Emodin Reverses Paclitaxel Resistance And Inhibits Invasive Activity Of Paclitaxel Resistant Ovarian Cancer Cells

Backgroud:Emodin is an effective component of traditional Chinese herbal medicine. It can be extracted from Rheum, Polygonum cuspidatum, Polygonum multiflorum, et al. Emodin has long been known to have a number of biological activities, such as anti-bacterial, immunosuppressive and anti-inflammatory effects, but its capability of anti-cancer and anti-invasion has not been adequately studied. Ovarian cancer has the highest mortality rate among gynecologic malignancies in the world. The standard treatment for ovarian cancer remains surgical debulking and chemotherapy with carboplatin and paclitaxel. However, the majority of ovarian cancer patients have one of the lowest 5-year survival rates, and the development of drug resistance is a major impediment toward successful treatment of the cancer. Thus, there have been a number of researches aimed at understanding the mechanisms of drug resistance, so as to develop strategies to overcome the disease.Paclitaxel is an effective chemotherapeutic agent and widely used for the treatment of ovarian cancer, whereas the development of drug resistance limits its availability. To date, several mechanisms of paclitaxel resistance in ovarian cancer cells have been proposed, including overexpression of ABC/MDR transporter family of proteins to increase the cellular efflux of paclitaxel, delayed G2/M transition, and alterations in apoptosis regulation. The overexpression of P-gp (P-glycoprotein), which is encoded by MDR-1 (Multidrug resistance-1) gene, plays a key role in the resistant mechanisms. P-gp functions as an efflux pump to decrease the intracellular accumulation of a variety of lipophilic drugs, including paclitaxel, leading to decresed therapeutic efficacy. Successful treatment with chemotherapeutic agents is largely dependent on their ability to trigger cell death in tumor cells; therefore, try to decrease the over-expression of anti-apoptotic molecules may affect the proximal level of apoptotic threshold. As direct caspase inhibitors and participants in a variety of survival signaling pathways, the inhibitor of apoptosis protein (IAP) family are important to the control of cell proliferation and drug resistance in multiple cancer types. As the important IAPs members, XIAP (X-linked inhibitor of apoptosis protein) and survivin up-regulated in most human tumor cells and make the caner cells escape from apoptosis. Evidence indicates that survivin and XIAP are correlated with chemresistance and decrease of these IAPs could induce apoptosis in chemoresistant human ovarian cancer cells. Down regulation of XIAP and survivin may enhance apoptosis in ovarian cancer cells.MIF (macrophage migration inhibitory factor, MIF) was one of the first cytokine activities to be described, originally being identified as a product of activated T lymphocytes that inhibited the random migration of cultured macrophages. Recently, MIF has also been implicated in many processes associated with tumor survival and invasion, such as stimulate cell proliferation, differentiation and migration, promote tumor angiogenesis. MIF expression is increased during the evolution of several malignancies, breast-, prostate-, colon-, lung- and ovarian-derived tumors have all been shown to contain significantly higher levels of MIF message and protein than their noncancerous cell counterparts. MIF has been reported to promote angiogenesis and the invasiveness and matrix degrading activity of cancer cells by stimulating mmp-2 and mmp-9. Taken together, MIF may be an important connective point in the process during invasive inhibition of emodin.MMPs (matrix metaloproteinases, MMPs), inuclding MMP-2 and MMP-9, play an important role in the degradation of basement membrane type IV collagen, which is associated with tumor cell invasion and metastasis. A role for MMPs in ovarian cancer development has been postulated based upon the observation that several members of the MMP family are up-regulated during ovarian cancer neoplastic progression. MMPs may play important role in the invasion and migration of tumor cells.Part I Emodin sensitizes human ovarian cancer cells topaclitaxel-induced apoptosisObjective:Ovarian cancer has the highest mortality rate among gynecologic malignancies in the world, and the development of drug resistance is a major impediment toward successful treatment of the disease. Emodin has been reported to sensitize human tumor cells to chemotherapeutic agents. In present study, whether emodin could overcome chemoresistance of paclitaxel resistant A2780/taxol cells and the mechanism were investigated.Methods:Cells were incubated and then treated with different concentration of emodin (10, 20, 40, 80μM) for 24h, respectively, then the viability of each group was determined by MTT assays. The apoptosis rate of cells treated with emodin or/and paclitaxel was determined by FCM. The expression of gene and protein level of MDR-1,XIAP and survivin were detected by QRT-PCR and western blot respectively. The function of P-gp was determined by FCM.Results:We found that emodin could induce apoptosis at a high concentration and enhance apoptosis induced by paclitaxel at a low concentration in both paclitaxel sensitive and resisitant ovarian cancer cell lines. QRT-PCR and western blot results indicated that the MDR-1 gene and P-gp, which was encoded by MDR-1 gene, were decreased after treatment with emodin. The function of P-gp is also attenuate. Meanwhile, the expression of XIAP and survivin were significantly down-regulated in cells treated with emodin. Furthermore, we found emodin could enhance the expression of cleaved fragments of caspase-3.Conclusion:Emodin could induce apoptosis alone and sensitize ovarian cancer cells to paclitaxel induced apoptosis. The results demonstrated that emodin could induce apoptosis of resistant ovarian cancer cells by increasing the cellular concentration of paclitaxel and decreasing the expression of anti-apoptotic molecules.Part II Emodin inhibits invasion activity of ovarian cancer cellsObjective:Ovarian cancer has the highest mortality rate among gynecologic malignancies in the world. Most of patients die of tumor invasion. Emodin has been reported to inhibit invasion activity in human tumor cells. In present study, whether emodin could inhibit cell invasion ability of paclitaxel resistant ovarian cancer cells were investigated.Methods:We checked the invasion ability of different ovarian cancer cells, and then checked the expression of invasion related factors MIF, MMP-2 and MMP-9 in A2780 and A2780/taxol cells. Then the high invasiveness cells were treated with different concentration of emodin (20, 40, 80 u M), then the cell invasion ability was measured by transwell assays. The changes of mRNA and protein expression of MIF, MMP-2 and MMP-9 were examined by QRT-PCR and western blot respectively.Results:A2780/taxol cells had high invasiveness than A2780 cells, and the expression of MIF, MMP-2 and MMP-9 were up-regulated in A2780/taxol cells. The invasive potential of both cell lines were reduced dramatically after treatment with emodin. QRT-PCR and western blot results indicated that the gene and protein expression of MIF, MMP-2 and MMP-9 were decreased after treatment with emodin. And the effect of emodin showed dose-dependent manner in ovarian cancer cells.Conclusion:A2780/taxol cells is more invasiveness than A2780 cells. The possible mechanisms are increased MIF, MMP-2 and MMP-9. Emodin could inhibit tumor invasion ability by down-regulation the expression of MIF, MMP-2 and MMP-9 in ovarian cancer cells.