Sivanesan Dhandayuthapani

Phycocyanin Induces Apoptosis and Enhances the Effect of Topotecan on Prostate Cell Line LNCaP

C-phycocyanin (C-PC) from Spirulina has been previously shown to have anticancer properties. Here, we report on anticancer activity of C-PC that was isolated from the novel cyanobacterium Limnothrix sp. 37-2-1. C-PC from this organism exhibited anticancer properties in our in vitro systems; however, the required doses were well above the range of anticancer drugs normally used. Therefore, we conducted several experiments to test whether lower-than-usual doses of the anticancer drug topotecan (TPT) can offer the same level of cytotoxic effects as normal doses when combined with C-PC. For this purpose, cytotoxicities of C-PC and TPT were tested using the LNCaP (prostate cancer) cells. We found that when only 10% of a typical dose of TPT was combined with C-PC, the cancer cells were killed at a higher rate than when TPT was used alone at full dose. Similarly, we were also able to detect an increased level of radical oxygen species (ROS) generation as well as an increase in activities of caspase-9 and caspase-3 when these two compounds were used in combination. Taken together, our findings suggest that combining C-PC from Limnothrix sp. with the lower dose of TPT can induce apoptosis through generation of ROS and activation of caspases. In that respect, we suggest that C-PC can potentially improve the efficacy of the currently available anticancer drug, and therefore diminish its harsh side effects in the patient.

Pro-angiogenic effects of MDM2 through HIF-1α and NF-κB mediated mechanisms in LNCaP prostate cancer cells

AbstractHypoxia stimulates several pathways that are critical to cancer cell growth and survival, including activation of vascular endothelial growth factor (VEGF) transcription. Overexpression of VEGF and the extent of neoangiogenesis are closely correlated with tumor development and cancer metastases. Recent studies suggest MDM2 as one of the major regulators of pro-angiogenic mechanisms. To assess the direct correlation of HIF-1α and NF-κB, and the actual mechanism of MDM2 involved in the control over VEGF transcription, we exposed the LNCaP and LNCaP-MST cells (MDM2 transfected) to hypoxia. Our experiments confirm that MDM2 activation can lead to significant decrease in the levels of p53 in MDM2 transfected LNCaP-MST cells than the wild-type LNCaP cells. The results further suggest that MDM2 can be a strong regulator of both p53 dependent and independent transcriptional activity. Similarly, an increased level of other transcription factors such as HIF-1α, P300, STAT3, pAKT and NF-κB was observed. As a point of convergence for many oncogenic signaling pathways, STAT3 is constitutively activated at high frequency in a wide diversity of cancers. Our results indicate that STAT3 can directly regulate VEGF expression that is controlled by MDM2. Furthermore, it is evident from our results that NF-κB may interfere with the transcriptional activity of p53, by downregulating its levels. On the other hand, several pro-angiogenic mechanisms, including VEGF transcription which is controlled by MDM2, seem to be mediated by NF-κB.

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 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

Abstract 4522: Pharmacokinetic evaluation of a novel anti-angiogenic molecule named JFD-WS in Balb/c mice

