Inhibition Of Antimicrobial Peptide Cecropin XJ Of Bombyx Mori(Bombycidae, Lepidoptera) On Human Gastric Cancer Cells

The cecropins, first isolated by Boman et al. from the Hyatophora cecropia pupae, are a family of antimicrobial peptides. To date, more than 20 cecropins were found. Cecropins have the ability to form specific amphipathic α-helices, which allow them to target nonpolar lipid cell membranes and exert strong antibacterial activities against gram positive and negative bacteria in the micromolar concentration. Many studies have indicated the antitumor activity of cecropins against various cancer cell lines, including leukemia, hepatocellular cancer cells, bladder cancer cells and gastric cancer cells. But cecropins have no toxicity to normal mammalian cells, suggesting that cecropins may be a promising candidate as a novel antitumor agent.CecropinXJ, a cationic antimicrobial polypeptide composed of 37 amino acid residues, molecular mass of 3.9 kDa, isoelectric point of 10.37, was first isolated from the larvae of Bombyx mori. CecropinXJ showed 98% homology with the cecropin B. We cloned CecropinXJ gene into the pYES2/CT/α Factor expression vector and expressed in the Saccharomyces cerevisia. Our previous studies have shown that CecropinXJ could inhibit the proliferation and induce apoptosis of various human cancers. However, the mechanism remains to be not determined. In this study, we investigated the cytotoxicity and the mechanism of CecropinXJ against the human gastric cancer BGC823 cells in vitro and in vivo. The anti-proliferation and anti-growth activity of CecropinXJ on BGC823 cells in vitro was assessed by the MTT and colony formation assays. Cell cycle distribution and cell apoptosis were detected by flow cytometry analysis. Cell morphology was detected by transmission electron microscope and inverted fluorescence microscope. The in vivo efficacy of CecropinXJ was evaluated using a BGC823 xenograft tumor and malignant ascite tumor models in nude mice and Balb/c mice, respectively. Tumor volume, tumor weight and body weight of mice were measured the inhibitory effects of CecropinXJ on the tumor growth of tumor-bearing mice. Histopathological changes of tumor tissues and organs of the mice were observed, the expression of apoptosis-related proteins, PI3K/Akt signaling pathway-related proteins and angiogenesis-associated proteins were analyzed by qRT-PCR, western blot and immunohistochemistry analysis. It is beneficial to further study on anti-tumor mechanism of CecropinXJ. The main results are as follows:1. CecropinXJ significantly inhibited the growth of human gastric cancer BGC823 cells in vitro and induced apoptosis through activating the mitochondrial-mediated pathway. MTT and colony formation assays indicated that CecropinXJ suppressed cell proliferation and reduced colony formation of BGC823 cells in a dose- and time-dependent manner, but without inhibitory effect on normal gastric epithelia GES-1 cells. S-phase arrest was observed after treatment with CecropinXJ. Flow cytometry and morphology analysis suggested that CecropinXJ induced both early and late phases of apoptosis. Further study found that CecropinXJ increased ROS production and disrupted mitochondrial integrity. The results of qRT-PCR and western blot demonstrated that CecropinXJ could up-regulate the expression of Bax and down-regulate the expression of Bcl-2, promote release of cytochrome c and activate caspase. Meanwhile, CecropinXJ promoted caspase-3/8/9 activity in a dose-dependent manner, and cell death ratio of BGC823 cells induced by CecropinXJ was significantly reduced by caspase-3 and caspase-9 specific inhibitors treatment. These results showed that CecropinXJ inhibited the growth of BGC823 cells in vitro and induced apoptosis of BGC823 cells through activation of caspase-dependent mitochondrial apoptotic pathway.2. CecropinXJ alone and in combination with LY294002 were inhibited the growth of gastric cancer BGC823 cells through blocking the PI3K/Akt signaling pathway. CecropinXJ decreased phosphorylated level of Akt and induced down-regulation of p-Bad and anti-apoptotic protein, up-regulation of pro-apoptotic protein. Therefore, CecropinXJ suppressed the growth of BGC823 cells mediated by blocking PI3K/Akt signal pathway. Furthermore, combined with LY294002 could enhance the blocking effect of CecropinXJ on PI3K/Akt signaling pathway and exhibite more effective in inhibiting growth and inducing apoptosis of BGC823 cells in vitro.3. The xenograft tumor derived from BGC823 cells was established in Balb/c nude mice and to investigate the anti-tumor activity of CecropinXJ on the growth and angiogenesis of tumor in vivo. The results showed that CecropinXJ significantly prevented the growth of xenograft tumor in the BGC823-bearing mice. Tumor tissue necrosis was observed and intercellular substance was expanded by CecropinXJ-treated. Further, apoptosis of cancer cells in CecropinXJ-treated tumor issues was detected. Besides, CecropinXJ inhibited the expression of CD31, CD34, VEGF, bFGF and vWF either in tumor or BGC823 cells. These results indicated that CecropinXJ could inhibit the growth of BGC823 cell xenografts in nude mice, induce apoptosis of cancer cells and inhibit angiogenesis.4. To investigate inhibition of CecropinXJ in vivo on gastric ascites tumor, the tumor ascites model were eastablished with gastric cancer BGC823 cells by intraperitoneal injection. Compared with BSA treatment, CecropinXJ and Dox significantly inhibited the formation and growth of malignant ascites. Furthermore, compared with Dox treatment, CecropinXJ induced more tumor cell apoptosis in ascites and improved survival condition of ascites tumor bearing mice.