Shankar Jayarama

Pro-apoptotic activity of imidazole derivatives mediated by up-regulation of Bax and activation of CAD in Ehrlich Ascites Tumor cells

SummaryIn this study we report that, imidazole derivatives can induce apoptosis in Ehrlich ascites tumor (EAT) cells, which is clearly evident from annexin-V staining, flow cytometric analysis of cell cycle phase distribution and DNA fragmentation. Delineating further into molecular mechanisms leading to apoptosis of EAT cells, we observed that imidazole derivatives induce tumor cell death by the up-regulation of proto-oncoprotein Bax, release of cytochrome c from the mitochondria which activates caspase-3 and activated caspase-3 activates CAD (Caspase Activated DNase) causes DNA fragmentation. The status of Bcl-2 remains unaltered in EAT cells, and the under expression of Bcl-2 and up-regulation of Bax resulted in the increase of Bax: Bcl-2 ratio suggesting that Bcl-2 family involved in the control of apoptosis. These results suggest a further possible clinical application of imidazole derivatives as pro-apoptotic agent in association with conventional chemotherapeutic agents.

Novel synthetic bisbenzimidazole that targets angiogenesis in Ehrlich ascites carcinoma bearing mice

Cancer is a leading cause of death in developed countries and second cause in developing countries. Herein we are reporting the synthesis of novel bisbenzimidazole derivatives and their anticancer properties. Among the newly synthesized bisbenzimidazoles, 3-(4-flurophenylsulfonyl)-1,7-dimethyl-2-propyl-1H,3H-2,5-bibenzo[d]imidazole (FDPB) presented as a potent antiproliferative agent against HeLa, HCT116 and A549 cells with selectivity over normal Vero cells (IC50 >50 μM). Additionally, we evaluated the efficacy of lead compound against Ehrlich ascites tumor (EAT) bearing mice for its antitumor and antiangiogenic properties. Our lead compound significantly reduced the cell viability, body weight, ascites volume and downregulated the formation of neovasculature and production of Vascular Endothelial Growth Factor (VEGF).

3,5-Disubstituted Isoxazole Derivatives: Potential Inhibitors of Inflammation and Cancer

The products of arachidonic acid metabolism by lipoxygenase (LOX) and cyclooxygenase (COX) significantly contribute to inflammation and carcinogenesis. Particularly, overproduction of leukotrienes and prostaglandins contribute to tumor growth by inducing formation of new blood vessels that sustain tumor cell viability and growth. Hence, search for novel anticancer drug via inhibition of LOX and COX enzymes constitutes an impressive strategy till date. In this context, a series of isoxazole derivatives were synthesized and screened for their anti-inflammatory activity via LOX and COX inhibition. Among these, 3-(3-methylthiophen-2-yl)-5-(3,4,5-trimethoxyphenyl)isoxazole (2b) showed significant inhibitory activity toward LOX and COX-2. Additionally, 2b showed a good inhibition of tumor growth, peritoneal angiogenesis, and ascite formation in Ehrlich ascites carcinoma (EAC) cell mouse model. Further, the in silico molecular studies also revealed that the compound 2b binds to the catalytic domain of LOX and COX-1 and COX-2 strongly with high atomic contact energy (ACE) score compared to standard drug. These initial pharmacological data support the fact that the compound 2b serves as the basis in developing anti-inflammatory and anticancer agents.

Synthesis, Characterization of 4-Anilino-6,7-Dimethoxy Quinazoline Derivatives as Potential Anti-Angiogenic Agents

