Studies On The Role Of Homeoboxa10 Gene And Medroxyprogesterone Acetate In The Ovarian Carcinogenesis

Epithelial ovarian cancer (EOC) is the most common tumor of genital system, and also the fifth leading cause of cancer death in women as well as the first lethal gynecological malignancy. Ovarian cancer has a high incidence of metastasis that generally remains localized within the peritoneal cavity. EOC is not a single disease entity, but instead comprises a heterogeneous group of tumors. These tumors are classified according to their patterns of histological differentiation. The most common histological subtype are-serous?mucinous?and endometrioid tumors. The mechanism of regulation of subtype is unknown, which attract much attention from researchers. During the past hundred years, changes in body structure and development, such as the replacement of antennae with legs, were observed in Drosophila melanogaster mutants. Hox genes would be confirmed and highly conserved homologues identified in most animals. In vertebrates, the Hox genes are located contiguously in clusters, with the number of clusters varying according to anatomic complexity. HOX compromises a series of advanced conserved genes. Hox genes are integral to normal temporospatial limb and organ development along the anterior-posterior (A-P) axis. First, the position of a Hox gene 3'to 5'within a Hox cluster corresponds to its expression in the animal along the A-P axis. One group of Hox gene expression according to the arrangement of A-P named I-group Hox; and another group is scattered on different chromosomes named II- group. Numerous examples of aberrant HOX gene expression have been found in different cancers. The oncogenesis is related to leukocythemia?breast cancer?colorectal carcinoma?prostatic carcinoma?and renal carcinoma (including Wilms tumor). According to our previous study, expression of HOXA9?HOXA10 and HOXA11 were not detected in normal human ovarian surface epithelium. However, coliner expression patterns of these HOX genes that normally occur in Mullerian-duct-derived epithelial were found to be recapitulated in serous?endometrioid and mucinous EOCs according to the pattern of Mullerian-like differentiation of these tumors. Importantly, HOXA10 is related to the ovarian endometrioid cancer specifically.Aberrations of DNA methylation are now believed to be an important epigenetic alteration occurring early in many cancers including ovarian ones. In general, DNA methylation is one of the best-understood epigenetic changes in human cancers and may play important roles in carcinogenesis. Carcinogenesis is associated with changes in the epigenetic phenomenon, including two distinct and seemingly opposing trends: global decrease in cytosine methylation (hypomethylation) and methylation of cytocine in CpG islands (hypermethylation). Yoshida et al have brought forward that HOXA10 is down-expressed in endometrial carcinoma, which is related to low grade tumor, and also with the methylation of HOXA10. In that way, we hypothesized that the high expression of HOXA10 in ovarian cancer is related to the methylation condition of HOXA10 CpG islands. This study is aiming to finding the underling relationship between the HOXA10 CpG islands methylation and the oncogenesis of ovarian cancer, further seek out the new idea for the prevention and curing the ovarian cancers.Prevention and treatment is the severe problems in the iateria of ovarian cancer. Ovaries are located deep in the pelvic, and because of the special anatomical location, the early detection and diagnosis of ovarian cancer become very difficult. Meanwhile laking of high sensitivity diagnostic indicators caused that only when the ovarian cancer is in a advanced stages, the exact diagnosis can be done. And it often leads to poor prognosis of patients. On the current international principles of treatment of advanced ovarian cancer is a comprehensive treatment including a cytoreductive surgery and paclitaxel and a platinum-based chemotherapy. Due to a high rate of recurrence and high resistance rates in recurrent tumors, five-year survival rate of ovarian carcinoma still in a low level.MPA is one kind of progestin drugs, the most common uses of MPA are as the progestin component of menopausal hormone replacement therapy and also as an oral or depot-injected contraceptive. Medroxyprogesterone is also used as a treatment for endometriosis, dysmenorrhea, and amenorrhea. Because MAP has anti-estrogen function and has capability to promote the proliferative endometrium transform to secretary endometrium, it has been widely used as an adjuvant treatment of estrogen and progesterone receptor-positive endometrial cancer. In recent years, some studies suggested that progesterone can inhibit ovarian cancer cell proliferation and induce cancer cell apoptosis. But the specific mechanism remains unknown.Some researchers pointed that MPA might induce apoptosis via PI3K/Akt pathway. Being a important regulatory sites in cell survival pathway, PI3K/Akt pathway play a vital role in cell's proliferation and apoptosis. Numerous reports suggest a role of PI3K signaling in invasion and metastasis in various kinds of carcinomas. Akt has been also shown to be amplified or over-expressed in ovarian cancer, implying that it also plays a role in ovarian oncogenesis. Survival factors induce Bcl-2 pathway activation via several protein kinase signaling pathways including activation of phosphatidylinositol 3-kinase (PI3K)-Akt, which directly determined the cell proliferation or apoptosis.Epidemiological studies and long-term clinical practice show that progesterone may antagonize the effect of estrogen to protect the ovary, preventing the occurrence of ovarian cancer. Currently, there is no consensus about whether progesterone can be applied as adjuvant treatment of ovarian cancer. The present study attempts to reveal the mechanisms of medroxyprogesterone acetate induced apoptosis in ovarian cancer cells, and