Anti-proliferative And Induced Apoptotic Effects Of Levonorgestrel On Human Uterine Leiomyoma Cells In Vitro

Uterine leiomyoma is benign smooth muscle cell tumor of the myometrium,occurring in as many as 30%adult women.Homeostatic control of the net growth of tumors is thought to be the result of the dynamic balance between cell proliferation and cell death;too much growth can come from too little death as well as from too much proliferation.Actually,apoptosis is known to occur in tumors either spontaneously or in response to treatment.Recently,it has been found that the use of LNG-IUS is effective in reduction of uterine myoma volumes.To explore the mechanisms of inhibitory effect of high level levonorgestrel on uterine leiomyomas growth,our researches obtained primarily cultured uterine leiomyoma cells,and then observed the effects of levonorgestrel on proliferation and apoptosis in it.一,Primary culture and identification of human uterine leiomyoma cellsBy explant culture,cells could be seen emigrating from the edge of tissue pieces after 7-10 days,and subcultured after about half of a month;Whereas cells obtained by digestive method having an initial viability of 90%by dye exclusion.Cells morphologically appeared spindle-shaped with a big oviform nuclei and grew in the "hill-valley" mode.There weren't significant differences in doubling time of cells at the first,third and ninth passages.Immunohistochemical study revealed strong expression ofα-SMA,showing that the purity of primary cells was above 95%.In conclusion,We have successfully developed a good in vitro model of primary leiomyoma cells with high purity and stability either by explant or digestive method.二,Effects of levonorgestrel on proliferation and apoptosis in human uterine leiomyoma cells in vitroMTT assay demonstrated that the inhibitory effect of levonorgestrel on UtLMCs growth developed when the concentration was up to 10μg/mL and was in a dose and time dependent manner.The resultes of FCM analysis showed that levonorgestrel treatment could induce a substantial apoptotic response in UtLMCs,which was enhanced with the raise of levonorgestrel.The earlier apoptotic changes,such as endoplasmic vacuolization and condensed chromatin,were observed under TEM in LNG treated cells.AO/EB double staining disclosed that there were more viable apoptotic cells in LNG treated groups than control group and the number of non-viable apoptotic cells and dead cells increased with dose increasing.Thus,certain concentration of levonorgestrel resulted in a decrease in proliferation and an increase in apoptosis in UtLMCs.三,The mechanism of anti-proliferative and apoptotic effects of levonorgestrel on human uterine leiomyoma cells in vitroSurvivin expression in LNG treated cells was decreased both at mRNA and protein levels according to RT-PCR and Western blot results. It had been observed by RT-PCR that the IGF-Ⅰexpression decreased with LNG addition.Western blot analyses showed that phosphorylated p38 and Caspase 3 expressions were up-regulated by 10μg/mL and 20 μg/mL LNG treatment.So,the molecular mechanism by which levonorgestrel participate in the inhibition of human uterine leiomyoma growth might be due to the marked down-regulation of IGF-Ⅰand Survivin,as well as the direct activation of p38 MAPK and Caspase 3.In summary,based on the model of primary cultured human uterine leiomyoma cells,we demonstrated that LNG could efficiently inhibit the proliferation of leiomyoma cells and induce cell apoptosis,either by down-regulating IGF-Ⅰ,Survivin expressions,or activating p38 MAPK and Caspase 3.