BACKGROUND Quinazolines are a big family of heterocyclic compounds with anti-cancer properties. OBJECTIVE The latest investigation was on synthesis, characterization of novel 4-anilinoquinazoline derivatives for their anti-angiogenic effect. METHOD A series of novel 4-anilino-6,7-dimethoxy quinazoline derivatives were synthesized and characterized using 1H, 13C NMR, FT-IR and LC-MS techniques. Cytotoxicity assays were performed for all compounds against different cell lines such as Human colon carcinoma (HCT116), Human chronic myeloid leukemia (K562) and Human breast cancer (SKBR3) cell lines using 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyl tetrazolium Bromide (MTT), Trypan blue and Lactose dehydrogenase release assay. The selected compounds were evaluated for their anti-tumor and anti-angiogenic effect on EAC tumor model. The molecular docking studies were drawn using maestro 2D sketcher and energy minimize was compounded by OPLS 2005. RESULTS Among all compounds, RB4 and RB7 showed moderate activity whereas RB1 showed most potent activity comparable with that of the standard drug cisplatin against all three cell lines. RB1 also inhibited the proliferation of tumor cells in three different cell lines. Further, in-vivo studies revealed that RB1 significantly reduced secretion of ascites, tumor cell proliferation and increased the life span of tumor bearing mice. The antiangiogenic effect of RB1 was revealed from the reduced vessel sprouting in the peritoneum region of treated mice and induced avascular zone in chorioallantoic membrane (CAM) model. The insilco molecular docking studies clearly demonstrate the dual inhibitory potential of RB1 against VEGFR-2 and EGFR from binding to the active site of its receptors. CONCLUSION However these studies clearly show that RB1 might be a potent antitumor and anti-angiogenic agent representing a promising lead for further optimization and elucidation of the mechanism of action.

Angio Suppressive Effect of Clitoria ternatea Flower Extract is Mediated by HIF-1α and Down Regulation of VEGF in Murine Carcinoma Model

Angiogenesis is a vital process in the progression of cancer as it also play a key role in tumor transition from its dormant state to a malignant stage. VEGF is key growth factor plays an important role in angiogenesis and is regulated by transcription factor HIF-1α. Natural compounds derived from plants have been a prime source for numerous clinically useful anti-cancer agents specially for targeting neo-angiogenesis. Medicinal plants continue to play a central role in the healthcare system of large proportions of the world’s population, particularly true in developing countries like India. In the current report, we studied the angio suppressive effect of aqueous extract of Clitoria ternatea in EAC cells induced angiogenesis. In vivo anti-angiogenic effect of C. ternatea was demonstrated by the down regulation of VEGF secretion from Ehrlich ascites carcinoma (EAC) cells and inhibition of blood vessels formation indicating the potential angio suppressive effect of plant. HIF-1α protein, a transcription factor known to be key a regulator in hypoxia-induced angiogenesis was also down regulated by C. ternatea. Our invetigation indicated that, HIF-1α nuclear sequestration is repressed by C. ternatea through inhibition of nuclear translocation. We hypothesize that decreased levels of HIF-1α in the nucleus of EAC cells upon MECT treatment could be responsible for decreased expression of VEGF which is also attributed to the angio-suppressive effects of MECB. C. ternatea promises to be a potential anti-angiogenic plant which can be exploited to treat cancer.

Anti-cancer and anti-angiogenic effects of partially purified lectin from Praecitrullus fistulosus fruit on in vitro and in vivo model

Praecitrullus fistulosus, belonging to the family of Cucurbitaceae is a tropical vegetable and medicinal plant, grown and consumed extensively in subtropical countries, including the subcontinent India. However, there are limited reports on the medicinal properties of the plant and need to be explored. The lectin identified from the fruit sap of Praecitrullus fistulosus, named as PfLP, possesses potent agglutinating activity against trypsinized rabbit erythrocytes and exhibited its functional role against tumor progression, on in vitro &in vivo models. Experimental results revealed that PfLP shows a promising cytotoxic effect against multiple cancer cell lines. Further, we examined the in vivo anticancer and anti-angiogenic properties of PfLP against EAC bearing mice. PfLP treatment resulted in tumor growth inhibition and increased the life-span of the EAC bearing mice, without showing any detectable side effects, as revealed by histological parameters. Further, a significant decrease in the ascites VEGF secretion level was parallel with a drastic reduction in tumoral neovasculature as evidence for angiogenic parameters. Gelatin zymogram study reveals that PfLP inhibits metalloproteinases (MMP-2 & MMP-9) activity in order to execute its anti-angiogenic effect. PfLP has also inducing apoptosis, in cancer cells was revealed by DNA fragmentation assay followed by Giemsa and AO/EBr staining method, showed the apoptotic bodies and condensed nuclei compared to control cells. More interestingly, PfLP did not exhibit any adverse side effects or secondary complications in normal mice. These results clearly exhibit the potential role of PfLP in regressing the tumor progression by targeting angiogenesis and inducing cell death in mouse transplantable tumor.

