The Study Of Gestrinone Microparticle On Pharmacodynamics And Mechanism For Its Action On Uterine Leiomyoma

AIMTo evaluate the inhibition of gestrinone microspheres on experimental guinea pig model andcultured cells of uterine leiomyoma, and study the action mechanism of gestrinone.METHODS1. We analyzed releasing time in M.fascicularis for single administration of gestrinonemicrioparticle.2. We observed the inhibition on sexual cycle and ovulation on SD rat for singleadministration of gestrinone micrioparticle.3. Guinea pigs were subcutaneously treated with estradiol benzoate (E₂) 50 or 100μg/day,twice or three times a week for 8, 12 and 16 weeks. After the animals were killed, theuteri were dissected out for calculating the weight and making ultrathin sections.Pathohistologic characteristics were observed under the microscope.4. After being oophorectomized, guinea pigs were randomized into groups. The modelgroup was treated with E₂ for 16 weeks. In gestrinone treated groups, animals weretreated with E₂ for 6 weeks in advance, and then in combination with gestrinonemicrioparticle (2, 4 or 8mg/kg) for 10 weeks. Histological examination was performedto evaluate whether there were leiomyoma features in these animals.5. Leiomyoma was obtained from hysterectomy specimens and digested with collagen.Then cells were cultured in medium contain 10% fetal bovine serum. The inhibitoryeffect of gestrinone on the cells was investigated.6. By immunohistochemistry (IHC), real-time RT-PCR and Western blot, we investigatedthe expression of ER, PR and its phosphorylation form in context of E₂ induced animalmodel and E₂-free leiomyoma cells. The effect of gestrinone on the expression of ER,PR and its phosphorylation form was investigated at the same time.7. By flowcytometer, we investigated the effect of gestrinone on cell cycle and apoptosis.8. By microarray with 15 signal pathway, we analyzed the gene expession in the cells ofleiomyoma and the matched myometrium from hysterectomy specimens. The effect ofgestrinone on the gene expression was also examined.9. By immunohistochemistry (IHC), real-time RT-PCR and Western blot, we examinedthe expression of c-Src and its phosphorylation form in context of E₂ induced animalmodel and E₂-free leiomyoma cells. The effect of gestrinone on the expression of ER,PR and its phosphorylation form was also investigated. 10. By immunopricipitaion (IP), we investigated whether there is a crosstalk between ERand c-Src. The effect of gestrinone on the crosstalk was also considered.RESULTS1. The releasing time in M.fascicularis for single administration of gestrinonemicrioparticle (0.6, 2 and 6mg/kg) was about 490hr. The effective concentration wouldmaintaine for 350hr.2. The inhibitory effect ofgestrinone micrioparticle (3.5, 7.0 and 14.0mg/kg) on ovulatingwould maintaine for 2 weeks.3.The uteri treated with estrogen were proliferated and deformated. Uterine nodules wereobserved in most of the tested animals. According to the results of pathohistology,these cells demonstrate the features of smooth muscle-like cells. Morphologicalchanges were observed in myometrium of the guinea pig model, including increasingof uterine weights, proliferation of uterine smooth muscles and the formation ofnodules. Pathologically, the characteristics of leiomyoma were observed in most ofanimals.4. In gestrinone micrioparticle (2, 4 and 8mg/kg, once ten days for 10 weeks ) treatedanimals, there was no nodule observed. The uterine weight decliend. The histologicalfeatures of myometrium are similar to that of the control group. Serm E₂ concentrationdecreased at gestrinone 4 and 8mg/kg.5. In vitro, gestrinone inhibited the growth of E₂-free cells of leiomyoma. The IC50 was26.7μmol/L.6.In cultured E₂-free cells of leiomyoma, ERmRNA and PRmRNA are lower inleiomyoma than that in matched myometrium. However, the protein expression of ER,PR, phospho-¹⁶⁷ER and phospho-¹⁹⁰PR are higher in leiomyoma than that in matchedmyometrium.7.In E₂ induced guinea pig model of uterine leiomyoma, high expressions of ER and PRand its phosphorylation form were observed in the uteri of model group. Lowexpressions of ER and PR and its phosphorylation form were observed in thegestrinone microparticle (2, 4 and 8mg/kg) treated group. In E₂-free cells, gestrinone(0.1～1μmol/L) down-regulated the expressions of PRmRNA, ERαmRNA andERβmRNA. Gestrinone (10μmol/L) down-regulated the expressions of PRmRNA andERαmRNA, while the expression of ERβmRNA up-regulated. Gestrinone(1～10μmol/L) down-regulated the protein expressions of ER, PR, phospho-¹⁶⁷ER andphospho-¹⁹⁰PR. 