Preliminary Exploration Of Molecular Mechanism Of Apoptosis Induced By Hemolymph Of Eupolyphaga Sinensis Walker In Human Erythroleukemia HEL Cells

Eupolyphaga sinensis Walker,a traditional Chinese medicine,is widely used in Chinese medicine prescription.Long-term practice has shown that the female insects are used in traditional prescriptions to prevent and treat many diseases,including promotion of fracture healing,health care,and enhancing human immune function and so on.The studies of modern pharmacology showed that,E.sinensis is useful for its antithrombotic,antioxidant,antitumor activities.Insect hemolymph is an important biological material.Studies have shown that the antibacterial peptides contained in insect hemolymph have antibacterial,antifungal,and anticancer effects,and have little toxic and side effects on mammals.Therefore,we studied influence of E.sinensis Walker hemolymph?ESWH?on proliferation of tumor cells.ESWH showed potent cytotoxicity against the human erythroleukemia HEL cells.To gain insight into ESWH exhibited a potent inhibition on HEL cells,we further investigated its effects on proliferation,apoptosis,cell cycle,migration of HEL cells.The molecular mechanism underlining its effects on HEL cells was also studied.The results will provide theoretical and experimental basis for further exploration of antitumor activity of E.sinensis.The main research findings were summarized as follows:1.Antitumor activities in vitro of ESWHTo study influence of ESWH on the proliferation of tumor cells?PC3,HEL,WM9,MDA-MB-231?in vitro.The hemolymph was collected by puncturing method and the influence of ESWH in proliferation of tumor cells was measured by MTT assay.ESWH showed antitumor activity on four cancer cells in vitro,and HEL cells were significantly inhibited in a dose-dependent manner.2.Antiproliferation of ESWH on HEL cells in vitroTo explore the effect of ESWH on the proliferation and apoptosis of human erythroleukemia HEL cells.Cells were treated with different concentrations?10-60 mg/L?of ESWH in vitro.Cell viability was assessed by MTT assay,morphological changes in apoptotic HEL cells were observed by Hoechst 33258 and Annexin V-FITC fluorescence staining,apoptosis rate was evaluated by Annexin/PI method.Furthermore,the changes of cell cycle distribution were monitored with flow cytometry;Transwell assay was used to explore the effect of ESWH on migration of HEL cells.The results showed that the IC₅₀values of ESWH inhibiting the growth of the two types of cells decreased gradually with the increase of the treatment time.The IC₅₀of ESWH at 24 h,36 h and 48 h were 22.72 mg/L,20.78 mg/L and 16.96 mg/L,respectively.We observed cell morphological change caused by ESWH,such as reduction of cell density,cell shrinkage and becomes smaller.The results of Hoechst 33258 staining and annexin V FITC staining showed that the cells showed a significant morphological change in nuclear chromatin.The nucleus of the untreated cells was less bright with more homogeneous color than the ESWH treated cells suggesting chromatin condensation which is the hallmark of apoptosis.The effect of ESWH on cell cycle of HEL cell was analyzed using PI staining and flow cytometry analysis.Our results demonstrated that ESWH induced cell cycle arrest at G2/M phase in HEL cells.In addition,treatment with ESWH caused decrease HEL cell migration in a dose-dependent manner.3.Molecular mechanism underlining its antiproflieration effects in vitro of ESWHTo investigate the molecular mechanism underlining the antiproliferation effects in vitro of ESWH.RT-q PCR and Western blot assay were employed to detect m RNA and protein expression levels of apoptosis-related genes and proteins.ESWH down-regulate the m RNA expression levels of PARP1 and Bcl-2,and up-regulate expression of Caspase-3,Caspase-8,Bax,Cytc and P53.The results of Western blot have showed the expression of Bax,Cyt C,Cleaved caspase-8,Cleaved caspase-3,Cleaved caspase-9,Cleaved PARP1,P53 were increased while the Bcl-2 and Fli-1 were decreased.To explore the mechanism for ESWH-induced G2-M phase arrest,Western blot analysis was used to examine the effect of ESWH treatment on the expression levels of cell cycle-related proteins,such as cyclin B1,Cdc2,cyclin D1 and CDK4.Results from western blot analysis suggested that ESWH dose-dependently downregulated expression of cyclin D1 and CDK4,and downregulated cyclin B1 and Cdc2 after treatment with 40 mg/L ESWH for 24 h.To explore the mechanism used by ESWH inhibited migration of HEL cells was also evaluated by checking the protein expression of CXCR4,MMP-2 and MMP-9.The results showed that ESWH dose-dependently downregulated CXCR4,and MMP-2 and MMP-9 expression were significantly downregulated in HEL cells treated with 40 mg/L ESWH.Moreover,the effect of ESWH on MAPK pathway was detected using Western blot.The results suggested that,the expression levels of P-ERK1 and P-P38 were increased in a dose-dependent manner with the increase of ESWH concentration,and expression of P-ERK1 and P-P38 were indeed significantly upregulated treated with 10 mg/L ESWH or higher concentration.