雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Inhibition of autophagy leads to increased apoptosis in LNCaP cells under androgen deprivation conditions

目的探讨雄激素剥夺条件下阻断自噬后LNCaP细胞凋亡变化与半胱天冬酶（caspase）激活的关系。方法应用激光共聚焦显微镜、RT—PCR方法观察雄激素剥夺致细胞自噬增加后，利用DAPI染色观察细胞凋亡变化及药物抑制caspase后对凋亡的影响。结果①雄激素去除后LNCaP细胞自噬体增加，标准培养基（CM）培养下LNCaP细胞自噬体数量为1．90分；无血清培养基（SF）中细胞自噬体数量增高为2．64分；加入双氢睾酮（SFA组）后细胞自噬体下降至1．85分（P〈o．01）。CM中LNCaP细胞LC3mRNA表达率为23％，血清饥饿12h后，LC3表达量上调至100％，而SFA组LC3mRNA表达量为86％；血清饥饿24h后，SF组LC3mRNA表达量为62％，SFA组为35％。②SF组和SFA组LNCaP细胞基础凋亡率分别为（3．19±1．09）％和（3．01士0．33）％，加入3-甲基腺嘌呤（3-MA）阻断自噬24h后，SF组凋亡率为（10．90±2．91）％，SFA组为（4．63±1．69）％。SF＋3-MA组中加入Z—VAD-FMK后，细胞凋亡减至（1．16±0．52）％，组间差异有统计学意义（P〈O．01）。结论剥夺雄激素后LNCaP细胞中自噬明显增加，阻断自噬后凋亡发生率增加。

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was determined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apoptosis of LNCaP cells after inhibition of autophagic by 3 MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results （1）Androgen deprivation led to increased autophagy in LNCaP cells. LNCaP cells cultured in complete medium（CM） presented low autophagic process with 1.9 scores. After 24 hours, the punctate GFP-LC3 structures were accumulated in the cells cultured in serum-free medi um （SF）（2.64 scores）. In contrast, the number of punctate GFP-LC3 remained at a very low level （1.85 scores）, when cells were incubated with DHT in SFA（serum-free medium＋DHT）. Statistical analysis showed the significant difference between SF and SFA （P〈0.01）. Semiquantitative RT-PCR was employed to examine the mRNA expression of LC3. Indeed, cells grown in the medium without serum had a higher LC3 mRNA expression with the highest at 12 hour time point as compared with the cells grown in CM. DHT treatment reduced the level of LC3 mRNA. （2）Blockage of autopbagy by 3 MA increased the apoptosis of LNCaP cells. LNCaP cells in SF and SFA just presented a basal level of apoptosis, which is （3. 19±1.09）% and （3.01±0.33）% , respectively. Under androgen free conditions, inhibition of autophagy by 3-MA could increase apoptosis significantly（10. 90 ± 2. 91%）. While Z-VAD-FMK, a pan Caspase inhibitor, was able to suppress this apoptotic process to the level of （1.16±0.52）%, which was statistically significant（P〈0.01）. Conclusions Androgen removal can lead to the increase of autophagy in LNCaP cells. Moreover, inhibition of autophagy promotes the occurrence of apoptosis.