Pro-apoptotic activity of novel 4-anilinoquinazoline derivatives mediated by up-regulation of bax and activation of poly (ADP-ribose) phosphatase in ehrlich ascites carcinoma cells

Quinazolines are very important class of heterocyclic compounds with antitumor properties. In search of novel anti-tumour agents, a series of 4-anilinoquinazolines were synthesized and characterized using proton and carbon-13 nuclear magnetic resonance, Fourier transform infrared and mass spectroscopic techniques. These compounds were evaluated for their cytotoxic effect on Ehrlish Ascities Carcinoma cells using 3-(4,5-Dimethylthiazol- 2-Yl)-2,5-Diphenyltetrazolium Bromide against Ehrlish ascities carcinoma cell lines. Among the tested compounds, compound N-(3-((6,7-dimethoxyquinazoline-4-yl)amino)phenyl)-4-nitrobenzene sulfonamide compound exhibited more potent activity with an IC50 value of 10.29 ± 1.14 μM against EAC cell line. In vivo studies using compound N-(3-((6,7-dimethoxyquinazoline-4-yl)amino)phenyl)-4-nitrobenzene sulfonamide showed that there was reduction in the body weight, ascites volume and decrease in cell number. Mice treated with compound N-(3- ((6,7-dimethoxyquinazoline-4-yl)amino)phenyl)-4-nitrobenzene sulfonamide showed higher survivability compared with that of control treated mice. The cells treated with compound N-(3-((6,7-dimethoxyquinazoline-4-yl)amino) phenyl)-4-nitrobenzene sulfonamide also exhibited typical morphological changes of apoptotic damages. Further, compound N-(3-((6,7-dimethoxyquinazoline-4-yl)amino)phenyl)-4-nitrobenzene sulfonamide induced tumour cell death by activating proapoptotic protein Bax which activates caspase-3 and activated caspase –3 cleaves poly (ADP- ribose) polymerase causes DNA fragmentation. Thus, our results strongly conclude that our compound 4G acts as a anticancer agent by inducing apoptosis in Ehrlish ascites carcinoma cells.

EVALUATION OF APOPTOGENIC EFFECTS OF AVERRHOA BILIMBI EXTRACT ON EHRLICH ASCITES CARCINOMA BEARING MICE

ABSTRACT Objective: The proapoptotic potential of aqueous methanol extract of Averrhoa bilimbi fruit (AMBE) in vivo against Ehrlich ascites carcinoma (EAC) bearing Swiss albino mice was studied. Methods: Cytotoxicity of the extract on the EAC cells was monitored by tumor growth response, trypan blue exclusion assay, Giemsa staining, DNA fragmentation, fluorescence-activated cell sorting (FACS) analysis, and reverse transcription-polymerase chain reaction (RT-PCR). The phytochemical screening using LC-MS and Fourier transform infrared (FT-IR) was conducted. Results: The extract at 100 mg/kg body weight was significantly cytotoxic toward the cells with approximately 73% growth inhibition on day 12. It markedly decreased the tumor volume by 65% and viable tumor cell by 72%. Giemsa staining of AMBE treated cells displayed apoptotic morphologies such as membrane blebbing, cytoplasmic condensation, and apoptotic bodies. Cytotoxicity of the extract to the carcinoma cells through apoptosis was further highlighted by DNA fragmentation in treated cells, while FACS analysis showed that growth arrest took place at G0/G1 phase. RT-PCR analysis displayed reduced level of Bcl-2/Bax ratio in test cells as compared to control cells. Phytochemical analysis of the extract using LC-MS and FT-IR studies showed that protocatechuic acid was the predominant component present in the extract. Conclusion: Our studies indicated that Averrhoa bilimbi extract expressed significant apoptogenic potential against EAC cells in vivo. Keywords : Ehrlich ascites carcinoma, Apoptosis, Fluorescence-activated cell sorting, Bax/Bcl-2, Protocatechuic acid.