mtt-formazan and Leiomyoma

The levonorgestrel intrauterine system (LNG-IUS) is a widely recognized intrauterine anti-fertility system, which can alleviate symptoms of uterine leiomyoma. This study aims to evaluate leimyoma cell growth inhibition induced by high concentrations of LNG.. After treatment with LNG, the growth rate of the cultured primary uterine leiomyoma cells was studied with methyl thiazolyl tetrazolium (MTT) assay. Cell apoptosis rate was determined by morphological changes and flow cytometry. Reverse transcription polymerase chain reaction (RT-PCR) and Western blotting were performed to measure the differential mRNA and protein expression levels.. The proliferation rate of uterine leiomyoma cells was suppressed after treatment with LNG at a minimum concentration of 10 mcg/mL. The inhibitive effect was positively correlated with the LNG concentration and with the incubation time. Flow cytometry showed that the apoptosis rate was increased with the LNG concentration. The mRNA levels of IGF-1, Bcl-2 and survivin were down-regulated significantly after treatment with 10 mcg/mL LNG. Western blot analysis confirmed that the expression of Bcl-2 and survivin was decreased significantly, and the p38 phosphorylation level was increased and caspase 3 was activated remarkably 72 h after treatment with 10 and 20 mcg/mL LNG.. This study demonstrated that LNG may suppress the proliferation and induce apoptosis of the uterine leiomyoma cells.

Topics: Adult; Apoptosis; Cell Proliferation; Contraceptive Agents, Female; Female; Formazans; Histocytochemistry; Humans; Inhibitor of Apoptosis Proteins; Insulin-Like Growth Factor I; Leiomyoma; Levonorgestrel; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Middle Aged; Proto-Oncogene Proteins c-bcl-2; Reverse Transcriptase Polymerase Chain Reaction; RNA, Neoplasm; Survivin; Tetrazolium Salts; Tumor Cells, Cultured; Uterine Neoplasms

The objectives of the present study were to evaluate the expression level of ATP-sensitive potassium (K(ATP)) channels in smooth muscle cells in human uterine leiomyoma and the involvement of the channel in potentiating effect of estrogen on leiomyoma growth.. Reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR and Western blot were used for the identification and quantification of K(ATP)-channel subunits in the control myometrial and leiomyoma cells. Furthermore, we measured the K(ATP)-channel activity in enzymatically isolated single uterine smooth muscle cells by whole-cell patch-clamp recordings. The estrogen-induced cell proliferation in leiomyoma was measured by the MTT assay.. The subunits of K(ATP) channels (Kir6.1, Kir6.2, SUR2B) were more highly expressed in leiomyoma cells than in control cells. The whole-cell currents mainly through K(ATP) channels were also greater in the leiomyoma cells. Estrogen applied in the bath solution could acutely enhance the channel activity. Estrogen-induced proliferation of the leiomyoma cells was inhibited by pretreatment with glibenclamide, a K(ATP)-channel inhibitor.. Estrogen may induce the proliferation of leiomyoma cells, at least in part, by activating the K(ATP) channel. Increased expression of the K(ATP) channel may be a causal factor for the high growth rate of uterine leiomyoma.

Topics: Adult; Cell Growth Processes; Estradiol; Estrogen Receptor alpha; Estrogen Receptor beta; Female; Formazans; Glyburide; Humans; Immunoblotting; KATP Channels; Leiomyoma; Middle Aged; Patch-Clamp Techniques; Pinacidil; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazolium Salts; Up-Regulation; Uterine Neoplasms