The Clinical And Experimental Research On The Treatment Of Endometriosis With Thiostrepton

BackgroundEndometriosis is a chronic estrogen-dependent disease described as the presence of epithelium and stroma outside the uterine cavity. As the most common gynecological disorder, it affects at least 10%-15% women in their reproductive-aged and is associated with chronic pelvic pain, dysmenorrheal and infertility. 35% to 50% of chronic pelvic pain and infertile women were diagnosed as endometriosis and the incidence of endometriosis is rising in recent years. A large number of evidence indicates that oestrogen plays an important role in the establishment and development of ectopic endometrium. However, the pathogenesis of endometriosis remains unclear.Variety of theories have been proposed, including:(1) endometrium implantation theory,which consists of retrograde menstruation and the spread of lymphatic and venous. However, retrograde menstruation occurs in 76%～90% of women with menstruation eddy through fallopian tubes and only a few of these women have endometriosis. The theory is failed to explain the endometriosis of adolescent and newborn. (2) coelomic metaplasia and induction theory, which postulates that endometriosis originates from the metaplasia of the cells which present in the mesothelial lining of the visceral and abdominal peritoneum. Hormonal or immunological factors are thought to stimulate the transformation of normal peritoneal tissue into endometrioid tissue. Nonetheless,oestrogen is not high in the adolescent girls and therefore it could explain part of them. (3) dysplasia of mulerian duct. (4) chromosome and gene abnormalities theory which is on the basic of familial aggregation of endometriosis, such as chromosome abnormality and abnormal epigenetic modifications have been found in ectopic tissue. (5) oxidative stress and inflmmation. Increased oxidation of lipoproteins has been associated with the pathogenesis of endometriosis, where reactive oxygen species causes lipid peroxidation that leads to DNA damage. The excess production of reactive oxygen species is also accompanied by a decreased level of antioxidants that lead to the delelopment of ectopic endometrium. The ectopic endometrium also secrete large amounts of macrophage, prostaglandin,cytokines, chemokine and so on. (6) other theories and factors such as stem cells and apoptosis suppression. The pathogenesis of endometriosis is so complicated that the theories above are unable to explain clearly.The histological connection between endometriosis and cancer has first been postulated long before 1925. Sampson based on microscopic observations, put forward that endometrial ovarian cancer may develop from endometriotic tissue and described the criteria for diagnosis of endometriosis-associated ovarian cancer,including:(1) evidence of endometriosis close to the tumor, (2) exclusion of invasion from other origins, (3) presence of tissue resembling endometrial stoma surrounding characteristic epithelial glands. Scott added a fourth criterion that histological evidence of transform from benign changes in endometriosis to malignant changes in cancer in 1953. Nowadays, all four criteria are still in use to define endometriosis-associated malignancy (EAM). Various of mutations in tumor suppressor genes and oncogenes have been discovered in the ectopic lesions, which strongly support endometriosis as the precancerous condition of endometriosis-associated ovarian cancer. Accumulation of evidence indicates the relationship between endometriosis and cancer,especially clear cell ovarian cancer. So the change of tumor suppressor genes and oncogenes may lead to abnormal proliferation in ectopic endometrial tissues. Numerous mutations in tumor suppressor genes linked to carcinogenesis have been identified in both endometriotic lesions and ovarian cancer, such as PTEN, p53, Bcl. ARID1A and TP53, therefore endometriosis is known as the instability of genetics.There have been growing evidences indicated abnormal epigenetic modifications plays an important role in the pathogenesis of endometriosis, which consist of dysregulated of DNA methylation, microRNA expression and histone modification. Take aberrant histone deacetylase modification for example, H3K27me3 is the mostly studied histone modification, which plays a significant role in silence gene transcription, stem cell differentiation and tumorigenesis. Janice B and his team put forward that high methylation level of histone 3 in ectopic endometrial tissues, which included of H3K4, H4K9, H3K27,and high acetoxylation of promoter in steroids generated factor 1 (SF1).Nowadays, there are two primary theories which attempt to explain the origin of endometriosis-associated malignancies. Scholars reported endometriosis-associated ovarian cancer (EAOC) may arise from atypical endometriosis of the ovary according to numbers of