| Background:Gastric cancer is one of the most common malignant tumors in China with a very high mortality.As a part of combined therapy,chemotherapy has exerted great role in treatment of gastric cancer.But nowadays it encounters a dilemma—multidrug resistance(MDR).Multidrug resistance(MDR)describes a phenomenon of cross-resistance of tumor cells to several structurally unrelated chemotherapeutic agents alter exposure to a single cytotoxic drug.Resistance to anticancer drugs is a major obstacle preventing the effective treatment of malignancy.Recently,it has been reported that transforming growth factor beta(TGFβ),a multi-functional cytokine,is involved in the development of malignancy.But it plays different even contradictory role in different tumors.The mechanism for the contradictory role of TGFβ1 is not clear.In our earlier studies,we found that TGFβ1 could enhance the invasion and metastasis of gastric cancer and induce the expression of GST-π,which indicate that TGFβ1 might involve in drug-resistance of gastric cancer.But there is no literature to investigate this phenomenon till now.Objectives:In this study we conducted MTT,siRNA,proteomics,western blotting,immunohistochemistry etc.to confirm the hypothesis and explore the mechanisms under this phenomenon in SGC-7901.Methods and Results:Firstly,we conducted MTT assay to detect the effect of TGFβ1 on sensitivity of SGC-7901 to mitomycin.As a result,we found that pre-treatment of TGFβ1 could induce the drug-resistance of SGC-7901 and the inhibition rate of mitomycin to SGC-7901 decreased from 89.5±10.6%to 35.0±4.1%.Secondly,we used Hoechst 33258 staining and FCM to test the alteration of apoptosis rate in SGC-7901 after treatment of TGFβ1.As a result,we found that it had nothing to do with the apoptosis of SGC-7901.Thirdly,we selected SGC-7901 cells to analyze the differential profiles between TGFβ1 treated and untreated groups with classic high throughput proteomic technology,two dimensional gel electrophoresis(2-DE),matrix-assisted laser desorption/ionization time of flight(MALDI-TOF)and bioinformatics.Locus repeat analysis of protein spots showed in IEF direction,the mean deviations of TGFβ1 treated group and untreated group were(0.908±0.102mm)and(0.838±0.116mm), respectively.In SDS-PAGE direction,the mean deviations of these two groups were 1.032±0.108 mm and 1.075±0.168mm,respectively.These results confirmed the establishment of well-resolved and reproducible 2-DE patterns in our study.Analysis of 2-DE patterns 20 spots were upregulated and 10 downregulated in TGFβ1 treated group.Subsequently,two interesting protein spots were excised from gels and then digested in-gel by trypsin.After that,the digest was determined by MALDI-TOF mass spectrometry.Then,the resulting peptide mass fingerprints(PMF)were used to search the MS Database with the software Mascot peptide mass fingerprint.GST-π, MDR1 were successfully identified.Fourthly,we investigated their expression in gastric cancer tissue with immunohistochemistry assay to explore the relation of TGFβ,GST-πand MDR1 in gastric cancer.We found that the expression of GST-πand MDR1 was correlated with that of TGFβ1,which indicated that GST-πand MDR1 may participate in the TGFβ1-mediated drug-resistance of gastric cancer.Fifthly,we used Smad4 siRNA and special inhibitor of MAPK signal pathway to explore the role of these signal pathway in this biological effect to investigate the mechanisms of TGFβ1-induced GST-πand MDR1.As a result,we found that expression of GST-πinduced by TGFβ1 could be inhibited by Smad4 siRNA and PD98059 but not SB203580 and SP600125,which indicated that TGFβ1 could induce expression of GST-πthrough Smad and ERK signal pathway.As to MDR1,we found that it could be induced through JNK and ERK signal pathway by TGFβ1.Conclusions:(1)We found that TGFβ1 might induce the drug resistance in SGC-7901,which might correlate with GST-πand MDR1;(2)TGFβ1 could induce through Smad and ERK signal pathway and the expression of MDR1 through JNK and ERK signal pathway.
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