor implanted athymic nude mice (This project was supported by The Royal Dames of Cancer Research Inc., Ft.Lauderdale, Florida). Citation Format: Thanigaivelan Kanagasabai, Janelle Alvarez, Meera Bhalani, Sivanesan Dhandayuthapani, Appu Rathinavelu. The in vivo activity of a novel anti-angiogenic compound, JFD-WS, in human breast adenocarcinoma xenograft implanted athymic nude mice. [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 1380. doi:10.1158/1538-7445.AM2015-1380

Abstract 5318: Effect of C-phycocyanin on the anticancer properties of taxol and topotecan in lung cancer implanted athymic nude mice

The biodiversity of marine environment and the associated chemical multiplicity offers unlimited resource for discovering new antitumor agents. For the first time we have tested the anticancer properties of C-phycocyanin (C-PC) isolated from a unique cyanobacterial strain named Limnothrix sp., which is found in Florida9s Everglades. Our preliminary studies originally confirmed antiproliferative activity of C-PC against LNCaP prostate cancer cells and subsequently against A549 lung cancer cells also. Therefore, we speculated that C-PC could potentiate the anticancer effects of certain therapeutic agents when used in combination treatments. For this purpose the cytotoxic effects of C-PC and topotecan were confirmed using cell viability assays in A549 lung cancer cells. Subsequently, our in vivo experiments confirmed the efficacy of C-PC in potentiating the anticancer effects of taxol and topotecan in athymic nude mice that were sub-cutaneously implanted with the xenograft tumors established from A549 lung cancer cells. During the treatment period the experimental mice had free access to C-PC that was dissolved in drinking water at a dose of 100 mg/kg body weight. The anticancer drugs were given as intraperitoneal injections at a dose of 1.0 mg/kg body weight twice a week for a period of 60 days. The tumor growth was assessed once in every two weeks using caliper measurements. At the end of the treatment period the levels of lung tumor biomarkers CYFRA 21-1 (Cytokeratin 19 fragments) and CEA (Carcino Embryonic Antigen) in serum were analyzed. As a result of the treatment, the tumor growth was found to be 54% and 46% less in taxol and topotecan treated groups compared to the control group. Interestingly C-PC + taxol combination showed 75% inhibition of tumor growth. Thus, the mice treated with the C-PC + taxol combination showed an additional 21% tumor growth inhibition when compared to the group treated with taxol alone. Furthermore, the levels of the tumor biomarkers CYFRA 21-1 and CEA in serum were significantly reduced by the treatment showing a good correlation with the inhibition of tumor growth in C-PC + taxol treated animals. The C-PC + taxol combination treated animals showed 91% decrease while C-PC alone was able to reduce the levels of CYFRA 21-1 by 85%. Similarly, the CEA levels were reduced by 97% following the C-PC + taxol combination treatment. It was further determined that C-PC treatment could significantly down regulate the levels of anti-apoptotic protein such as Bcl-2 to trigger apoptosis in lung cancer cells. Thus, results from our study confirm that C-PC from Limnothrix sp., can significantly enhance the anticancer activity of taxol against the A549 lung tumor in athymic nude mice possibly by decreasing the levels of Bcl-2. (Project was supported by the PFRDG grant of NSU, James & Esther King Biomedical Research Program of the State of Florida and The Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida). Citation Format: Sivanesan Dhandayuthapani, Miroslav Gantar, Thanigaivelan Kanagasabai, Manasa Subbarao, Appu Rathinavelu. Effect of C-phycocyanin on the anticancer properties of taxol and topotecan in lung cancer implanted athymic nude mice. [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 5318. doi:10.1158/1538-7445.AM2015-5318