| Objective:Studies have shown that curcumin has antitumor effects on various malignancies including lung cancer.However,whether curcumin as a chemotherapeutic drugs sensitizer can reverse chemoresistance and its molecular mechanism is not clear.The purpose of this study is:(1)to investigate the effect of curcumin on the proliferation,division and apoptosis for cisplatin resistant cell line(A549/DDP)in vitro;(2)to explore the effect of curcumin on the tumorigenicity of cisplatin resistant cell line of lung cancer(A549/DDP)in nude mice;(3)to screen the molecular markers of cisplatin resistance in human lung adenocarcinoma and to explore the molecular markers of cisplatin resistance in human lung adenocarcinoma and its molecular mechanism.Methods:(1)A549 and A549/DDP cells were routinely cultured in vitro and treated by different concentrations of cisplatin(1 μg/ml,2 μg/ml,4 μg/ml,8 μg/ml,16 μg/ml,32 μg/ml,64 μg/ml),different concentrations of curcumin(1.5625 μM,3.25 μM,6.5 μM,12.5 μM,25 μM,50 μM,100 μM)and different concentrations of cisplatin(1 μg/ml,2 μg/ml,4 μg/ml,8 μg/ml,16 μg/ml,32 μg/ml,64 μg /ml)combined with curcumin(12.5 μM)for 72 hours,MTT assay was used to detect the survival rate of A549 and A549/DDP cells;(2)A549 cells were divided into control group(blank culture medium),DDP group(2μg/ml cisplatin),CUR group(12.5μM curcumin),DDP+CUR group(2μg/ml cisplatin +12.5μM curcumin),A549-DDP cells were divided into control group(blank culture medium),DDP group(12 μg/ml cisplatin),CUR group(12.5 μM curcumin),DDP + CUR group(12 μg/ml cisplatin + 12.5 μM curcumin).After treating with different drug for 72 hours for each group,Crystal Violet staining was used to examine the proliferation rate of A549 and A549/DDP cells;(3)flow cytometric analysis was used to determine the apoptosis of A549 and A549/DDP cells.(4)Nude mice were implanted with 200 μL(about 2×106)A549 cells or A549/DDP cells on the right groin.When the volume of transplanted tumor reached 200mm3,the nude mice were randomly divided into control group,DDP group,CUR group and DDP+CUR group.Control group: saline solution(intraperitoneal injection,qw)+ Carboxymethyl solution(gavage,qd);DDP group: Cisplatin solution(3mg/kg,intraperitoneal injection,qw)+ carboxymethyl solution(gavage,qd);CUR group: Curcumin suspension(100mg/kg,gavage,qd)+ saline solution(intraperitoneal injection,qw);DDP+CUR group: Cisplatin solution(3mg/kg,intraperitoneal injection,qw)+ curcumin suspension(100mg/kg,gavage,qd).After 25 days of continuous administration with different drugs,the length,diameter and weight of the tumor were measured every other day.The volume of the transplanted tumor was measured.(5)A549 and A549/DDP cells were routinely cultured in vitro.Cell viability,growth curve,and doubling time were measured,and total RNA,including mi RNA,was extracted and purified.mi RNA chip was used to detect the difference of mi RNA expression profiles between A549 and A549/DDP cell lines.Results: 1.MTT assay results showed that cisplatin remarkably inhibited the survival rate of A549 and A549/DDP cells in a concentration-dependent manner.The IC50 of cisplatin in A549 and A549/DDP cells was 2.3 μg/ml and 11.23 μg/ml,respectively.Curcumin inhibited the survival rate of A549 and A549/DDP cells in a concentrationdependent manner.The IC50 of curcumin in A549 and A549/DDP cells was 45.41 μM and 46.25 μM,respectively.Curcumin had no significant inhibitory effect on the suivival of A549 and A549/DDP cells.The IC50 of cisplatin in combination with curcumin in A549 and A549/DDP cells was 2.12 and 3.44,respectively.Curcumin combined with cisplatin significantly increase inhibitory effect of cisplatin on the survival rate