A novel molecular mechanism for anticancer drug-induced ovarian failure: Irinotecan HCl, an anticancer topoisomerase I inhibitor, induces specific FasL expression in granulosa cells of large ovarian follicles to enhance follicular apoptosis

Clinical use of CPT-11 combination chemotherapy frequently induces ovarian dysfunction in premenopausal and perimenopausal cancer patients, but its mechanism remains unclear. Mouse experiments were performed to clarify the molecular mechanism of CPT-11-induced ovarian dysfunction. Clinically therapeutic doses of CPT-11 were injected intraperitoneally into 8-week-old female MCH mice, and their ovaries were examined by the TUNEL assay to detect dead cells. Immunohistochemical examinations were simultaneously performed to detect the expression of activated caspase 3, Fas antigen and Fas ligand (FasL). Furthermore, normal murine ovarian tissue fragments were incubated with recombinant soluble FasL in organ cultures and stained by the TUNEL assay to detect apoptotic cells. Intraperitoneal CPT-11 injections induced specific TUNEL-positive cells and cell death with cleaved caspase 3 expression among large ovarian follicular granulosa cells. Apoptotic follicles (follicles containing >/=10 TUNEL-positive cells per ovarian section) were only found among large follicles. The final apoptotic follicle ratios were approximately 30% of the total follicles independent of the CPT-11 dose, while CPT-11 dose-dependently enhanced apoptotic processes in murine ovarian follicles. Fas antigen was expressed in most ovarian cells, with extremely high expression levels detected in luteal cells. CPT-11 injections did not significantly increase the Fas expression levels in ovarian cells. Although no FasL expression was detected in normal ovarian tissues, CPT-11 injections significantly induced specific FasL expression in granulosa cells. Incubation of organ-cultured normal murine ovarian tissue fragments with recombinant mouse soluble FasL significantly increased the numbers of TUNEL-positive granulosa and luteal cells. In conclusion, CPT-11 dose-dependently induced specific FasL expression in granulosa cells of developing ovarian follicles. The induced FasL reacted with the Fas antigen constitutively expressed on granulosa cells, such that apoptosis can only be enhanced and induced in granulosa cells in an autocrine and/or paracrine manner. This cell lineage-specific and differentiation stage-specific apoptosis in granulosa cells is thought to be the main molecular mechanism of the ovarian dysfunction induced by CPT-11 combination chemotherapy.