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

Ryu, Ki Sung; Seo, Mi Young; Jo, Yun Sung; Kim, Mee Ran; Kim, Jin Woo; Han, Goo Taik; Lee, Joon Mo; Kim, Jang Heub

Abstract

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.