In Vitro Investigation Of Cytotoxicity And Apoptosis Induction By Methanol Extract Of Calophyllum Inophyllum Fruit In Mcf-7 Human Breast Cancer Cell Line
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Date
2016-07
Authors
Shanmugapriya
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Abstract
Calophyllum inophyllum is an exquisite plant species with rich ethnomedicinal values have been diversely utilized to heal several diseases. In spite of its long-established sophisticated traditional medicinal properties, only a few investigations have reported its cytotoxicity especially fruit extract, with absolutely no means of scientific evidence of its comprehensive molecular mechanism. Hence, this study was conducted to determine the in vitro cytotoxicity of C. inophyllum fruit extract against MCF-7 human breast cancer cells with an intricate elucidation of the molecular mechanism of the cell death. In this study, authentication of the plant species and the determination of total phenolic content were carried out. The cytotoxic effect of C. inophyllum fruit extract against MCF-7 cancer cells was evaluated through MTT and CyQuant assays which demonstrated the inhibition of cell viability with the IC50 values 19.63 μg/mL and 27.54 μg/mL respectively. The preliminary time-based morphological investigation of MCF-7 cells treated with the IC50 value (23.59 μg/mL) of C. inophyllum fruit extract revealed prominent histological characteristics of apoptosis. Flow cytometric analysis of Annexin V/ Propidium Iodide assay ascertained the induction of apoptosis in C. inophyllum-treated MCF-7 cells in a dose-dependent manner. Moreover, flow cytometric cell cycle analysis demonstrated cell cycle arrest at G0/G1 and G2/M phases simultaneously. Reactive oxygen species (ROS) activity revealed that C. inophyllum fruit extract induces the generation of ROS in treated MCF-7 cells when compared to
the untreated cells. Furthermore, C. inophyllum fruit extract dose-dependently decreases the mitochondrial membrane potential (Δѱm) in MCF-7 cells. The enzymatic activity of caspase-3 in MCF-7 cells treated with the IC50 value (23.59 μg/mL) of C. inophyllum fruit extract in comparison with untreated cells revealed an elevated hydrolysis of the substrate in treated cells, upholding that the extract induced apoptosis via activation of caspase-3. In addition, comet assay showed a dose-dependent genomic DNA fragmentation indicated by the evaluation of tail length, tail moment and tail DNA collectively. Conclusively, based on the data obtained from this overall study not only determined the in vitro cytotoxicity of C. inophyllum fruit extract but also shed light on its comprehensive molecular mechanism which clearly indicated a caspase-dependent intrinsic apoptotic pathway of cell death. Considering apoptosis as a clinically admissible approach of cancer cell death, C. inophyllum fruit extract could be a promising novel anticancer drug candidate.
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The in vitro cytotoxicity of C. inophyllum fruit extract against , human breast cancer cells.