JNJ-7706621对人乳腺癌细胞周期及凋亡的影响

  目的   探讨周期素依赖性蛋白激酶（cyclin-dependent kinases，CDKs）抑制剂JNJ-7706621对人乳腺癌细胞周期及凋亡的影响。  方法  常规培养乳腺癌T47D、MD-MB-231细胞，MTT法检测不同浓度JNJ-7706621（0、1、2、4 μM）对乳腺癌细胞增殖的影响；流式细胞术检测JNJ-7706621对细胞周期的影响，Annexin Ⅴ-FITC/PI双染法检测JNJ-7706621对细胞凋亡的影响；West. ern blot检测JNJ-7706621对凋亡及周期相关蛋白表达的影响。  结果  JNJ-7706621能够抑制乳腺癌T47D、MDA-MB-231细胞增殖，具有浓度和时间依赖性；流式细胞术显示，JNJ-7706621可将T47D、MDA-MB-231细胞的周期阻滞于G2/M期，具有浓度依赖性，与对照组相比差异有统计学意义（P < 0.05）；Annexin Ⅴ-FITC/PI双染法示随着JNJ-7706621浓度增加或作用时间的延长，T47D、MDA-MB-231细胞凋亡所占比例逐渐增加，与对照组相比，差异有统计学意义（P < 0.05）；Western blot结果显示，随JNJ-7706621浓度增加，Bcl-2的表达明显下调，Caspase3、PARP的剪切明显增高，而p53的表达无明显变化；周期相关蛋白CDK1的表达无明显变化；CDK1在Thr161位点的磷酸化水平降低，而在Tyr15位点的磷酸化水平增高；  结论   在体外条件下，JNJ-7706621可通过阻滞细胞周期与诱导细胞发生凋亡的方式，有效抑制乳腺癌细胞增殖，可能与抑制CDK1在Thr161位点的磷酸化水平有关。 

Effect of JNJ-7706621 on cell cycle and apoptosis of breast cancer cell lines

  Objective   This study aimed to investigate the effects and the molecular mechanism of JNJ-7706621 on cell cycle and apoptosis of breast cancer cell lines.   Methods   Breast cancer cell lines T47D and MDA-MB-231 were cultured in vitro and treated with JNJ-7706621 at varying concentrations. MTT assay was used to measure cell proliferation. Flow cytometry was applied to analyze the distribution of cell cycle and apoptotic rates. Western blot was performed to analyze the expression of cyclin B1-CDK, the phosphorylation levels of CDK1Thr161 and CDK1Tyr15, as well as the expression of p53, Bcl-2, caspase3 and poly(ADP-ribose) polymerase (PARP).   Results   JNJ-7706621 inhibited the proliferation of the T47D and MDA-MB-231 cells in a dose- and time-dependent manner. Flow cytometry showed that the T47D and the MDA-MB-231 cells in the G2/M-phase significantly increased after treatment at varying concentrations (0, 1, 2, 4 μM) of JNJ-7706621: the G2/M-phase rates at the corresponding concentrations were (12.66±1.55)%, (20.63± 1.32)%, (23.20±1.82)%, and (32.19±2.37)%, respectively, for the T47D cells (P < 0.05), and the G2/M-phase rates were (16.22±1.48)%, (21.45±0.85)%, (25.25±1.26)%, and (31.08±1.16)%, respectively, for the MDA-MB-231 cells (P < 0.05). The rates of apoptotic cells were also significantly increased (P < 0.05). Western blot results indicated that JNJ-7706621 exerted a slight effect on the expression of CDK1 and p53 and that the phosphorylation level of CDK1 decreased at the Thr161 site but increased at the Tyr15 site. In addition, cleaved caspase3 and PARP increased, whereas Bcl-2 and cyclinB1 decreased significantly.   Conclusion  JNJ-7706621 can significantly inhibit the proliferation of breast cancer cell lines T47D and MDA-MB-231 by inducing cell cycle arrest and apoptosis, which may be associated with the downregulation of the CDK1 phosphorylation at the Thr161 site.