[The role of mitogen-activated protein kinase cascades in inhibition of proliferation in human prostate carcinoma cells by raloxifene: an in vitro experiment].
OBJECTIVE
To investigate the role of mitogen-activated protein kinase (MAPK) in the apoptosis and cell cycle arrest of human prostate carcinoma cells induced by raloxifene (RAL).


METHODS
Human prostate carcinoma cells of the line PC3 were cultured. RAL of the concentrations of 10(-4), 10(-5), 10(-6), and 10(-7) mol/L were added into the culture fluid. MTT method was used to detect the inhibitory rate of the PC3 proliferation. RAL of the concentrations of 10(-6) mol/L or 10(-6) mol/L +10 micromol/L PD98059, a MEK1/2 inhibitor, and 10(-6) mol/L RAL +10 micromol/L SB203580, JNK inhibitor, and RAL + SB203580, a p38 inhibitor were added respectively for 48 h, and the flow cytometry (FCM) was used to detect the cell cycle. The cell apoptosis percentage was measured by TUNEL staining. The activation of extra cellular regulated protein kinases (ERK1/2), c-Jun N-terminal protein kinase (JNK), and p38 mitogen-activated protein kinase (p38), and the expressions of Bcl-2, Bax, phospho-Bcl-2 (p-Bcl-2), and caspase-3 were determined by Western blotting. The expressions of estrogen receptor (ER) alpha, ERbeta, cyclin dependent kinase inhibitor (P21(WAF1)), and cyclinD1 mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR).


RESULTS
A dose-dependent proliferation inhibition of RAL was demonstrated in the PC3 cells. A G(1) cell cycle arrest and apoptosis were induced in the PC3 cells exposed to 10(-6) mol/L RAL. 10(-6) mol/L RAL induced the activation of ERK1/2 and p38 with different time courses, but it did not induce the activation of JNK. Suppression ERK1/2 activation by treatment with PD98059 or p38 activation by treatment with SB203580 attenuated the cell-cycle arrest at the G(1) phase respectively. 48 h after the treatment of 10(-6) mol/L RAL the PC3 cells was arrested at G(1) stage, however, 48 h after the treatment of 10(-6) mol/L RAL +10 micromol/L PD98059 and 10(-6) mol/L RAL +10 micromol/L SB203580 the degree of PC3 cell arrest at the G(1) stage was lower. 18 h after the treatment of 10(-6) mol/L RAL, the expressions of cyclinD1 and P21(WAF1) gene were (0.50 +/- 0.02) and (4.48 +/- 0.12) times that of the control group, and the expressions of cyclinD1 and P21(WAF1) gene of the SB203580 pretreatment group and SB203580 pretreatment groups were (2.36 +/- 0.08) and (4.50 +/- 0.03) times, and (0.49 +/- 0.02) and (1.77 +/- 0.06) times respectively. 48 h after treatment with 10(-6) mol/L RAL, the apoptosis rates were 22.9% +/- 1.5%, significantly higher than those of the 10(-6) mol/L RAL +10 micromol/L PD98059 and 10(-6) mol/L RAL +10 micromol/L SB203580 groups (15.2% +/- 1.8% and 9.7% +/- 0.6% respectively, both P < 0.05). The expression of Bcl-2, Bax and caspase-3 (control, 10(-6) mol/L raloxifene, 10(-6) mol/L raloxifene +10 micromol/L PD98059 and 10(-6) mol/L raloxifene +10 micromol/L SB203580) was 1, 0.33 +/- 0.02, 0.34 +/- 0.01, 0.81 +/- 0.05; 1, 3.14 +/- 0.02, 1.67 +/- 0.11, 3.15 +/- 0.03; 1, 4.16 +/- 0.02, 2.66 +/- 0.03, 1.80 +/- 0.06, respectively. Western blotting showed that 48 h after the treatment of 10(-6) mol/L RAL the expression of Bcl-2 decreased, and the expression of caspace-3 increased. Pretreatment with PD98059 inhibited the increase of caspase-3 and Bax expression induced by RAL. 1.5 h after the treatment with RAL the p- Bcl-2 expression increased remarkable, pretreatment with SB203580 inhibited the p- Bcl-2 expression induced by RAL, and however, PD98059 did not show such effect.


CONCLUSIONS
RAL inhibits the proliferation and induces the apoptosis and G(1) cell cycle arrest via MAPK cascades in human prostate carcinoma cells. Up-regulation of P21(WAF1) mRNA expression by activating ERK1/2 and down-regulation of cyclinD1 by activating p38 induces G(1) cell cycle arrest in the human prostate carcinoma cells. The ERK1/2 or p38 cascade is involved in the induction of apoptosis by RAL. The activation of ERK1/2 increases the expression of Bax. The activation of p38 phosphorylates Bcl-2 then inactivates Bcl-2.
Zhang
Yu-Xi
YX
Department of Urology, First Affiliated Hospital, China Medical University, Shenyang 110001, China.
Kong
Chui-Ze
CZ
D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D050071:Bone Density Conservation Agents / Q000494:pharmacology; D053148:Caspase 3 / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects*; D019938:Cyclin D1 / Q000235:genetics; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology; D020287:In Situ Nick-End Labeling; D048031:JNK Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D011471:Prostatic Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D011725:Pyridines / Q000494:pharmacology; D020849:Raloxifene Hydrochloride / Q000494:pharmacology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D050071:Bone Density Conservation Agents; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004791:Enzyme Inhibitors; D005419:Flavonoids; D007093:Imidazoles; D019253:Proto-Oncogene Proteins c-bcl-2; D011725:Pyridines; D019938:Cyclin D1; D020849:Raloxifene Hydrochloride; D048031:JNK Mitogen-Activated Protein Kinases; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D053148:Caspase 3; C093642:SB 203580; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one