The Mechanism Of Glucocorticoids Enhancing Chemotherapy Resistance Of Human Ovarian Cancer Cell

Ovarian carcinoma is the leading cause of death due to gynecologicalmalignancy.This is usually partly due to the advanced disease at the time ofdiagnosis but also due to the fact that most tumours become resistant tochemotherapy quickly. The primary therapy comprises cytoreductive surgeryas well as adjuvant or neoadjuvant chemotherapy using schemes consisting ofa platinum and a taxane derivative. Glucocorticoids (GCs) such as thesynthetic corticoid, dexamethasone (DEX), are given as part of anti-emeticprophylaxis or the prevention of allergic reactions to taxanes. Newer agents,such as gemcitabine, are currently investigated in sequential therapy to avoidthe development of drug resistance.DEX and similar GCs were firstintroduced to tumour therapy on the basis of pro-apoptotic effects inlymphoid cells and on their effectiveness in treating tumour related edema,inflammation, pain, and electrolyte imbalance as well as stimulating theappetite and, most importantly, preventing the nausea and emesis caused bycytotoxic drugs.However, controlled randomized trials evaluating thepotential impact of GCs on the growth of solid tumours and patient survivalhave never been performed. Concerns about the widespread use of GCs in thetherapy of solid tumours have been expressed repeatedly, Recent studiessuggest the inhibition of cytotoxic therapy-induced apoptosis by DEX inestablished lung, cervical and breast carcinoma cell lines in vitro, ex vivo andin vivo. More recent data indicate that GCs can inhibit apoptosis induced bychemotherapy not only in established cancer cell lines and tumor xenografts,but also in the freshly isolated cells from surgical resections from tumors ofvarious origins, including ovary, breast, prostate, pancreas, liver, colon,brain,cervix, bone, skin, and nervous system In addition, the anti-chemotherapeutic effect of GCs can be seen in several anticancer drugs includingcisplatin,paclitaxel,5-fluorouracil, adriamycin,actinomycin D, doxorubicin, andgemcitabine. The GC-induced pro-survival effects should be of importantclinical relevance when they interfere with the effect of chemotherapeutics.In our previous work, we found that Dex could dramatically prolong thedetachment time of the cells digested with trypsin in human ovarian cancercell line HO-8910, suggesting that Dex may be able to increase the celladhesion ability to extracellular matrix (ECM). Since cell adhesion to ECM ispivotal for the survival and growth of most of the solid cancer cells derivedfrom epithelium, we hypothesized that the increase in cell resistance tocytotoxic therapy induced apoptosis by Dex may be due to its promotioneffect in cell adhesion to ECM. Therefore, in the present study, weinvestigated the effect of Dex on cell adhesion to ECM of two human ovariancancer cell lines, HO-8910and SKOV3, and examined the relationshipbetween Dex's effect of enhancing adhesion and Dex-induced cell resistanceto chemotherapeutic agents. We further explored the mechanism of Dex'saction and mainly focused on the adhesion molecular integrin b1subfamily,as well as CD44. Our results provide new evidence that Dex's role inpro-adhesion through the enhancement of integrin b1signaling andCD44signaling is one of the basic mechanisms responsible for Dex-inducedapoptotic resistance against chemotherapy in ovarian cancer cells. Part I glucocorticoids enhance human ovarian cancer cellresistance to chemotherapeutic drugsObjective:TO demonstrate Whether the DEX resistance to chemotherapydrugs on ovarian cancer cells apoptosis.Methods: We first detected the effect of cisplatin or paclitaxel alone or plusDex on the survival of HO-8910and SKOV3cells using MTT assay.Wefurther used Annexin V-FITC staining to detect the change in apoptosis.Wealso detect the expression of activated form of caspase-3using westernblotting.Results:(1) Cisplatin alone can significantly reduce cell number in adose-dependent manner.Co-treatment of cells with Dex and different doses ofcisplatin significantly increased the survival cell number.A similar protectiveeffect of Dex was also seen when cells were treated with paclitaxel, anotherchemotherapeutic drug.(2) We further used Annexin V-FITC staining todetect the change in apoptosis. Cisplatin resulted in remarkable cell apoptosiswith18.38%(P<0.05). However, co-treatment of cells with Dex and cisplatinsignificantly diminished the apoptosis of cells to10.1%(P <0.01).(3) We alsodetect the expression of activated form of caspase-3using western blottingand the result show that cisplatin significantly induced the activation ofcaspase-3, but Dex could inhibit the activation of caspase-3protein bycisplatin in HO-8910cells.(4) MTT assay showed that Dex couldsignificantly enhance the adhesion of the HO-8910and SKOV3cells onFN-coated culture plates in a time and dose-dependent manner.Conclusion:our studies suggest the inhibition of cytotoxic therapy-inducedapoptosis by DEX in established ovarian carcinoma cell lines(HO-8910andSKOV3), Dex enhances the ovarian cancer cells and extracellular matrixadhesion ability. Part II The mechanism of glucocorticoids enhance humanovarian cancer cell resistance to chemotherapeutic drugsObjective: TO dicuss The mechanism of glucocorticoids enhance humanovarian cancer cell resistance to chemotherapeutic drugs.Methods: Cells were cultured in the medium containing100nM Dex fordifferent time.The expression of Collagen type I mRNA,Collagen typeIIImRNA,Collagen type IV mRNA,FN mRNA,FN mRNA were detected byReal-time PCR.The lever of LN and HA secerted were detected by ELISA.Examined the influence of the integrin b1-blocking antibody on the celladhesion and cell survival by assay. The effects of Dex on the level of FAKphosphorylation detected by Western Blotting method. The effects of Dex onthe level of Akt phosphorylation detected by Western Blotting method.Results；(1)Dex has no effect on the expression of the mRNA of Col-I,Col-III, Col-IV and LN, and using ELISA assay we next found that LN andHA are also no change in HO-8910cells with or without Dex. FN mRNA wasslightly enhanced by Dex administraton for0~48h (about1.7-fold ofcontrol).(2) MTT assay showed that Dex could significantly enhance theadhesion of the HO-8910and SKOV3cells on FN-coated culture plates.in atime and dose-dependent manner The integrin family of cell surfacereceptor.(3) The results show that preincubation of the cells withintegrinβ1-blocking antibody partially blocked the Dex-induced adhesionbetween cells and FN, in a dose-dependent manner. The cell survivalprotective effect of Dex was also decreased when cells were treated with Dex combined with integrinβ1-blocking antibody.(4) CD44and integrin familyequivalent to a membrane receptor protein is also involved in the regulationof adhesion between cells and extracellular matrix. Preincubation of the cellswith CD44-blocking antibody partially blocked the Dex-induced adhesion ina dose-dependent manner.(5) results showed that Dex increased the level ofphosphorylated FAK in time and dose dependent manner.(6) the level of AKTphosphorylation can be improved in time-dependent manner. Dex canpromote PKB/AKT activation, and rapid activation of FAK may be involvedin the process of the Dex impact of HO-8910cell adhesion.(7) AKT inhibitorcan partially block the Dex anti-killing effects of chemotherapy drugs.Conclusion:These results show that the effect of Dex enhanced cell adhesionto ECM is not achieved by changing the composition of the extracellularmatrix. Dex-enhanced cell attachment and cell survival are partially reversedby integrin b1-blocking antibody.FAK and PI-3K-Akt Participated in the Dexanti-apoptotic effect...