Apoptosis Induction and Clusterin Expression of NRP-152 Cells by Tamsulosin

PURPOSE: The aim of this study was to know whether and how tamsulosin induces apoptosis of normal rat prostate cells, and the relationship between apoptosis and clusterin expression. MATERIALS AND METHODS: We used a prostate cell line, NRP-152 cells which are the basal epithelium cell originated from rat prostate. The NRP-152 cells were treated with various concentrations(50, 100, 200, 400 uM) of tamsulosin for 24 h. To evaluate apoptosis, the cultured NRP-152 cells were stained with Heochst 33258 and Propidium Iodide (PI) without fixation. We also examined DNA fragmentation analysis to confirm apoptosis. In addition, to elucidate the signal transduction pathway by which apoptosis is induced, we examined Bcl-2 family proteins such as Bcl-2, Bax, Bad, Bcl-xL, and Bim by real-time RT-PCR. RESULTS: After tamsulosin treatment, the rate of apoptosis was 25% at 100 micrometer, 50% at 200 micrometer, and 63% at 400 micrometer, whereas the rate of necrosis was 10% at 100 micrometer, 38% at 200 micrometer, and 56% at 400 micrometer. DNA fragmentation was also gradually increased and the highest at 400 micrometer, similar to apoptotic cell rates. As a result of real-time RT-PCR, there was significant difference of Bcl-2 and Bim mRNA expression among the groups. Expression of clusterin protein was significantly increased after treatment of tamsulosin, even as low as 50 micrometer concentration. CONCLUSION: These results demonstrate that tamsulosin causes the cell death of NRP-152 cells, displaying low concentration of tamsulosin induces apoptosis, but high concentration occurs necrosis. Bim, a proapoptotic factor of the Bcl-2 family, expression was increased in the cells treated with tamsulosin, whereas Bcl-2 expression was decreased. The present study suggests that clusterin may play a role in the process of apoptosis induced by tamsulosin and Bim could be involved in the apoptosis.

Apoptosis and Peripheral Benzodiazepin Receptor (PBR) Expression in Human Granulosa-Luteal Cells by GnRH-agonist / 대한불임학회지

OBJECTIVE: To investigate whether GnRH-agonist (GnRH-Ag) using in IVF-ET affects apoptosis of human granulosa-luteal cells and expression of peripheral benzodiazepine receptor (PBR) protein involved in the apoptosis of the cells. METHODS: Granulosa-luteal cells obtained during oocyte retrieval were cultured and treated with 10(-5) M GnRH-Ag. Apoptosis of the cells by the treatment was confirmed using DNA fragmentation analysis 24 h after culture. The presence of PBR protein within the cells was examined by immunofluorescence staining and the expression of the protein was analyzed by Western blotting. In addition, it was measured for progesterone and nitric oxide (NO) produced by granulosa-luteal cells after GnRH-Ag treatment. To evaluate the relationship between NO production and PBR expression, sodium nitroprusside (SNP) as a NO donor was added in media and investigated the expression of PBR protein by Western blotting. RESULTS: Apoptosis increased in the granulosa-luteal cells 24 h after GnRH-Ag treatment, whereas the expression of PBR protein significantly decreased. Furthermore, the production of progesterone and nitric oxide (NO) by the cells significantly fell from 12 h after the treatment. In the results of Western blotting after SNP treatment, the expression of PBR protein increased in the treatment with SNP alone to the granulosa-luteal cells, but was suppressed in the treatment with GnRH-Ag and SNP. Additionally, the staining result of PBR protein in the cells showed the even distribution of it through the cell. CONCLUSION: These results demonstrate that GnRH-Ag treatment induces apoptosis, decreasing expression of PBR protein and NO production in human granulosa-luteal cells. The present study suggests that one of the apoptosis mechanism of human granulosa-luteal cells by GnRH-Ag might be a signal transduction pathway via NO and PBR.

GnRH-agonist Induces Apoptosis of Human Granulosa-luteal Cells Via Caspase-3 and -9 and PARP Cleavage / 대한산부인과학회잡지

OBJECTIVE: GnRH-agonist (GnRH-Ag) used in controlled ovarian hyperstimulation (COH) for IVF-ET has been known to affect directly on apoptosis of human ovarian cells, but its mechanism is not clearly understood. Therefore, the purpose of the present study was to investigate whether caspase-3 and -9 activation and poly-(ADP-ribose)-polymerase (PARP) cleavage are involved in the mechanism(s) by which GnRH-Ag induces apoptosis in human granulosa-luteal cells. METHODS: Human granulosa-luteal cells collected from IVF-ET patients were cultured and treated with 10(-6) M GnRH-Ag or saline as a control. To access apoptosis in the cells, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labeling (TUNEL) assay and DNA fragmentation analysis were preformed 24 h after the treatment. Activity of caspase-3 and -9 in the cells was examined by using a fluorogenic substrate. Caspase-3 and -9 activation and poly (ADP-ribose) polymerase (PARP) cleavage were analyzed by Western blotting. RESULTS: DNA fragmentation in the cells increased in the higher concentration over 10(-6) M GnRH-Ag. In the result of TUNEL assay, the rate of apoptotic cells in GnRH-Ag treatment increased significantly compared with that of saline treatment (p<0.05). The activity of caspase-3 and -9 investigated by using a fluorogenic substrate increased only in the apoptotic cells. In Western blot analysis, the cells treated with GnRH-Ag revealed an increase in active forms of caspase-3 and -9 compared with those of the saline treatment. In addition, cleavage of PARP also increased in the cells treated with GnRH-Ag. CONCLUSION: These results suggest that activation of caspase-3 and -9 and cleavage of PARP might be involved in apoptosis of human granulosa-luteal cells induced by GnRH-Ag.