histopathological studies. This heterogeneous expression is histologically characterized by proliferation of endometrial glands with cytological heterogeneity or presence of atypical hobnail cells within ovarian endometriosis.A direct link between atypical endometriosis and ovarian cancer was first reported by La Grenade and Silverberg in 1988. Five cases of ovarian cancer,which included of three clear cell carcinomas and two endometrioid carcinomas, accompanying atypical endometriosis was found in the ovary and in four of the cases atypical changes were in contiguity with ovarian cancer.Besides that, a significantly higher rate of cytological atypia within endometriotic tissue was demonstrated in cases of endometriosis-associated ovarian cancer compared with endometriosis alone.Although the hypothesis that atypical endometriosis may be a premalignant condition is supported by the fact that it can be detected in up to 80% of endometriosis-associated ovarian cancer, there are still not enough evidence to prove this conclusion. Some researches suggest that endometriosis and endometriosis-associated ovarian cancer share the similar mediums and cytokines in their own microenvironment. Whether ndometriosis-associated malignancy rises by malignant transformation of endometrial cells through intermediary state, called atypical endometriosis, or the association of similarity of microenviroment between them have not been definitely clarified.Although endometriosis is considered to be a benign lesion, it shares malignant biological behavior similar to tumours, such as proliferation and metastases, attachment, invasion and destroy of adjacent tissues subsequently. Unlike tumours, endometriosis does not cause metabolic change or even lead to death. Studies had showed that female who suffer from endometriosis increases the risk of endometrial ovarian cancer and clear cell carcinoma.FOXM1 is affluent in epithelial ovarian cancer and is associated with the low survival rate and drug resistance. Current treatment modalities may be divided into symptomatic and fertility-based therapies, such as surgical and conventional hormonal treatments. All of these treatment modalities have limited success and various side effects. However, the rates of relapse are high after the cessation of such therapies. Forkhead Box M1 (FOXM1) is a transcriptional factor that plays an important role in cell proliferation, differentiation and transformation. As one of the early up-regulated proteins in cancerogenesis, FOXM1 has been demonstrated to contribute to all hallmarks of cancer. FOXM1 is overexpressed in a large variety of solid cancer and hematologic carcinomas, such as hepatocellular carcinoma, acute myeloid leukemia, pancreatic cancer and ovarian cancer.The FOXM1 expression seems to be controlled by the Ras/MAPK(Ras, ERK) pathway,AKT and p38 signaling pathway,which are frequently activated in tumors.Thiazole antibiotics, small molecule inhibitors of the transcription factor FOXM1, such as siomycin A and thiostrepton possess antitumor activity in various cancers in vivo and vitro.These inhibitors present high specificity inhibitory activity of the transcription of FOXM1, without any effect of other transcription factors. Thiostrepton is not only a thiazole antibiotic but also a proteasome inhibitor which regulate FOXM1 transcriptional activity and expression. Thiostrepton exerts antineoplastic activity in a wide variety of tumor cells via different pathways. Maqbool reported that thiostrepton suppressed tumor development by down-regulating FOXM1, MMP-2 and MMP-9 level in PTC cell line xenografts and attenuating migration and invasion of PTC cells in vivo and vitro.In the current study, we study the expression of FOXM1 and then investigate the mechanism of estrogen in ERK/FOXM1/MMP9 pathway activation in the endometriosis. Finally, we evaluate the therapeutic potential of thiostrepton to determine the ability of this agent to down-regulate FOXM1 and MMP-9 expression, promote apoptosis and reduce the ectopic growth of endometrial tissue in an animal model.Methods1. Vaginal smears were performed daily at fixed period to determine if the animals have normal oestrous cycle. Rats with two consecutive normal oestrous cycle were included in the experiment.Fifty female Wistar rats were surgically induced with endometriosis via autotransplantation.2. After 4 weeks of observation, all the rats were anaesthetized for the second exploratory laparotomy to examine endometrial implants. The implant volumes were measured using a caliper (length, width, and height, in millimeters) and calculated with the formula (V=0.52*length*width* height).3. Twenty and thirty rats were randomly allocated to an ovariectomized(OVX) group and a treatment group, respectively. The OVX group was ovariectomized and randomly divided into OVX-estrogen group and control group. All rats were allowed a