Exploring mechanisms for regulating growth and survival of ectopic endometriotic cells using comprehensive DNA microarray analysis

Abstract

OBJECTIVE: We previously reported that TNFα induced IL-8 expression in endometriotic stromal cells (ESCs). Bacterial endotoxin lipopolysaccaride (LPS), as an inducer of inflammation, enhanced TNFα and IL-8 production as well as cell proliferation in ESCs. On the other hand, altered apoptosis in endometriotic tissues is an aspect that illuminates the pathophysiology of endometriosis. To further define the nature of inflammatory reaction and abnormal survival of endometriotic cells, we sought to reveal the molecular mechanisms with regard to (a) the LPS induced-cell proliferation and (b) the resistance to apoptosis using cDNA microarray. DESIGN: Prospective study. MATERIALS AND METHODS: ESCs were obtained from the ovarian chocolate cysts. Eutopic endometrial stromal cells (EMSCs) were isolated from the endometrial tissue of premenopausal women who underwent hysterectomy for uterine myoma. LPS and staurosporine (SS) were exposed the cultured cells as the inflammatory reaction- and the apoptosis-inducing agent, respectively. Signaling pathway- or apoptosis related-gene expression profiles were analyzed using the pathway-focused cDNA microarray. Based on these microarray data, siRNAs directed against the target genes were transfected. The level of various gene and protein expression was evaluated by realtime RT-PCR, ELISA and western blotting. Cell proliferation was assessed by BrdU incorporation and WST-8 assay. RESULTS: ESCs exhibited the TNFα-induced cell proliferation and the ability to survive against SS exposure compared to EMSCs. In pathway finder array, knockdown of TNFα gene in ESCs repressed LPS-induced cIAP (inhibitor of apoptosis protein) -2 and IL-8 expression, the NFκB pathway responsive genes. After LPS addition, cIAP-2 and IL-8 expression in ESCs was higher than that in EMSCs. TNFα silencing also attenuated the LPS-induced ESCs proliferation. Adding NFκB inhibitor abolished these effects induced by TNFα silencing. On the other hand, in apoptosis gene array, cIAP-1, XIAP, and especially survivin, IAP family members, were highly expressed in ESCs. Survivin expression in ESCs after SS treatment was enhanced, whereas it was repressed in EMSCs, suggesting aberrant survivin expression in ESCs may be involved in the resistance to drug-induced apoptosis. EMSCs showed the potent activation of SS-induced caspase-3 or -7, whereas it was not detected in ESCs. CONCLUSIONS: TNFα and IAP family gene expression may be attributed to the mechanisms regulating mitosis and resistance to apoptosis in endometriotic tissues.