| BackgroundGastric cancer is considered to be the second cause of the whole deathsdue to cancer, and its incidence is still rising.The prognosis of gastric cancer ispoor, because the most of patients occurred metastases when the diagnosis ismade. Traditional treatment of gastric cancer include surgery, radiation therapy,chemotherapy and supportive treatment. Of cause, surgical resection is the mosteffective treatment, however, to achieve therapeutic purposes, surgical resectionis not of such efficient, and some patient can not even receive the surgery.Adjuvant chemotherapy is used as a common therapy of advanced gastric cancer.5-FU is an analogue of uracil, which can block the progress of deoxyriboseuridine converting to thymidylate by synthase thymidylate, so as to interfere thesynthesis of DNA.5-FU is one of the first chemotherapy drugs used in treatinggastric cancer. In recent years, it is found that gastric cancer tuned tochemotherapy resistance, while its mechanism is still unclear.Enhance thesensitivity of gastric cancer cells to chemotherapy will contribute to theprognosis and prolong survival of the patients with gastric cancer.Cordyceps militaris is one of the most important traditional herb medicine,which has the effect of anti-tumor, inhibiting tumor metastasis,immunomodulatory, anti-oxidation, inhibit inflammation, insecticide, anti-microbial, lowering blood pressure, anti-aging, kidney and nerve protectionfunction and other effects. Polypeptides, referred peptide involved in cell growth, development, physiological function and proliferation in all aspects. Recentstudies have found that some peptides can promote the body’s lymphocyteproliferation, enhance macrophage phagocytosis, thereby improving the body’sability to resist external pathogens, and also anti-tumor functions. These maylead us to a new therapy of gastric cancer.MethodsPart I: Lyophilized powder of Cordyceps militaris was made by enzymaticextraction, ultrafiltration, and Lyophilization in progress. Tricine-SDS-PAGEelectrophoresis was used to the preliminary identification of Cordyceps militarispolypeptide.Part II: Cell culture was done to describe the growth curve of SGC-7901,thus to determine the growth characteristic. the chemosensitivity of SGC-7901with different concentrations of5-FU and Cordyceps polypeptide (5mg/ml,10mg/ml and20mg/ml) were tested by MTT assay, results were estimated asIC50. We got an appropriate concentrations of5-FU an Cordyceps peptide forthe later experiments.Part III: The most appropriate concentration of Cordyceps polypeptide and5-FU were used to study the mechanism of SGC-7901chemosensitivity on5-FU.Four groups were set as follow:1) control group: SGC-7901parent strain;2)group1: treat with5-FU (1200μmol/L);3) group2: treat with5-FU (1200μmol/L) and LY294002(20μmol/L);4) group3: treat with5-FU (1200μmol/L)and Cordyceps polypeptide20mg/ml; The proliferation of tumor cell was tested by EdU detection; Protein synthesis of the components of PI3K/Akt/mTOR andNF-κB/IκB were tested by WB;and flow cytometry were used to apoptosisdetection.ResultPart I:Cordyceps polypeptide was identified by Trincine-SDS-PAGEelectrophoresis and its molecular weight is mainly between4.1~66KDa.Part II:1. Cordyceps polypeptid of2.5mg/ml had no significant inhibition ongastric cancer cell line SGC-7901(P=0.621), the concentration of5mg/ml,10mg/ml,20mg/ml Cordyceps polypeptide can inhibit gastric SGC-7901cellline proliferation (P <0.05); the difference between different concentrations ofpolypeptide is of significance(P<0.05), and showed a dose-dependent inhibitionon the proliferation of SGC-7901(P <0.05).2. IC50of SGC-7901on5-FU chemotherapy was1206.3±53.2μmol/L;experimental group with5mg/ml,10mg/ml,20mg/ml Cordyceps polypeptide,which IC50are1069.4±79.1μmol/L,987.8±40.5μmol/L, and943.1±49.5μmol/L respectively; the IC50of experiment groups was significantlylower than with5-FU chemotherapy group (P <0.05), and the value of IC50fallswith the concentration Cordyceps peptide (P <0.05).Part III:1. EdU detect cell proliferation activity showed, the proliferative activity of tumor cells in each experimental group was significantly lower than the controlgroup (P <0.05); in group2and group3, the proliferation of tumor cells wassignificantly lower than that of group1(P <0.05); the proliferation of tumorcells of group3was significantly lower than that of group2(P <0.05)2. The protein expression of p-PI3K, p-Akt, and p-mTOR in eachexperimental group were significantly lower than that of the control group (P<0.05), the protein expression of p-PI3K, p-Akt,and p-mTOR in group2andgroup3were significantly lower than group1(P <0.05), the protein expressionof group2and group3showed no significant difference (P=0.073,0.898and0.844).3. The expression of NF-κB p50and NF-κB p105in each experimentalgroup, were significantly lower than the control group (P <0.05), the proteinexpression of NF-κB p50and NF-κB p105in group2and group3weresignificantly lower than group1(P <0.05), the protein expression of NF-κB p50and NF-κB p105in group3were significantly lower than group2(P <0.05);The expression of IκB-β in each experimental group, were significantly higherthan the control group (P <0.05), the protein expression of IκB-β in group2andgroup3were significantly higher than group1(P <0.05), the protein expressionof IκB-β in group3were significantly higher than group2(P <0.05).4. The incidence of early gastric apoptosis, late apoptosis and totalapoptosis were higher in all experimental groups compared with control group(P <0.05), the incidence of early apoptosis, late apoptosis and total apoptosis in experimental group2and3were higher than group1(P <0.05), the incidence ofearly apoptosis, late apoptosis and total apoptosis in experimental group3werehigher than group2(P <0.05),.Conclusion1. Cordyceps polypeptide was identified by Trincine-SDS-PAGEelectrophoresis as the methods in this study and its molecular weight was mainlybetween4.1~66KDa.2. SGC-7901gastric cancer cell lines went into the logarithmic growthphase after cultured48-72hours, and the fifth days went into the plateau phase.Among the doses used in this study, Cordyceps peptides can inhibt theproliferation of gastric cancer cell line SGC-7901, the higher the concentrationof the Cordyceps peptides, the less the proliferation of the gastric cancer(P<0.05).3. Cordyceps peptide can enhance the chemosensitivity of gastric cancercell line SGC-7901and inducing tumor cell apoptosis, which may be relatedto the inhibition of PI3K/Akt/m-TOR pathway and the regulating NF-κB/IκBexpression. |
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