Abstract Glioblastoma multiforme (GBM) is one of the most aggressive and lethal types of brain cancers with low 5-year survival rate. Therefore, search for better drugs and effective treatment strategies for GBM to improve patient outcomes is continuing. Since GBM is one of the most highly vascularized solid tumors and its growth is angiogenesis-dependent, antagonizing tumor angiogenesis by using angiogenesis inhibitors seems to be one of the promising approaches. In this context, intensive pre-clinical evaluation of our patented anti-angiogenic compound, code-named F16, has exhibited potent anti-angiogenic and anti-tumor activities via selectively antagonizing VEGFR-2 in both in vitro and in vivo models. More importantly, assessment of biochemical parameters that are reflecting the safety of F16 in the in vivo system showed no alterations in the toxicological parameters of heart, liver, kidney, and pancreas, after 30 days of treatment, at the dose of 100 mg/kg body weight. Hence, we tested the direct effects of F16 for inhibiting the growth, angiogenesis and the migratory abilities of the U87MG glioblastoma cells, which are known to express high levels of VEGFR. Our in vitro studies have confirmed potent inhibitory effects of F16 towards the migration and invasion of U87MG cells, and also revealed potent cytotoxic effects (IC50 26 µM) against U87MG cells in comparison to Temozolomide (IC50 430 µM) treatment. In addition, F16 inhibited the phosphorylation of VEGFR-2 through competitive binding and also induced cell cycle arrest and apoptosis by activating p53 pathway in U87MG cells. Furthermore, our in vivo results with ectopically implanted xenograft model confirm the fact that F16 can significantly inhibit tumor growth in the mice implanted with U87MG glioblastoma cell line. Based on our preliminary results, we are proposing that F16 could be a potential candidate for treating GBM, either in monotherapy or in combination with a cytoreductive drug. (This research was supported by the generous funds provided by the Community Foundation of Broward, Florida and also by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida) Citation Format: Mohammad Algahtani, Khalid Alhazzani, Thiagarajan Venkatesan, Ali Alaseem, Sivanesan Dhandayuthapani, Appu Rathinavelu. Direct cytotoxic effect of a novel anti-angiogenic drug F16 towards U87MG glioblastoma cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 185.
Abstract A method has been developed for quantitative determination of cyclopiazonic acid, a mycotoxin produced by a common food contaminant, Penicillium cyclopium. The organism was grown successively in synthetic minimal medium, rice, corn, and wheat for 15 days. The toxin was extracted with chloroform followed by separation by thin layer chromatography. A colorimetric assay procedure has been successfully developed for the analysis of cyclopiazonic acid present in infected rice, corn, and wheat. The sensitivity of the method was tested by using recovery experiments.
Epigenetic modifications can lead to significant influence on cancer development, growth and progression. The main epigenetic modifications observed in human are methylation and acetylation. Histone acetylations are largely regulated by histone deacetylases (HDACs) and, several HDAC inhibitors (HDACi) such as SAHA (Vorinostat), which are already approved for human use or currently in different stages of clinical trials, belong to the new class of drugs with promising effects on the cancer growth and metastatic process. The small molecule RG7388 is a newly developed inhibitor that is specific for an oncogene‐derived protein called MDM2, which is also in clinical trials for the treatment of various types of cancers. One of the unique characteristics of these two drugs is their ability to induce p21 expression through distinct mechanisms. A clear understanding related to the molecular mechanism whereby SAHA can induce cell cycle arrest and trigger necrosis, apoptosis or necroptosis is still evolving. Similarly, the ability of RG7388 for producing anticancer effect is undergoing thorough investigation, since it can produce p53 dependent and p53 independent effects. So far, our experiments with MCF‐7 breast cancer cells and LNCaP prostate cancer cells have confirmed the abilities of these two drugs to induce p21 expression through