莪术醇对人胃癌细胞株BGC823增殖的影响及其机制

Effect of curcumol on proliferation of human gastric carcinoma cell line BGC823 and mechanism

目的观察莪术醇对人胃癌细胞株BGC823细胞周期的影响，并探讨其作用机制。方法体外培养BGC823细胞，分别加入12．5、25．0、50．0、100．0mg/L莪术醇，对照组加入10ml／L无水乙醇，分别孵育24、48h。采用噻唑蓝（MTT）法、克隆形成实验分析莪术醇对BGC823细胞增殖的影响。流式细胞术测定细胞周期的变化。100mg／L莪术醇孵育BGC823细胞48h后，收集细胞，反转录-聚合酶链反应（RT-PCR）测定增殖细胞核抗原（PCNA）、细胞周期蛋白D1（CyclinD1）、细胞周期蛋白依赖性激酶4（CDK4）和p16mRNA的表达，Westernblot测定PCNA、CyclinD1、CDK4和p16蛋白的表达水平。结果MTT结果显示，莪术醇剂量和时间依赖性抑制BGC823细胞增殖，莪术醇为12．5、25．0、50．0、100．0mg／L时对BGC823细胞抑制率在24h分别为（13．59±2．74）％、（22．51±4．62）％、（46．09±6．14）％、（73．67±8．24）％，在48h分别为（15．02±2．26）％、（25．31±4．79）％、（53．62±6．37）％、（82．14±9．65）％；克隆形成实验结果显示，莪术醇具有剂量依赖性抑制BGC823细胞克隆形成率的能力，莪术醇为12．5、25．0、50．0、100．0mg／L作用14d时BGC823细胞克隆形成率为（0．88±0．14）％、（0．57±0．09）％、（0．36±0．06）％、（0．11±0．02）％；流式细胞结果显示，莪术醇处理48h后，G0／G1期的细胞均显著增加，S期细胞明显减少，且随浓度增高变化更为明显（P〈0．05）；RT-PCR和Westernblot结果显示，100mg／L莪术醇孵育BGC823细胞48h后，PCNA和CyclinD、CDK4mRNA和蛋白表达显著下降，p16mRNA和蛋白表达显著上升（P〈0．05）。PCNA、CyclinD1、CDK4、p16的mRNA在对照组相对表达量为0．64±0．16、0．57±0．12、0．46±0．06、0．26±0．04，在莪术醇组为0．31±0．07、0．19±0．05、0．15±0．04、0．59±0．09；各指标蛋白的相对表达量在对照组为0．84±0．21、0．62±0．17、0．74±0．19、0．35±0．06，在莪术醇组为0．43±0．09、0．26±0．07、0．22±0．06、0．64±0．16。结论莪术醇可使BGC823细胞周期阻滞于Gn／G，期而抑制其增殖，其主要机制可能与下调PCNA、CyclinD和CDK4表达、上调p16表达有关。

Objective To investigate the effect of curcamol on cell cycle of human gastric carcino- ma cell BGC823 and the mechanism. Methods Cultured BGC823 cells were treated with different concen- trations of curcumol （ 12. 5, 25.0, 50.0 and 100.0 mg/L） for different durations （24 and 48 h）, and the growth inhibition was tested by methyl thiazol tetrazolium （MTT） assay and colony formatting assay. Flow cytometry （FCM） was used to measure cell cycle of BGC823 cells. After treatment with 100 mg/L curcu- mol and culture for 48 h, cells were collected and subjected to reverse transcriptase - polymerase chain re- action （ RT - PCR） and Western blotting for detection of the mRNA and protein expression of proliferating cell nuclear antigen （PCNA）, Cyclin D1, cyclin - dependent kinase 4 （CDK4） and p16. Results The MTY assay showed that curcumol could greatly inhibit the growth and proliferation of BGC823 cells in a dose - and time - dependent manner （ P 〈 0.05 ）. When the concentration of curcumol was 12. 5, 25.0, 50.0 and 100.0 mg/L, the inhibition rate of BGC823 was （13.59 2.74）%, （22.51 ±4.62）%, （46. 09 ± 6. 14） % and （73.67± 8.24） % at 24 h, and that was （ 15.02± 2.26） %, （25. 31 ± 4. 79） %, （53.62 ±6. 37）% and （82. 14 ±9. 65）% at 48 h, respectively. Clone formation assay revealed that cur-cumol could greatly inhibit the colony formation rate of BGC823 cells in a dose - dependent manner （ P 〈 0. 05 ）. When the concentration of eurcumol was 12. 5, 25.0, 50. 0 and 100. 0 mg/L, the colony formation rate was （0. 88±0. 14）%, （0. 57 ±0. 09）%, （0. 36 ±0. 06）% and （0. 11 ±0. 02）% respectively at 14 day. FCM showed that the cell number of G0/G1 phase was increased, while that of S phase was decreased after BGC823 cells were treated with eurcumol for 48 h （ P 〈 0. 05 ）. Reverse transcriptase - polymerase chain reaction （RT- PCR） and Western blotting analyses indicated that curcumol decreased PCNA, Cyc- lin D1 and CDK4 expression as well as increased p16 expression （P 〈 0. 05 ）. The relative expression of PCNA, Cyclin D1, CDK4, and p16 mRNA was 0. 64 ±0. 16, 0. 57 ±0. 12, 0. 46±0. 06 and 0. 26±0. 04 in control group, and that in curcumol group was 0. 31 ± 0. 07, 0. 19± 0. 05, 0. 15 ± 0. 04 and 0. 59 ±0. 09 respectively. The relative expression of PCNA, Cyclin D1, CDK4, and p16 protein was 0. 84 ± 0. 21,0. 62± 0. 17, 0. 74 ± 0. 19 and 0. 35 ±0. 06 in control group, and that in eurcumol group was 0.43 ±0.09, 0.26 ±0.07, 0.22 ±0.06 and 0.64 ±0. 16 respectively. Conclusion Curcumol induces BGC823 cell cycle arrest in G0/Gl phase, which may be related with the down - regulation of PCNA, Cyc- lin D1 and CDK4 as well as the up - regulation of p16.