(2)プロゲステロン受容体モデュレーターならびにGnRH antagonistによるヒト子宮筋腫細胞発育制御機構の解析(<特集>第59回シンポジウム1「子宮筋腫の発育制御とその管理」)

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  • Analysis of the Mechanisms by Which Selective Progesterone Receptor Modulator and GnRH Antagonist Regulate the Growth of Cultured Human Uterine Leiomyoma Cells

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The objective of the study was to elucidate the effects of gonadotropin-releasing hormone(GnRH) antagonist and selective progesterone receptor modulator(SRPM) on the growth of cultured human uterine leiomyoma cells. Isolated leiomyoma cells and myometrial cells were subcultured in phenol red-free DMEM supplemented with 10% FBS for 120h, and stepped down to serum-free conditions with or without GnRH antagonist and SPRM. We examined the effects of GnRH antagonist Cetrorelix on the proliferation, apoptosis, and epidermal growth factor(EGF) expression in cultured leiomyoma cells. GnRH receptor mRNA was found to be expressed in the cells by using RT-PCR. Compared with untreated control cultures, the treatment with Cetrorelix significantly decreased the number of viable cells and proliferating cell nuclear antigen(PCNA) levels in cultured leiomyoma cells. Treatment with 10^<-5>mol/L Cetrorelix significantly increased the TUNEL-positive rate in the cells at 24 and 48h. Treatment with 10^<-5>mol/L Cetrorelix decreased immunoreactive EGF protein levels in the cells at 2 and 4 days. We next examined the effects of SPRM(asoprisnil) on the growth of cultured leiomyoma and myometrial cells. Treatment with asoprisnil decreased the viable cell number and increased the TUNEL-positive rate in cultured leiomyoma cells. Treatment with asoprisnil significantly decreased Bcl-2 and increased cleaved caspase-3 protein contents in cultured leiomyoma cells. In cultured myometrial cells, asoprisnil did not affect cell proliferation and apoptosis. We examined whether asoprisnil activates tumor necrosis factor-related apoptosis-inducing ligand(TRAIL)-mediated apoptotic pathway in cultured uterine leiomyoma and myometrial cells. Asoprisnil treatment significantly increased TRAIL, death receptor(DR)4, and DR5 protein contents in cultured leiomyoma cells with a cleavage of caspase-8, -7, and -3, and a decrease in X-linked chromosome-linked inhibitor of apoptosis protein(XIAP) contents. In cultured myometrial cells, asoprisnil treatment did not affect TRAIL signaling pathway and XIAP protein contents. We examined whether asoprisnil induces endoplasmic reticulum(ER) stress in cultured leiomyoma cells. RNA interference of growth-arrest-and DNA-damage-inducible gene 153(GADD153) was performed using small interfering RNA. The treatment with 10^<-7>M asoprisnil significantly increased the protein contents of phospho-double-stranded RNA-activated protein kinase-like ER kinase, phosphoeukaryotic initiation factor 2α, activating transcription factor 4, glucose-regulated protein 78KDa at 4h, followed by the significant increase in GADD 153 protein content from 6h. RNA interference of GADD 153 suppressed the levels of asoprisnil-induced poly (adenosine 5'-diphosphate-ribose) polymerase, Bax, and Bak in the cells, and attenuated asoprisnil-induced reduction in Bcl-2 protein content. These results suggest that GnRH antagonist directly inhibits leiomyoma cell growth by downregulating proliferation in association with a decrease in EGF expression and by up-regulating apoptosis, and that SPRM inhibits the growth of cultured leiomyoma cells by inducing apoptosis through activating TRAIL-mediated pathway and ER stress, and by down-regulating Bcl-2 and XIAP protein contents.

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