distinct mechanisms. In addition, these two drugs were expected trigger cell cycle arrest and cell death through mechanisms that are similar. Therefore, we performed experiments to measure the cell cycle arrest effects of SAHA and RG7388 by using the MCF‐7 and LNCaP cells. The cytotoxicity, cell cycle arrest and apoptosis/necroptosis effects of the treatments were assessed by using Trypan Blue Dye Exclusion (TBDE) method, and fluorescence assay with caspase 3/7 specific DEVD‐ amc fluorogenic substrate. In addition, cell cycle and apoptosis related proteins were analyzed by immunoblotting methods. To this point, our results from MCF‐7 and LNCaP cells suggest that the cell death caused by SAHA treatment in both cell lines are through induction of p21 WAF1/CIP1 and p27 Kip1 levels, that could be independent of p53. On the other hand, treatment of LNCaP cells with RG7388 was able to induce p21 WAF1/CIP1 expression through inhibition of MDM2 that was also causing significant cell death. However, the RG7388 treatment was not able to elevate p21 WAF1/CIP1 in MCF‐7 cells, even though there was clear evidence of p53 elevation in these cells. Hence, we are suspecting that, there is some level of uncoupling of p53 mediated transcriptional induction of p21 WAF1/CIP1 in MCF‐7 cells. As of now, our studies confirm that two different drugs, which have the ability to induce p21 expression, exhibit distinct mechanisms of cell death through p53 dependent and independent pathways. Since it points to an interesting interplay between multiple pathways, confirmation of the actual cell death mechanism induced by RG7388 in MCF‐7 and LNCaP cancer cells requires additional exploration. Support or Funding Information This research was supported by the Royal Dames of Cancer Research Inc., Fort Lauderdale, Florida This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .
Abstract 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 Recent studies have shown the crucial role of miRNAs in regulating cancer initiation, progression and metastatic process via influencing gene expression patterns. However, the distinct mechanism by which miRNA regulates tumorigenesis remains as an area requiring detailed research investigations. In our earlier studies, we have demonstrated a positive correlation between MDM2 expression and hypoxia inducible factor -1alpha (HIF-1α) levels in both normoxic and hypoxic conditions using prostate cancer cell lines. Subsequently, we postulated that the upregulation of HIF-1α is most likely regulated by MDM2 in cancers cells that are positive for this gene expression. Hence, to confirm our hypothesis, we utilized miScript miRNA PCR Array analysis (Qiagen, USA) and compared the gene expression profile of the hypoxia signaling pathway in LNCaP (prostate cancer cells) and LNCaP-MST (MDM2 transfected prostate cancer cells) under normoxic conditions. This study was expected to outline how MDM2 impacts the differential expression of miRNAs and leading to enhancement of tumor angiogenesis via increase in HIF-1α or other genes of the hypoxia signaling pathway. Our preliminary data indicate that hsamiR99a5p, hsamiR125b5p, hsamiR1385p, hsamiR199b5p and hsalet7c5p are significantly upregulated in LNCaP-MST cells compared to LNCaP. In addition, some of the miRNAs such as hsalet7d5p, hsalet7e5p, hsalet7f5p, hsalet7g5p and hsalet7i5p are significantly downregulated in the same cells. Among the upregulated miRNAs, hsamiR125b5p emerged as an important gene that is known to downregulate the protein levels of hypoxia inducible factor 1alpha inhibitor (HIF1AN or FIH), which can normally inhibit the transcriptional activity of HIF-1α in both normoxic and hypoxic conditions. Therefore, overexpression of hsamiR125b5p seems to be associated with the increase in HIF-1α level. From the aforementioned findings, it is confirmed that the MDM2 overexpression is impacting miRNA levels such as hsamiR125b5p that are involved in hypoxia related signaling pathway. Thus, our results offer a convincing evidence that MDM2 can promote angiogenesis and increase metastatic potential of cancers through altering the miRNA expression levels. (The financial support from the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida is gratefully acknowledged). Citation Format: Saad Ebrahim Alobid, Thiagarajan Venkatesan, Ali Alaseem, Khalid Alhazzani, Appu Rathinavelu. Analysis of human hypoxia related miRNAs 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 467. doi:10.1158/1538-7445.AM2017-467