of A549/DDP cells,but not A549 cells.2.Crystal Violet staining results showed that 12.5μM curcumin could not inhibit the proliferation of A549 cells,but 2μg/ml cisplatin and 2μg/ml cisplatin combined with 12.5μM curcumin could remarkably inhibit the proliferation of A549 cells.12.5 μM curcumin and 12 μg/ml cisplatin could not inhibit the proliferation of A549/DDP cells,but 12 μg/ml cisplatin combined with 12.5 μM curcumin could significantly inhibit the proliferation of A549/DDP cells.Curcumin combined with cisplatin significantly increased inhibitory effect of cisplatin on the proliferation rate of A549/DDP cells,but not A549 cells.3.Flow cytometry results showed that the apoptosis rate of A549 cells in the control group,DDP group,CUR group,and DDP+CUR group were 1.92±0.88%,12.90±0.33%,10.40±1.49%,and 14.88±1.00%,respectively.The apoptosis rate of A549 cells in DDP group,CUR group,and DDP+CUR group were significantly higher than that in the control group(P<0.05).There was no significant difference in the apoptosis rate of A549 cells between DDP+CUR group and DDP group.The apoptosis rate of A549/DDP cells in the control group,DDP group,CUR group,and DDP+CUR group were 1.69±0.80%,8.83±1.04%,7.70±0.32%,and 22.83±0.81%,respectively.The apoptosis rate of A549/DDP cells in DDP group,CUR group and DDP+CUR group was significantly higher than that in control group.The apoptosis rate of A549/DDP cells in DDP+CUR group was significantly higher than that in DDP group(P<0.05).4.Nude mice were subcutaneously transplanted with A549 cells to construct xenografts,and each group was treated with corresponding drugs for 25 days.Control(1309.75 + 594.52)mm3,DDP(980.46 + 143.64)mm3,CUR(1209.55 + 67.65)mm3 and DDP+CUR(973.25 + 84.23)mm3,the volume of tumor in DDP and DDP+CUR group was significantly smaller than that of the control group(p<0.05).There was no significant difference between the CUR group and the control group.There was no significant difference between the DDP+CUR group and the DDP group.The tumor volume of the nude mice with A549/DDP cells in each group were as follows: the control group(1712.45 + 483.39)mm3,DDP(1572.33 + 263.11)mm3,CUR(1585.93 + 427.64)mm3,DDP+CUR(1088.25 + 360)mm3,and there was no significant difference in tumor volume between the DDP group,CUR group and the control group.However,the tumor volume of DDP+CUR group was significantly lower than that in the control group,DDP group and CUR group(P<0.05).It is suggested that curcumin can not inhibit the growth of A549 cell and A549/DDP cell xenografts,and curcumin can not enhance the inhibitory effect of cisplatin on the growth of A549 cell xenografts,but can significantly enhance the inhibitory effect of cisplatin on the growth of Cisplantin resistance lung cancer cell line,A549/DDP cell,nude mouse xenografts.5.Results of mi RNA chip :(1)there was significant difference in mi RNA expression profile between A549 and A549/DDP;(2)there were 17 differential expression mi RNAs between A549 and A549/DDP cells,which 12 were up regulated and 5 were down regulated.(3)the regulation of mi RNA on the cisplatin resistance of lung cancer cells is a complex biological process involving multiple mi RNAsConclusions:(1)Curcumin significantly enhanced the sensitivity of A549/DDP cells to cisplatin and reversed cisplatin resistance in A549/DDP cells;(2)Curcumin can significantly enhance the sensitivity of A549/DDP cells to cisplatin and enhance the inhibitory effect of cisplatin on the growth of A549/DDP cell xenografts.(3)the differential mi RNAs between the A549 cells and A549/DDP cells might be related to the Cisplantin resistance of lung cancer. |
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