Evaluation of Cytotoxicity and Anti-Cancer Effect of Gleditsia Caspica Pod’s Extract on Lung Cancer A549 Cell-Lines

Abstract

Cancer causes significant morbidity and mortality and is a major public health problem worldwide. Lung cancer has the most mortality rate among other types of cancer in the world. According to American Cancer Society (ACS) in 2007, 31% all of cancer death in males and 26% in females, were related to lung cancer. Plant-derived compounds have been an important source of several clinically useful anti-cancer agents. Among these, Gleditsia species have been used in various traditional medicines around the world. In the current study we aimed to assess cytotoxic and possible anti cancer effects of Gleditsia caspica, a traditionally used herbal medicine, on A549 cell line. Materials and MethodsIn this study, we studied the growth inhibitory effect of G. caspica's anti-tumor activity using human lung cancer cell line A549. The cells were exposed to methanol extract at different concentration and for different time durations. Along with morphologic evaluation of the cultured cells, MTT assay were carried out to characterize the cytotoxicity of the extract used. Expression of apoptosis related genes such as bcl-2,bcl-XL, bax, p53 and Caspase-9 as well as GAPDH housekeeping gene was studied using reverse transcriptase-QPCR. ResultsOur results showed that methanol extracts of G. caspica greatly inhibited A549 cell proliferation in a concentration-dependent manner. Upon treatment with extract, a clear difference was observed in cellular morphology; the cells acquired more attenuated architecture. Although we did not observe any cytotoxic effect of the plant seeds extract; our MTT assay results for plant pods extract showed very strong cytotoxicity effect in a time and concentration dependent manner. For the concentrations less than 400 إg/ml almost no growth inhibition occurred in comparison to the untreated control after 48 and 72 hr. But 500 إg/ml had 50% inhibitory effect on the proliferation of A549 cell lines. In 48 and 72 hr post exposure, the extract at the concentrations of higher than 500 إg/ml showed significant toxicity (the highest inhibitory effect) on A549 cells and reduced the cell viability to about 3% of un-treated control cells after 48 and 72 hr. The RT-QPCR results also elaborated the observed results of MTT assay in the molecular level in which G. caspica's extract had altered the expression of different anti apoptosis, pro apoptosis and tumor suppressor genes. Taken together, the up-regulation of P53, Bax and caspase-9 in a time and concentration dependent manner, confirms the hypothesis that multiple apoptotic pathways can be considered as potential mechanism of anti-proliferative action of G. caspica's plant extract on human adenocarcinoma A549 cell-line.DiscussionBased on the results of cellular (microscopic and MTT assay) as well as molecular experiment (RT-QPCR) carried out in this study we could demonstrate thatG. Caspica methanol extracts had in vitro cytotoxic activity and inhibitory effect on the growth of human lung cancer cell line A549.As evidenced from these preliminary results, this medicinal plant and its bioactive constituents may be considered as attractive alternatives to serve as lead compounds in drug development for human lung cancer as an adjuvant therapy. Final conclusion Collectively, our results, suggest that the G. caspica pods methanol extract has a cytotoxic effect on human lung adenocarcinoma cell, A549.Although, in presence of the extract both anti-apoptotic and pro-apoptotic genes are elevated, overall balance of them has a role in cell proliferation inhibition or maybe other mechanisms apart from apoptosis are involved. However, much remains to be done before such agent could be introduced to the clinic.