provide a new theoretical basis for the treatment of ovarian cancer.Part I The effects of hypomethylation of HOXA10 CpG promoter in ovarian cancerObjective The purpose of this study was to find the highly expression of HOXA10 in ovarian carcinoma and to determine the relationship between hypomethylation of HOXA10 gene promoter and highly expression in malignant ovarian tissues, which could induce the ovarian carcinogenesis, and finally to confirm the function of 5-Aza-dC on epithelial ovarian cell lines to provide the evidence of the treatment of 5-Aza-dC in clinic.Methods We performed the methylation status of 29 samples from ovarian carcinomas and 16 from normal tissues by methylation-specific polymerase chain reaction (MSP). Then, we evaluated the expression of mRNA and protein of HOXA10 in all samples to work out the relationship between the methylation status of HOXA10 and its expression in transcriptional and translational levels. We then confirmed our present study using SKOV-3 and HEY ovarian cancer cell lines treated with the demethylation agent 5-aza-2-deoxycytidine (5-Aza-dC) to detect whether the expression of HOXA10 in the two cell lines was altered. And the cell proliferation was determined by using methyl thiazolyl tetrazolium (MTT) colorimetric assay.Results 1. HOXA10 is highly expressing in ovarian cancer tissues. HOXA10 expression was examined by RT–PCR in 29 malignant ovarian tissues and 16 normal ovarian tissues. Western Blot analysis of the tissues confirmed that HOXA10 protein was present in malignant ovarian tissues. The real-time PCR assay further confirmed the quantitative relationship between the hypomethyaltion status and HOXA10 expression.2. HOXA10 is hypomethylated in epithelial ovarian carcinomas. We determined the methylation status of the region in normal and malignant ovarian tissues using MSP, which showed that the promoter of HOXA10 gene in 17 out of 29 (58.62%) malignant cancers and in 4 out of 16 (25%) normal tissues was hypomethylated, and the result shows significant difference (p < 0.05). Combining the research data above, we arrived at three conclusions: (1) HOXA10 promoter is normally methylated in most normal ovarian samples, whereas hypomethylated in abundant epithelial ovarian cancers. (2) HOXA10 is highly expressed in epithelial ovarian cancers; nevertheless, the expression level is low in normal tissues. (3) Comparing the methylated and hypomethylated status of HOXA10 gene in different tissues (including normal and malignant ones), the HOXA10 expression is lower in former status and higher in latter status.3. Rescue of HOXA10 expression by 5-Aza-CdR treatment. The results showed that after the treatment of the SKOV-3 cell line with 5-Aza-dC, demethylation of HOXA10 and concomitant increasing HOXA10 expression occurred.Conclusion:1. In epithelial ovarian carcinoma, the HOXA10 gene promoter hypomethylation condition could bring about the highly expression of HOXA10 both in the transcript and translate levels, which could induce the ovarian carcinogenesis.2. By 5-Aza-dC treatment, the HOXA10 gene promoter could be demethylated, then concomitant increasing HOXA10 expression occurred, further stimulate the ovarian cell lines to proliferate and differentiate.Part II Medroxyprogesterone Acetate Induce Human Ovarian Cancer Cell Line SKOV-3 Apoptosis in VitroObjective 1. The purpose of this study was to observation of MPA depressed SKOV-3 cells proliferation in vitro.2. Explore the mechanism of MPA induced SKOV-3 cells apoptosis in vitro. 3. Reveal the functions of progesterone membrane receptors in human ovarian cancer cells apoptosis.Methods 1. Epithelial ovarian cancer SKOV-3 cells at exponential phase were subcultured at 7×103 cells/well into 96-well microplates and cultured overnight. Cells were treated with serial concentrations (0.1, 1,10,100?mol/l) of MAP for 12, 24 or 48h. And the cytotoxicity was determined by using methyl thiazolyl tetrazolium (MTT) colorimetric assay2. SKOV-3 cells were treated with serial concentrations (1, 5, 12.5, 25?mol/L)LY294002 for 1h before treated with different concentrations of MAP. And the cytotoxicity was determined by using methyl thiazolyl tetrazolium (MTT) colorimetric assay.3. SKOV-3 cells treat with serial concentrations MAP for 24h, lysed cells and collection of protein, Akt, pAkt, pBad and Bcl-2 protein expression was detected by Western blotting.4. SKOV-3 cells were treated with different concentrations of MPA role of 24, 48 h; Annexin V / PI double staining flow cytometry to detect cell apoptosis.Result 1. Medroxyprogesterone acetate inhibited the growth of SKOV-3cell line effectively, in a time-and-dose dependent way (P<0.01).2. MPA inhibition of SKOV-3 cell proliferation effect could be blocked by LY2940023. The expression of p-Akt and Bcl-2 protein in the SKOV-3 cells were decreased in all medroxyprogestogen groups and significant decreased in high dose medroxyprogestogen group (P<0.05). The expression of p-Akt protein was decreased to 72% in the low dose group and 65% in high dose group. The levels of Bcl-2 protein were descended to 90% in low dose group and 73% in high dose group. The expression of Akt protein was no significant difference in both medroxyprogestogen group and control group.4. MPA induced SKOV-3 cell apoptosis in a time-dose dependent manner. Comparison of different time statistics can be found, early apoptosis rate significantly rised(P< 0.05), when the time was 24h; extension of time to 48h, late apoptosis rate increased, and early apoptosis rate increased slowly.Conclusion 1. MPA induced SKOV-3 cell apoptosis in a time-dose dependent manner.2. MPA can inhibit the PI3K/Akt signal pathway, reducing the level of Bad phosphorylation, induced SKOV-3 cells apoptosis in vitro.3. The cells most sensitive to the drug, when the drug effects time was 24h and MPA concentration from 1?mol L to 10?mol / L, increase the drug concentration or drug effect can not be extended significantly improve the cell apoptosis.