Inhibitory Effects Of Bombyx Mori Antimicrobial Peptides On Esophageal Cancer Cells

Esophageal cancer is one of the most common malignancies worldwide.Chemotherapy is a common method for treating esophageal cancer.However,the resistance of tumor cells,the indiscriminate lethality of chemotherapeutics,and the various toxic and side effects of chemotherapeutics sometime accelerate the development of the disease.Therefore,it is urgent to find new drugs that are safe but effective in curing esophageal cancer.Bombyx mori Antibacterial peptides(Bm AMPs)are extremely important immune effectors in the silkworm's innate immune system and can kill a variety of bacteria and fungi.Recent studies have shown that Bm AMPs can inhibit a variety of tumor cells.In this study,we explored its inhibitory effect on two types of esophageal cancer cells Eca109 and TE13 through in vitro experiments,and explored the mechanism of the inhibition.In a mouse model,the ability of BmCecA to inhibit esophageal cancer in vivo was investigated through in vivo inhibition experiments,and its biosafety was evaluated.The synergistic effect of BmCecA and BmCecD with curcumin was evaluated for treating esophageal cancer.In our preparative experiments of cell proliferation,BmCecA and BmCecD were found among the Bm AMPs,obtained by solid-phase synthesis,to be effective in inhibiting proliferation of esophageal cancer cells.In the precent study,proliferation experiments,clone formation experiments,wound healing experiments,and invasion experiments were used to explore the inhibitory effects of the BmCecA and BmCecD on esophageal cancer cells Eca109 and TE13 in vitro.The results showed that the BmCecA and BmCecD inhibited the proliferation,clone formation,migration,and invasion of the two types of esophageal cancer cells in concentration dependent model,indicating that BmCecA and BmCecD can effectively affect activity of esophageal cancer cells.To study the mechanism of BmCecA in inhibiting cancer cells,flow cytometry was used to detect the apoptosis of Eca109 cells after 12 h of BmCecA treatment.The results showed that BmCecA induced apoptosis of Eca cells and the apoptosis rate increased with the increase of BmCecA concentration,indicating that BmCecA can promote the apoptosis of Eca109 cells.Furthermore,real-time fluorescence quantitative PCR(q RT-PCR)and Western-Blot results showed that BmCecA treatment down-regulated expression of Bcl-2 and up-regulated Bax in Eca109 cells,which activated caspase-3 through the release of cytochrome C into the cytoplasm,resulted in apoptosis.This indicates that BmCecA inhibits esophageal cancer cells by activating the mitochondrial-mediated apoptosis pathway.A tumor-bearing mouse model of Eca109 cells was successfully constructed for in vivo experiment.Tumor-bearing mice were treated by intratumoral injection of BmCecA.The results showed that the BmCecA treatment could inhibit the tumor growth in the mice but had no effect on the body weight.Furthermore,the blood was biochemically analyzed and several organs hischemically detected.The results showed that there were no significant differences in the blood indexes compared with the control group,and there were also no significant pathological changes in the tested organs of heart,liver,spleen,lung,and kidney samples.This indicates that BmCecA has good biological safety in a certain range.In order to increase the activity of Bm AMPs on esophageal cancer cells,synergistic effect of BmCecA and BmCecD with curcumin was evaluated.After combined treatment with curcumin and BmCecA or BmCecD for 12 h,the proliferation inhibition rate of esophageal cancer cells was analyzed by CCK-8 and Q value of the King's formula was used to judge the combined effect of curcumin and BmCecA or BmCecD.The result showed that compared with curcumin treatment alone,the combined treatment has an enhanced inhibitory effect on the proliferation of the two types of esophageal cancer cells.According to the King's formula,the synergy index Q is greater than 1.15,indicating that both BmCecA and BmCecD exhibited synergistic effect with curcumin in inhibiting esophageal cancer cells.These results provide a helpful experimental basis for further research on the antitumor effects and corresponding mechanism of Bm AMPs and the development of new antitumor drugs with Bombyx mori antimicrobial peptides in the future.