Abstract HDM2 is a master regulator and plays an important role in Tumor angiogenesis. To confirm this hypothesis, we used wild type LNCaP and HDM2 transfected LNCaP-MST prostate cancer cells for our study. In the HDM2 transfected cells we observed 96% higher HDM2 protein expression compared to LNCaP cells. In general, HDM2 over expression has been reported to be a potential cause for p53 degradation in many cancer cells. To confirm this negative correlation, p53 level was observed in HDM2 transfected cells. Compared to the wild type cells, only 10% of the p53 level was found in the HDM2 transfected LNCaP-MST cells. This confirms our speculation that, HDM2 mediated degradation of p53 may occur in LNCaP-MST cells. Following HDM2 transfection, the VEGF level was also increased by 180% in LNCaP-MST cells compared to LNCaP cells. Since, previous studies have indicated that HIF-1α has a major role in inducing VEGF transcription, we determined the level of HIF-1α in both cells. In LNCaP-MST cells, nearly 90% higher HIF-1α protein level was observed, this might be due to the inhibition of both prolyl hydroxylase and factor inhibiting HIF (FIH) activity by HDM2, which are essential to hydroxylate HIF-1α in critical position. Once, hydroxylated, HIF-1α can no longer bind to p300 and induce VEGF transcription. In addition, an increase in the activated form of STAT3 (p-STAT3) causing elevated VEGF transcription has been previously reported in many cancer cells. Accordingly, the p-STAT3 protein expressions observed in HDM2 transfected LNCaP-MST cells were 15 folds higher compared to the non-transfected wild type LNCaP cells. A negative regulatory role of p53 in controlling the levels of active STAT3 has also been previously reported in prostate cancer cell lines. Hence, it is suggested that HDM2 might be inducing the expression of active STAT3 by degrading p53 protein in LNCaP-MST cells. Overall, our results demonstrate that HDM2 has an important role in regulating the VEGF transcription via modulating HIF-1α and STAT3 protein expressions. We thank the Royal Dames of Fort Lauderdale, Inc. for their support. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B4.
Background: Prostate cancer is the second most common cancer in American men. The development of alternative preventative and/or treatment options utilizing a combination of phytochemicals and chemotherapeutic drugs could be an attractive alternative compared to conventional carcinoma treatments. Genistein isoflavone is the primary dietary phytochemical found in soy and has demonstrated anti-tumor activities in LNCaP prostate cancer cells. Topotecan Hydrochloride (Hycamtin) is an FDA-approved chemotherapy for secondary treatment of lung, ovarian and cervical cancers. The purpose of this study was to detail the potential activation of the intrinsic apoptotic pathway in LNCaP prostate cancer cells through genistein-topotecan combination treatments. Methods: LNCaP cells were cultured in complete RPMI medium in a monolayer (70-80% confluency) at 37ºC and 5% CO2. Treatment consisted of single and combination groups of genistein and topotecan for 24 hours. The treated cells were assayed for i) growth inhibition through trypan blue exclusion assay and microphotography, ii) classification of cellular death through acridine/ ethidium bromide fluorescent staining, and iii) activation of the intrinsic apoptotic pathway through Jc-1: mitochondrial membrane potential assay, cytochrome c release and Bcl-2 protein expression.Results: The overall data indicated that genistein-topotecan combination was significantly more efficacious in reducing the prostate carcinoma’s viability compared to the single treatment options. In all treatment groups, cell death occurred primarily through the activation of the intrinsic apoptotic pathway.Conclusion: The combination of topotecan and genistein has the potential to lead to treatment options with equal therapeutic efficiency as traditional chemo- and radiation therapies, but lower cell cytotoxicity and fewer side effects in patients. Key words: topotecan; genistein; intrinsic apoptotic cell death
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