The growth of cancer can control by inhibiting
The growth of cancer can control by inhibiting the rate of cell proliferation or increasing the rate of selective apoptosis and effectiveness of anticancer agents varies according to genetic constitution of cancer. Hence, we investigated the anticancer activity of arginase against different kinds of cancer cell lines and lowest IC50 value (0.8 IU ml−1) was recorded with human promyelocytic leukemia cell line HL-60. Notably, purified arginase did not exert cytotoxicity on human normal cells till at 3 IU ml−1. In the contrary, at 3 IU ml−1, approximately 68–85% proliferation of LS-180, HCT-116, MCF-7, HL-60, MOLT-4, K-562, BT-549, and PC-3 cells were inhibited. Thus, this confirms that purified arginase exhibited selective toxicity to neoplastic cells and non-toxic for normal cells. To evaluate the cell death kinetics of arginase, we performed time dependent MTT assay. We have found that arginase inhibited the proliferation of HL-60 cells and initiate apoptosis in concentration as well as time dependent manner. These findings indeed suggest that arginase from P. aeruginosa IH2 could be considered as an effective therapeutic agent. Growing evidences suggest that deregulation of apoptosis is the hallmark of cancer cells and induction of selective apoptosis in cancer cells is the major focus for development of anticancer therapeutics . Several morphological and biochemical changes such as membrane blebbing, cell shrinkage, chromatin condensation, DNA fragmentation and formation of apoptotic bodies are considered as biological end-points of the apoptosis . By performing phase contrast microscopy, DAPI staining, comet assay, AO/EB staining and DNA fragmentation assay, we observed that these apoptotic end-points were significantly increases with an increasing the concentration of arginase. Therefore, these observations are collectively indicated that purified arginase selectively induced apoptosis in cancer cells. Translocation of membrane phosphatidylserine from inner side to outer surface is one of the early events of apoptosis . We observed that upon treatment with arginase, majority of cells were dead due to apoptosis and minor population were underwent to necrosis indicating that arginase treatment induced cell death by activation of apoptosis. The flavin adenine dinucleotide mg is a set of coordinated events occurs in sequential fashion that culminates in the formation of two daughter cells from one parental cell. Deregulation of cell cycle specifically at cell cycle check points is the heart key of leukemic malignancies. In order to understand the effect of purified arginase on cell cycle phase distribution, we have investigated cell cycle profile by FACS analysis. We found that upon treatment with arginase, cell cycle was arrested in G0/G1 phase with reduced cells in S phase. Hence, here authors suggest that the quiescent of cell cycle progression in the G0/G1 phase may developed due to inhibition of enzymic machinery which is required for cell proliferation by breakdown of essential nutrient (l-arginine), which leads to apoptosis. These observations are also supported by the previous findings demonstrating that arginase treatment arrest cell cycle in G0/G1 phase . ROS is a byproduct of the normal metabolism of cell but excessive generation of ROS plays a critical role in activation of apoptosis. As we know that mitochondrion is very dynamic cell organelle, actively involved in ROS generation and dysfunctioning of mitochondrion is an important event of apoptosis . Several previous studies suggested that the amount of MMP loss is directly related to the ROS production , . Hence, in the present study, we have tested the MMP and ROS generation and found that arginase treatment reduced the MMP while enhanced the intracellular ROS generation in a concentration dependent manner. The activation of caspases-8 and 9, downstream caspase-3, elevated level cleavage of PARP, up-regulation of pro-apoptotic proteins (Bax) and down-regulation of antiapoptotic proteins (Bcl-2) suggested that arginase treatment triggers the activation of mitochondrial pathway of apoptosis. The growing evidences indicated that for the mitigation of harmful effects of ROS, cells activates enzymic (SOD, CAT and GPx) and non-enzymic (GSH) antioxidant defence mechanism, which converts ROS into non-toxic products and maintained the physiological balance . In our study, we have found that arginase treatment dramatically reduced the expression levels of both enzymic and non-enzymic antioxidants. Therefore, here authors suggest that starvation stress induced by arginase treatment exert ROS production, which promote mitochondrial dysfunctioning, reduced expression levels of endogenous antioxidants which led to mitochondrial mediated apoptosis.