JFD is a novel anti-angiogenic compound which mediates its action by blocking Vascular Endothelial Growth Factor Receptors and associated kinase activity, thereby arresting tumor angiogenesis and growth. The original JFD has been modified into a water soluble form (JFD-WS) to increase bioavailability and distribution during the in vivo pre-clinical testing in mouse model. Through both in vitro and in vivo testing, we have confirmed its anti-angiogenic and cytoreductive properties and are currently establishing its pharmacokinetic (PK) properties. The absorption, distribution and elimination of JFD-WS were determined by measuring the plasma and urine concentrations after intraperitoneal (i.p.) and oral administration in Balb/c mice. Following i.p. administration of JFD-WS (100 mg/kg body weight), the plasma samples were collected at 2.5, 5, 10, 15, 30, 60, 120, 1440 mins and the urine samples were collected at 15, 30, 60, 120, 1440 mins. The JFD-WS was extracted using a simple liquid extraction procedure and the concentrations in the plasma and urine were analyzed using 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 hours. Further analysis of plasma levels of JFD-WS using standard PK parameters has led us to hypothesize that JFD-WS may follow a two compartment kinetic model, according to which the molecule may enter the liver through the portal vein after absorption from the peritoneal cavity. During drug distribution, JFD-WS seems to enter the first compartment, including blood circulation, and then reaches the second compartment i.e., the rest of the body till equilibrium is attained. It is further speculated that the first phase of CYP450 system mediated metabolism may not be very extensive since our compound is in water soluble form. Finally, a majority of the molecule is excreted out by glomerular filtration in urine while a small portion is removed through the feces as well. Derived from an established PK formula, the half-life (t½) of our JFD-WS molecule was found to be around 33 mins. A high volume of distribution calculated for JFD-WS leads us to assume that it may distribute largely in extravascular regions such as muscles and tissues, while a high clearance rate could contribute to the lower toxicity of the compound. This hypothesis is further supported by our observations found in the in vivo systems, wherein JFD-WS is highly effective in causing tumor inhibition with minimal toxicity. Thus, the results of our study confirm that i.p. administration allows for reasonable distribution of JFD-WS to the tumor sites within a shorter duration of time that can produce cytoreductive effect. (The Royal Dames of Cancer Research Inc., Ft. Lauderdale Florida is gratefully acknowledged for their generous financial support). Citation Format: Manasa Subbarao, Sivanesan Dhandayuthapani, Mortatha Albassam, Appu Rathinavelu. Pharmacokinetic evaluation of a novel anti-angiogenic molecule named JFD-WS in Balb/c 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 4522. doi:10.1158/1538-7445.AM2015-4522

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 2365: Analysis of angiogenesis pathway-related gene expression impacted by MDM2 using PCR array

The Murine Double Minute 2 (MDM2) gene encodes an oncogenic MDM2 protein, which is a negative regulator of the p53 tumor suppressor. MDM2 amplification or over expression is found in many tumors that eventually lead to the inactivation of the cell cycle control and loss of pro-apoptotic functions that are typically exerted by wild type p53. Many of the oncogenic effects of MDM2 are resulting from the inactivation of p53 through direct binding or degradation of p53 following E3 ligase mediated ubiquitination. However, our recent findings have indicated that MDM2 over expression can also promote tumor angiogenesis through p53 independent pathways and thereby enhance the metastatic potential of cancer cells. In this respect increasing the levels of pro-angiogenic factors such as VEGF has been confirmed as one of the mechanisms regulated by MDM2. However, the actual cluster of genes or pathways regulated by MDM2 leading to such increase in the metastatic potentials of cancer cells have not been clearly identified. Therefore, the main purpose of our study was to understand the impact of MDM2 over expression on the levels of angiogenesis related genes that can also increase growth rate and metastatic potential. Hence, for our study we used the Human Angiogenesis PCR Array to compare the gene expression profile of LNCaP (prostate cancer cells) and LNCaP-MST (MDM2 transfected prostate cancer) cells. The MST cells expressed at least 10 fold increase in the levels of MDM2 after transfection. As a result of the elevated levels of MDM2 in LNCaP-MST cells the expression of genes such as THBS1 (125 fold), TNF-α (14 fold), MMP-9 (1.4 fold) were found to be significantly up-regulated compared to the MDM2 non-transfected cells. Cluster analysis further confirmed that these gene expression profiles are positively correlated with the pro-angiogenic potential of the LNCaP-MST cells compared to the non-transfected cells. In addition, the levels of EREG, TIMP1 and CXCL3 were down regulated significantly that favors the suppression of anti-angiogenic mechanisms. Our results offer a conclusive evidence that MDM2 can impact the cancer growth and metastatic potential of cancer cells by shifting the balance towards pro-angiogenic mechanisms (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged). Citation Format: Thiagarajan Venkatesan, Corine Stinson, Sivanesan Dhandayuthapani, Appu Rathinavelu. Analysis of angiogenesis pathway-related gene expression impacted by MDM2 using PCR array. [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 2365. doi:10.1158/1538-7445.AM2014-2365