Nature has endowed plants with the power to cure multitude of diseases. In case of cancer treatment, the search for natural agents with potential antitumor effect has been intensified with time. Here we are reporting analysis of methanol extract of Caesalpinia bonducella (MECB) for its anti-proliferative and pro-apoptotic effect on Ehrlich ascites tumor (EAT) model in-vivo. MECB at 200 mg/kg concentration was responsible for decrease in the total percent of viable EAT cells (51.6%) and ascites volume (65%) in treated mice. MECB increased the rate of apoptosis with more number of cells exhibiting characteristic features such as membrane blebbing, apoptotic body formation and fragmented DNA which was evident by Giemsa and Acridine orange/Ethidium bromide (AO/EB) staining. Alongside, the mice survival time was increased upon MECB treatment. In addition, FACS data suggests that death of MECB treated EAT cells was due to apoptosis and not by necrosis. Furthermore the analysis for molecular mechanism revealed MECB decreased the level of anti-apoptotic Bcl-2 expression while increasing pro-apoptotic Bax level. The immunoblot analysis confirmed the activation PARP (Poly (ADP-ribose) polymerase), a substrate for executioner caspase-3, which brings about subsequent DNA fragmentation in apoptotic process. These results confirm the antiproliferative and pro-apoptotic activity of MECB, which may further translated into therapeutic drug development to combat cancer. Department of Biotechnology and Zoology, University of Mysore, Mysore, India. Ehrlich ascites tumor (EAT) cells were routinely maintained in our laboratory through in-vivo transplantation. Antibodies against Bcl2, Bax, PARP (Poly (ADP-ribose) polymerase) and β-actin were purchased from Cell Signaling Technology, USA. TRIzol reagent and Super-script III one-step RT-PCR system were obtained from Invitrogen, USA. Tetramethylrhodamine methyl ester (TMRM, ≥ 95%) was from Sigma-Aldrich. DMSO (Dimethyl sulphoxide) (cell culture grade) and all other chemicals, reagents were obtained from Hi-media, India. Plant extract preparationC. bonducella leaves were collected from Western Ghat region of Shimoga district in Karnataka. Dried leaf powder (25 g) was extracted in methanol at 50°C for 12 h (Soxhlet extraction). The solvent was subjected for evaporation at room temperature to get concentrated plant extract and the final yield was noted (4.1 g). Finally, the extract was dissolved in 1% DMSO for the treatment.
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.
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