雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate 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. ②Blockage of autophagy 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 con-ditions, 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.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate 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. ②Blockage of autophagy 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 con-ditions, 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.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate 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. ②Blockage of autophagy 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 con-ditions, 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.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate 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. ②Blockage of autophagy 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 con-ditions, 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.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate 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. ②Blockage of autophagy 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 con-ditions, 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.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate 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. ②Blockage of autophagy 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 con-ditions, 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.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate 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. ②Blockage of autophagy 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 con-ditions, 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.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate 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. ②Blockage of autophagy 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 con-ditions, 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.     

雄激素剥夺条件下阻断自噬增加LNCaP细胞凋亡

Objective To investigate the change of apoptosis in LNCaP cells after inhibition of autophagic process under androgen removal conditions. Methods The autophagic level was deter-mined by using confocal microscopy and RT-PCR. The DAPI staining was used to indicate the apopto-sis of LNCaP cells after inhibition of autophagic by 3-MA. Also, Z-VAD-FMK was used to extend the apoptosis results. Results ①Androgen deprivation led to increased autophagy in LNCaP cells. LN-CaP cells cultured in complete medium(CM) presented low autophagic process with 1.9 scores. After 24 hours, the punetate 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. ②Blockage of autophagy 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 con-ditions, 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.     

Apogossypolone, a novel inhibitor of anfiapoptotic Bcl-2 family proteins, induces autophagy of PC-3 and LNCaP prostate cancer cells in vitro

Limited treatment options are available for aggressive prostate cancer. Gossypol has been reported to have a potent anticancer activity in many types of cancer. It can increase the sensitivity of cancer cells to alkylating agents, diminish multidrug resistance and decrease metastasis. Whether or not it can induce autophagy in cancer cells has not yet been determined. Here we investigated the antiproliferative activity of apogossypolone （ApoG2） and （-）-gossypol on the human prostate cancer cell line PC3 and LNCaP in vitro. Exposure of PC-3 and LNCaP cells to ApoG2 resulted in several specific features characteristic of autophagy, including the appearance of membranous vacuoles in the cytoplasm and formation of acidic vesicular organelles. Expression of autophagy-associated LC3-Ⅱ and beclin-1 increased in both cell lines after treatment. Inhibition of autophagy with 3-methyladenine promoted apoptosis of both cell types. Taken together, these data demonstrated that induction of autophagy could represent a defense mechanism against apoptosis in human prostate cancer cells.