Antiproliferative Effects of Mullerian Inhibiting Substance on Human Ovarian Cancer Cell Lines / 대한산부인과학회지

OBJECTIVE: In order to explore Mullerian inhibiting substance (MIS) effects on the ovarian neoplasia, the expression and localization of the MIS type II receptor (MISR II), the growth inhibitory effects of MIS, and the underlying molecular mechanisms were investigated in the ovarian cancer cell lines. METHODS: Expression of MISR II were studied in SKOV-3, OVCAR-3, and OVCAR-8 cell lines by immunohistochemical staining. The antiproliferative effects of MIS in these cell lines were investigated by methylthiazoletetrazolium (MTT) assay, fluorescence-activated cell sorting (FACS) analysis, annexin-V-FITC binding, and western blot analysis. RESULTS: All cell lines showed strong specific staining for MISR II, although staining in OVCAR-8 cells was more intense than that in SKOV-3 and OVCAR-3. Treatment of OVCAR-8 cells with MIS led to a dose- and time-dependent inhibition of cell growth and survival was determined use by MTT assay. But OVCAR-3 cells exhibited growth inhibition at higher doses after 48 hours of treatment and SKOV-3 cells did not demonstrate response. Using FACS analysis, exposure of OVCAR-8 cells to MIS (71 nM) resulted in G1 arrest after 24 hours of treatment. This pattern was changed by time-dependent increase in the percentage of cells with a sub G0G1 DNA content, suggesting apoptosis, after 48 hours of treatment. These results suggested that cell death be preceded by cell cycle arrest. Time-related induction of apoptosis was also observed in this cell line as measured by annexin-V-FITC binding. In OVCAR-8 cells, the growth inhibitory effects of MIS were mediated through specific induction of CDKI p16 protein expression and via regulation of E2F1 in the absence of detectable levels of pRb. We estimated that OVCAR-3 cells were affected by MIS through p16-independent, alternative mechanistic pathways, since the growth inhibitory effects of MIS were minimal. SKOV-3 cells did not express p16 protein. CONCLUSION: We have demonstrated that ovarian cancer cells express the MISR II. Epithelial ovarian cancer cells respond to MIS by growth inhibition. Although the precise mechanisms of MIS mediated inhibition of ovarian cancer cell growth have not been fully defined, these data suggest that MIS has activity against ovarian cancers in vitro and may also be an effective targeted therapy for ovarian cancer.

Expression of Mullerian Inhibiting Substance Type II Receptor during Follicular Development in the Human Ovary / 대한산부인과학회잡지

OBJECTIVES: This study was aimed to obtain information on the ontogeny of the production profile of MIS type II receptor (MISR II) and the pattern of its localization according to follicular development METHODS: Expression of MISR II were studied in 21 ovarian specimens from adult normal cycling women by RT-PCR and in situ hybridization of the MISR II mRNA and immunohistochemical staining of the MISR II. RESULTS: The first staining for MISR II and MISR II mRNA were detected in the granulosa cells in primary follicles. The granulosa cells of multiple layered growing follicles showed strong specific staining for MISR II and MISR II mRNA. Among the growing follicles, large follicle stained more intensely than small one. Expression of the MISR II and MISR II mRNA were also seen in the granulosa cells and theca cells of antral follicles. The expression levels of MISR II and MISR II mRNA in mature follicles were lower than those in growing follicles and were even further reduced, but still detectable, in corpus luteum. There was a decreased level of MISR II and MISR II mRNA expression when follicles become atretic. Both expressions were eventually lost from atretic follicles. And the MISR II and MISR II mRNA staining were not found in primordial follicles, oocytes, interstitial cells, ovarian epithelium, and corpus albicans. CONCLUSION: The production and localization of MISR II in granulosa cells, theca cells, and corpus luteum in normal reproductive ovary indicate that actions of MIS via MISR II are autocrine and paracrine in nature. The pattern of MISR II and MISR II mRNA expression according to follicular development indicate that MIS function in the ovary is turned on in primary follicles, increases to maximal levels in large growing follicles, and decreases just before ovulation. These experiments suggest that MIS may play an important role in follicle maturation and follicle selection during the adult reproductive cycle. And this study may yield important information to direct the development of newer contraceptive strategies.

Expression of Mullerian Inhibiting Substance and Its Receptor in the Human Ovary During Menstrual Cycle / 대한산부인과학회잡지

In this study, in order to further understanding of function of Mullerian inhibiting substance (MIS) and the ontogeny of the production profile of biologically active MIS and MIS type II receptor (MISR II), the patterns of their localization according to the follicular development in 21 ovarian specimens from women in reproductive age were studied by immunohistochemical staining. The flattened granulosa cells in primordial follicles failed to stain for MIS and MISR II, but the first staining was detected in the cuboidal granulosa cells in primary follicles. MIS and MISR II were detected specifically and exclusively in the cytoplasm of granulosa cells. The granulosa cells of both single and multiple layered growing preantral follicles showed strong specific staining for MIS and MISR II. Among the growing follicles, large follicle stained more intensely than small one. Within the multiple layers of granulosa cells, the innermost cells, closer to the oocyte, stained more intensely for MIS than those near the basement membrane, but MISR II was evenly distributed. In antral follicles, expression of the MIS was only seen in the granulosa cells, but MISR II was seen in the granulosa cells and theca cells. In large antral follicles, cumulus cells and periantral granulosa cells stained more intensely for MIS than those in the periphery. MIS staining waned in the mature follicles just before ovulation and could not be found in atretic follicles, corpus luteum, and corpus albicans. The expression levels of MISR II in mature follicles was lower than those in growing follicles and were even further reduced, but still detectable, in corpus luteum. There was a decreased level of MISR II expression when follicles become atretic and eventually lost from atretic follicles. The MIS and MISR II staining were not found in primordial follicles, oocytes, interstitial cells, ovarian epithelium, and corpus albicans. It is concluded that actions of MIS via MISR II are autocrine and paracrine in nature. The pattern of MIS and MISR II expression according to the menstrual cycles and development suggest that MIS may act as an intraovarian regulator of follicle maturation, selection and ovulation during the adult reproductive cycle.