| Background:Non-small cell lung cancer accounts for about 80%of lung cancer,with high malignancy.The main therapies for it are surgery,radiotherapy and chemotherapy.Different from the two former,chemotherapy is suitable for NSCLC patients in various clinical stages,due to its characteristic that could kill tumor cells anywhere in the body.However,traditional chemotherapeutics also kill normal cells not specific when applying it which brings inevitable side effects to patients.Its clinical application is restricted.In recent years,molecular targeted drugs with specific targets and lower side effects have gradually entered the ranks of mainstream.The most common molecular targeted drug for NSCLC was the epidermal growth factor receptor tyrosine kinase inhibitors(EGFR-TKIs)represented by gefitinib.Researches had shown that Asian women lung cancer patients without smoke would benefit from the EGFR-TKI easily,and later it is proven that tumors sensitive to EGFR-TKI usually carried EGFR gene mutations on exons 18,19 or 21.However,the most of NSCLC patients would develop acquired drug resistance(ADR)to EGFR-TKI after they had received the drug treatment for 6-12months.The resistance greatly limited the use of EGFR-TKI clinically.Now more established mechanisms related to ADR of EGFR-TKI were following:EGFR gene’s second mutations like T790M;Key proteins’ activation(like KRAS)on EGFR pathway;some phenotypic changes inducing tumor cell proliferate without the EGFR’s activation,such as transforming to small cell lung cancer or mesenchymal cells.About half of NSCLC patients’ tumor with ADR to EGFR-TKI was carrying the T790M.Therefore,T790M might be considered as the molecular basis of ADR to EGFR-TKI.T790M’s role in the process that NSCLC patients obtain acquired drug resistance to EGFR-TKI is still not clear completely.Research found that the tumor cells carrying T790M of NSCLC patients often emerged transition to the new tumor cells with other phenotypes when the ADR to EGFR-TKI developed.The phenomenon hints that some factors which could promote transformation of cell phenotype might induce the tumors to develop ADR to EGFR-TKI.At present the most clearly transformation factor was transforming growth factor β(TGF-β)which represented by TGF-β1.There are a variety of cells in the body secrete it and express its receptors,which shows that TGF-β1 could contribute to cell phenotypic transitions in different cells.We speculate that TGF-β1 might promote the NSCLC cells carrying T790M to develop ADR to EGFR-TKI.Objective:In the study,we detected TGF-βs effect on sensitiveness of NSCLC cell line NCI-H1975 to gefitinib in order to prove that TGF-β1 could play a catalytic role in process of NSCLC cells’ developing ADR to EGFR-TKI.Methods:1.Cell line and Culture Conditions(1)NSCLC cell line NCI-H1975 with T790M was selected for the research.(2)Culture conditions:Temperature is 37℃;CO2 concentration is 5%;Humidity is saturated;Medium is RPMI-1640 complete medium containing 10%fetal bovine serum and 1%penicillin standard streptomycin mixture.2.Selection of gefitinib concentration and action timeMTT was used to assay IC50 of gefitinib on NCI-H1975,alternative gefitinib concentration range:40μmol/L to 1.25μmol/L(6-fold dilution concentrations in all).3.Selection of TGF-β1 concentration and action time(1)TGF-β1 was put into the NCI-H1975 cells with the treatment of 40,20,10,5 and Ong/ml gefitinib;(2)The growth state of the cells was observed under an inverse microscope;(3)The IC50 of gefitinib to living cells was detected by MTT assay;(4)The concentration of TGF-β1 was decided according to the growth state of living cells and the IC50 of gefitinib to living cells.4.Detection of TGF-β1’s effect on sensitivity of NCI-H1975 to gefitinib(1)According to whether TGF-β1 or gefitinib were put into culture conditions,the cells are divided into four groups:NCI-H1975:NCI-H1975 parental cell lines in independent culture condition;NCI-H1975/TGF:NCI-H1975+TGF-β1;NCI-H1975/G:NCI-H1975+Gefitinib;NCI-H1975/G/TGF:NCI-H1975+Gefitinib+TGF-β1.