| Research Background Gastric cancer(GC)is a common malignant tumor of digestive system neoplasm in clinic,with high morbidity and mortality,which seriously threatens human life and health.At present,surgical resection with chemotherapy is the main treatment of gastric cancer.However,the existing chemotherapy drugs are not specific to tumors.It will cause damage to normal cells while killing tumor cells,thus causing serious side effects.Therefore,solving the toxic and side effects of chemotherapeutic drugs has been an important issue for research.As it is known to all,the toxic and side effects of chemotherapy drugs are generally proportional to the dose used.If a compound that has a synergistic effect and is not toxic itself is used in combination with chemotherapy drugs,the dosage of chemotherapy drugs can be reduced.Thereby reducing the side effects and adverse reactions of chemotherapy drugs without affecting the efficacy.Fluorouracil(5-fluorouracil,5-FU)is one of the most commonly used classic chemotherapy drugs for clinical treatment of gastric cancer and other digestive tract malignant tumors.It has a significant effect,and the price is low.However,after using this drug,patients with gastric cancer are prone to more serious gastrointestinal side effects such as nausea,vomiting,diarrhea,stomatitis,gastritis,and bone marrow suppression and other symptoms,which seriously affect the life of patients.Therefore,it is an important task to actively explore Chinese medicine or active ingredients that have attenuated or enhanced effects on 5-FU.Curcumin(CUR)is a phenolic compound derived from Chinese herbal medicine plant turmeric.It has anti-inflammatory,antioxidant,anti-tumor and other biological activities.In recent years,numerous experimental studies in-vitro by researchers around home and abroad had shown that curcumin has a growth inhibitory effect on gastric cancer cells and induces apoptosis.On the other hand,some researchers have used a certain dose of curcumin in combination with different doses of 5-FU for gastric cancer SGC-7901 cells cultured in vitro.The result showed that curcumin and 5-FU have a synergistic anti-tumor cell effect.Curcumin can enhance the effect of 5-FU in growth inhibitory and apoptosis induction on SGC-7901 cells.In other words,the combination of lower dose 5-FU and curcumin can achieve the effect of higher dose 5-FU.But the specific mechanism of the combination of the two drugs inducing apoptosis in SGC-7901 cells remains to be revealed.Objective In this project,we will observe the effect of CUR combined with 5-FU on gastric cancer SGC-7901 cells at the cell level,investigate whether the combination of CUR and 5-FU has anti-gastric cancer cell synergy,and reveal the possible mechanism for its function.This may provide a preliminary theoretical basis for the use of CUR to reduce the dosage of 5-FU and reduce the toxic and side effects of 5-FU during the clinical chemotherapy of gastric cancer.Methods Inoculate gastric cancer SGC-7901 cell line into a culture bottle or Petri dish containing 9% newborn bovine serum 1640 medium,put it into a carbon dioxide incubator,and culture the cells under the conditions of 37 ° C and 5%CO2.During the growth period,CUR and 5-FU with different concentration gradients were co-cultured to make the drug act on the cells for 24 h or 48 h.Then use the following different methods to detect the relevant indicators of the effect of drugs on the growth and apoptosis of gastric cancer SGC-7901 cells.1.The MTT method was used to detect the effect of CUR and 5-FU series concentrations on the growth of SGC-7901 cells,and the IC50 of the half-inhibitory concentration(half-inhibition rate)of the two drugs on SGC-7901 cells was calculated.2.The MTT method was used to detect the inhibitory effect of CUR and 5-FUon cell growth,and the cell growth inhibition rate was calculated.3.The effect of CUR and 5-FU on the division and proliferation of SGC-7901 cells and the formation of clones was tested by plate cloning experiments.The cell cloning rate of the experimental group and the control group was calculated.4.Fluorescent microscopy based on AO/EB double staining method was used to observe the morphological changes of gastric cancer SGC-7901 cell apoptosis induced by combined use of CUR and 5-FU,and the apoptosis rate in every group was calculated.5.Flow cytometry based on Annexin V-FITC/PI double staining was used to detect the effect of combined use of CUR and 5-FU on apoptosis of gastric cancer SGC-7901 cells,and the apoptosis rate was calculated.6.Western blot method was used to detect the expression levels of Fas,FADD,caspase-8 and caspase-3,the key proteins of the signaling pathway of apoptosis mediated by death receptor of SGC-7901 cells.Results1.The MTT results showed that the IC50 of CUR and 5-FU on SGC-7901 cells were 44.08 ?M and 60.13 ?M,respectively.2.The MTT results showed that CUR and(or)different doses of 5-FU acted on SGC-7901 cells for 24 h,36 h and 48 h,the cell growth inhibition rate of each experimental group was significantly different compared to the control group(P<0.05),and it is time-dependent.Meanwhile,there was nosignificant difference in cell growth inhibition rates between CUR combined 5-FU(L)group and 5-FU(M)group or between CUR combined5-FU(M)group and 5-FU(H)group(P> 0.05).3.The results of colony formation experiments showed that CUR and(or)different doses of 5-FU acted on SGC-7901 cells for 24 h,the colony formation rate of each experimental group was significantly different compared to the control group(P<0.05).Meanwhile,there was no significant difference in the colony formation rate between CUR combined5-FU(L)group and 5-FU(M)group or between CUR combined 5-FU(M)group and 5-FU(H)group(P> 0.05).4.AO/EB fluorescence staining and flow cytometry results showed that CUR and(or)different doses of 5-FU acted on SGC-7901 cells for 24 h and 48 h,the apoptosis rate of each experimental group was significantly different compared to the control group(P<0.05),and it is time-dependent.Meanwhile,there was no significant difference in the apoptosis rate between CUR combined 5-FU(L)group and 5-FU(M)group or between CUR combined 5-FU(M)group and 5-FU(H)group(P> 0.05).5.Western Blotting results showed that CUR and/or different doses of 5-FU acted on SGC-7901 cells for 24 h,the protein expression of caspase-8,caspase-3,Fas and FADD in each experimental group was significantly different compared to the control group(P<0.05).Meanwhile,there was no significant difference in the expression of caspase-8,caspase-3,Fas andFADD between CUR combined 5-FU(L)group and 5-FU(M)group or between CUR combined 5-FU(M)group and 5-FU(H)group(P> 0.05).Conclusion1.Curcumin and 5-FU both inhibit the proliferation of SGC-7901 cells in vitro and are time-dependent.At the same time,curcumin can enhance the proliferation inhibition of 5-FU on SGC-7901 cells.2.Curcumin and 5-FU can promote the apoptosis of SGC-7901 cells in vitro.At the same time,curcumin can enhance the pro-apoptosis effect of 5-FU on SGC-7901 cells.3.The fact that curcumin enhances the growth inhibition effect of 5-FU on human gastric cancer SGC-7901 cells in vitro is related to the apoptosis mechanism.Furthermore,it is suggested that apoptosis induced by the Fas signaling pathway plays an important role in the expression of Fas?FADD caspase-8 and caspase-3,.Therefore,the synergistic inhibition of curcumin and 5-FU on SGC-7901 cells was induced by apoptosis through Fas signaling pathway. |
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