8. In E₂ induced guinea pig model of uterine leiomyoma, comparing with the normalcontrol group, there was no decreased apotosis rate observed in model group, and nosignificant increased apotosis rate in gestrinone treated group. As showed in E₂-freecells as well. However, there was markedly decreased apotosis rate observed inmefepristone treated group.9.In cultured E₂-free cell of leiomyoma, there was 22 pieces of genes showedup-regulated and 25 pieces of gene down-regulated. The obviously up-regulatedpathway involved hypoxia, inflammation, androgen, WNT, Hedgehog and STAT. Therewas no obvious change on survive (PI3K / AKT) and DNA damage.10. After treatment with gestrinone (3μmol/L), 11 pieces of genes were showedup-regulated and 46 pieces of gene down-regulated. The markedly down-regulatedpathway involved estrogen, hypoxia, inflammation, WNT, Hedgehog and STAT. Therewas no obvious change on survive (PI3K / AKT) and DNA damage.11. Comparing with the normal myometrium, there was obvious positive expression ofc-Src in E₂ induced guinea pig model or cultured E₂-free cell of leiomyoma. Afterbeing treated with gestrinone, the expression of c-Src down-regulated in transcriptionaland protein level.12. Comparing with the normal myometrium, there was marked positive expression ofphospho-⁴¹⁶Src in E₂ induced guinea pig model. After being treated with gestrinone, theexpression of phospho-⁴¹⁶c-Src down-regulated in protein level.13. The preliminary result showed that there may be a cross-talk between ER and c-Src,and gestrinone may interfere with the cross-talk.CONCLUSION1. The animal model for uterine leiomyoma was established successfully after castratedguinea pig was subcutaneously injected E₂ 100gg, twice a week for 12～16weeks.Thepathology of myometrium in guinea pig is similar to that of human being.2. Gestrinone microparticle delayed release in M.fascicularis and SD rat.3.Gestrinone 2, 4 and 8mg/kg (once ten days, for 10 weeks) are capable of inhibiting thegrowth of uterine leiomyoma in guinea pig model.4. In cultured E₂-free cell of leiomyoma, gestrinone is capable of inhibiting the growth ofuterine leiomyoma cell in vitro.5. In E₂ induced guinea pig model of uterine leiomyoma, the expression of ER, PR andPRB decreased in gestrinone treated group. In cultured E₂-free cell of leiomyoma,phosphorylation of ER and PR are independent of E₂. Gestrinone is capable of inhibiting the transcriptional and protein expression of ER, PR, phospho-¹⁶⁷ER andphospho-¹⁹⁰PR.6. The inhibition of gestrinone on leiomyoma may independent on the process ofapoptosis.7. The mechanism for gestrinone's action may relates to signal pathway, such as estrogenpathway, hypoxia pathway, inflammation pathway, WNT, Hedgehog and STAT. therewas no obvious change on survive (PI3K / AKT) and DNA damage.8. The up-regulation of c-Src in E₂ induced guinea pig model of uterine leiomyoma andcultured E₂-free cell of leiomyoma indicate that c-Src is associated with the formationof uterine leiomyoma. The expression of c-Src is independent of E₂, while theactivation of c-Src may dependent on E₂. During the growth of leiomyoma, theexpression location of c-Src may move from cytoplasm to nuclear.9. Gestrinone markedly suppresses the protein expression of c-Src andautophosphorylation of c-Src in the E₂-induced leiomyoma model of guinea pig andthe cultured E₂-free cell of leiomyoma.10. There may be a cross-talk between ER and c-Src, and gestrinone would interfere withthe cross-talk.