resting period of 3 days prior to any operation. The rats in the estrogen group were given estradiol (10ug/0.1ml /d) by subcutaneous injection while the control group was treated with an equivalent amount of seasame oil for 7 days.The treatment group was randomly divided into three groups to receive the following medication:thiostrepton at 150mg/kg,ip.; thiostrepton at 250mg/kg,ip.; sterile normal saline;ip.The groups received these dosages every two days for two weeks.4. Caliper measurement was used to evaluate the lesion growth after the medication. We applied histology and immunostaining to comfirm the model and detect FOXM1 position and expression. mRNA level was examined by real-time PCR. We researched the associated protein expression of ERK/FOXM1/MMP9 signalling pathway and Bcl-2 by western blot.Results1. Histopathologic staining:Histopathologic comfirmation was performed at Day 28 after the first operation. Macroscopically, the transplanted uterine tissue of all groups had developed to typical endometriotic lesions containing kiyosuke liquid, independently of the localization on the peritoneal wall. The cyst-like dilated tissue and its surrounding were abundant with neovascularization. The histologic examination confirmed that ectopic endometrium has similar endometrial epithelial cells and glands.2. Immunostaining of FOXM1:Immunofluorescence staining showed that FOXM1 was predominantly localized in the nucleus and cytoplasm of epithelial cell and mesenchyme. FOXM1 expression significantly increased in the OVX-estrogen group when compared with the OVX-oil group,and express more in nucleus.3. mRNA expression of FOXM1 and MMP9:The results of real-time PCR indicated that both FOXM1 and MMP9 were significantly higher in the estrogen group while compared with the control group(P<0.05).4. Western blot analysis of EKR/FOXM1/MMP9 signaling pathway:Western blot analysis revealed that phosphorylation of ERK,FOXM1 and MMP9 level elevated significantly in OVX-estrogen group as compared with the OVX-oil group.5. Evaluation of the effect of thiostrepton in the experimental endometriosis rat:(1) Pre-and posttreatment volumes of the endometriotic implants:As expected, peritoneal lesions of thiostrepton-treated animals exhibited a markedly decreased size when compared with vehicle-treated controls.Macroscopic analysis of the endometriotic implants showed that there was no significant difference among the pretreatment volumes in all treaing groups (48.91±22.29 mm3 for 150mg/kg thiostrepton group,45.01± 16.37 mm3 for 250mg/kg thiostrepton group,45.75±15.61 mm3 for the control group; P> 0.05). Likewise, no significant change was observed between the pre-and posttreatment volumes of the endometriotic implants in the saline-treated group (45.75±15.61 mm3 vs 36.09±19.46 mm3). Amazingly, there was a marked reduction in the volume of the endometriotic implants from 45.01±16.37 mm3 to 15.52±6.93 mm3 in 250mg/kg thiostrepton treating group (P< 0.01), which showed statistical differences compared with the other two groups. Furthermore, the volumes of the endometriotic implants were significantly smaller than in the control group (23.37±13.65 mm3 vs 36.09±19.46 mm3; P<0.05), following the dose of 150mg/kg thiostrepton administration.(2) Protein expression of FOXM1 and MMP9:FOXM1 protein level was significantly decreased in thiostrepton-treating group than saline-treating group. There was significant difference among the three groups respectively (P<0.05). The expression of MMP9 of 250mg/kg thiostrepton-treated group showed a significantly reduction when in comparison with the other two groups, but there were no difference between the other two groups. Thiostrepton exerted a significant inhibitory impact on the development of endometrial implants in this rat model of endometriosis by decreasing the total volume of lesions per rat and the protein expression of FOXM1 and MMP9.(3) Antiapoptotic protein Bcl-2 expression:The expression of Bcl-2 of 150mg/kg thiostrepton-treated group and 250mg/kg thiostrepton-treated group was significantly down-regulated when in comparison with the control group. There was no difference between the two treatment groups.Conclusion1. FOXM1 is affluent in the nucleus of ectopic endometrium in the OVX-estrogen group, especially in the nucleus of endometrial columnar epithelium cells.2. Protein of ERK/FOXM1/MMP9 signalling pathway were significantly higher after estrogen administration, which demonstrated that this signalling pathway might contributed to the establishment and development of endometriosis.3. Thiostrepton dose-dependently decreased the expression of FOXM1 and MMP9 in endometriotic lesions of the treated rats. We concluded that thiostrepton may be expected to be a drug in the treatment of endometriosis.4. Thiostrepton promoted the apoptosis of ectopic lesions by down-regulating antiapoptotic protein Bcl-2 expression.