(TGF-β1 concentration:10ng/ml;reaction time:48h Gefitinib concentration:10μmol/L;reaction time:24h)(2)The growth state of the cells was observed under an inverse microscope;(3)The proportion of viable cells was obtained with cytometry;(4)The cell growth curve was drawn according to cytometry of cells;(5)The IC50 of cells to gefitinib was detected by MTT assay;(6)The cells’ growth inhibition curves in the presence of gefitinib were drawn according to trypan blue assay.5.TGF-β1’s effect on the presence of EMT in NCI-H 1975 cells in the presence of gefitinib(1)The expression of EMT marker proteins(E-cadherin and Vimentin)in cells was detected by immunocytochemistry;(2)The changes of expression levels of E-cadherin and Vimentin in the total cellular protein were determined by western-blot semi-quantitative method.Results:1.NSCLC cell line NCI-H1975 was fusiform with plump cytoplasm,smooth membrane and adherent growth.2.The IC50 of gefitinib on NCI-H1975 was 12.61±1.33μmol/L.The 10μmol/L as gefitinib concentration was used.3.The growth states of the cells and the IC50 of gefitinib to living cells show:(1)The NCI-H1975 cells with the treatment of TGF-β1 were growing better than those without TGF-β1 and had a high density of living cells;(2)The IC50 of gefitinib to living cells of NCI-H1975 with the treatment of 40,20,10 and 5 ng/ml TGF-β1 were 57.84±1.38μmol/L,51.36±2.66μmol/L,39.3811.74βmol/L and 29.59±1.81μmol/L,the resistance index were 4.57,4.07,3.12 and 2.35.The IC50 of living cells with TGF-β1 were larger than that of living cells without TGF-β1(P<0.05)and it showed a positive correlation with the concentration of TGF-β1;(3)The TGF-β1 concentration with 10ng/ml was decided.4.Detection of the changes about the sensitivity of NCI-H1975,NCI-H1975/TGF,NCI-H1975/G and NCI-H1975/G/TGF to gefitinib(1)Compared with the NCI-H1975/G,The number of surviving cells of NCI-H1975/G/TGF was larger and NCI-H1975/G/TGF cells have full cytoplasm,clear and smooth membrane;(2)The living cell density of NCI-H1975/G/TGF was higher significantly being(0.76±0.063)×105/ml than that of the NCI-H1975/G being(0.24±0.015)×105/ml(P<0.05);(3)Cell growth curves showed that proliferation of NCI-H1975/G/TGF was faster than that of NCI-H1975/G.The population doubling time of NCI-H1975,NCI-H1975/TGF,NCI-H1975/G/and NCI-H1975/G/TGF were 46.54±3.79h、48.22±2.08h、46.45±4.27h and 44.58±2.17h respectively.The population doubling time of NCI-H1975/G/TGF was 1.87h(P>0.05)shorter than that of NCI-H1975/G;(4)The IC50 of gefitinib on NCI-H1975,NCI-H1975/TGF,NCI-H1975/G and NCI-H1975/G/TGF were 12.61±1.33μmol/L,16.98±0.50μmol/L,12.43±1.19μmol/L and 29.75±2.30μmol/L.The IC50 of gefitinib on NCI-H1975/G/TGF was improved 17.32μmol/L(P<0.05),compared with that of NCI-H1975/G;(5)The results of Trypan blue assay showed that the viable cell numbers of NCI-H1975/G/TGF treated with 10 and 20μmol/L gefitinib did not significantly decrease on the 1st day to the 3rd(P>0.05);at any time the number of viable cells of NCI-H1975/G/TGF was larger than that of NCI-H1975/G.The tolerance of NCI-H1975/G/TGF to gefitinib was enhanced;5.The results of Immunocytochemistry and Western-blot showed that the expression level of E-cadherin of tumor cells in NCI-H1975/TGF/G was lower than that of NCI-H1975/G(P<0.05),while the expression level of Vimentin of tumor cells in the former group was higher than that of the latter group(P<0.05).Conclusion:1.TGF-β1 could promote NSCLC cell line NCI-H1975 cell to develop the acquired resistance to EGFR-TKI.2.TGF-β1 has Protective effects on the NSCLC cell line NCI-H1975 cell from gefitinib with a concentration-dependent manner.3.TGF-β1 could induce NSCLC cells to EMT in the presence of gefitinib which might be the mechanism of the acquired resistance of NSCLC cells to EGFR-TKI. |
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