| Ovarian cancer(OC)is a common malignant tumor of female reproductive organs,The incidence rate is only third to the incidence of cervical and uterine cancer.Due to the lack of specific symptoms in early-stage and lack of effective screening strategies,most of cases have progressed to an advanced stage at the time of primary diagnosis.Because of the difficult treatment,high recurrence rate,fast metastasis,ovarian cancer is regard as the first leading cause of death from female reproductive system malignant tumor.The effective treatment for OC is the surgery and the platinum-based chemotherapy,Though it improved the prognosis of patients to some extent,However,The majority of patients ultimately experience chemoresis--tance,and the 5-year survival rate of patients is stay for 30%-40%.Thus,Ovarian cancer become the serious threatening diseases to gynecologists and researchers.Multi-drug resistance of tumor(MDR)refers to the tumor cells are in contact with a kind of antitumor drug resistant,have cross resistance with other antitumor drugs that have not come into contact with、 have different structures and different mechanisms.The mechanism of multidrug resistance of tumor is complex,one of the important reasons is that the drug efflux in cells by P-gp.P-gp uses intracellular adenosine triphosphate(ATP)to remove a variety of cytotoxic drugs outside the cell,increasing the resistance of tumor cells to drugs.Over the years,around the MDR1/ P-gp as the research target of the reversal of drug resistance include the use of P-gp competitive inhibitors,monoclonal antibodies,antisense oligonucleotides and RNA interference technology began to emerge,but the formation of drug resistance is a polygenic,multi-factor complex process,nonspecific,side effects,safety and economic problems limiting its application.Therefore,To expore new strategies of reversing drug resistance become the hot spot and the difficulty of ovarian cancer.Traditional Chinese medicine has been widely used in China.In recent years,a variety of anti-tumor components extracted from traditional Chinese medicine and natural plants have been proved to be effective antitumor agents.Naringin,called the new hesperidin naringenin glycoside,is a kind of flavanone compounds,mainly found in the Rutaceae plants,such as grapefruit,lime peel and its variants,with advantages of rich resources,clear chemical structure,low price of raw materials.the existing research indicates that naringin has anticancer、 anti-inflammatory and other pharmacological effects of prevention of atherosclerosis.As other flavonoids,naringin also has antitumor effects on common tumors such as breast cancer、colon cancer、bladder cancer and liver cancer etc,research in gynecological malignant tumor is rare,early results of our research group suggested that naringin can significantly inhibit the proliferation of human ovarian cancer cell line SKOV3 cells in vitro,along with the extension of the time of drug action and the increase of the concentration of naringin,the proliferation inhibition rate increased gradually.Further study showed that the proliferation of naringin on human ovarian cancer cell resistant to cisplatin SKOV3/DDP cells also has obvious inhibitory effect,and can enhance the sensitivity of SKOV3/DDP cells to DDP,the reversal mechanism may be associated with the down-regulation of multidrug resistance gene MDR1 and multidrug resistance related protein(MRP2)gene expression,provides preliminary of the idea of antitumor and reversal of cisplatin resistance to the role of naringin in ovarian cancer.Recent studies have shown that nuclear factor kappa B(NF-κB)is closely related with tumor occurrence、development and metastasis,many of its encoding protein can promote tumor growth,the study found that cisplatin resistant ovarian cancer cells contain high expression of NF-κB,and at the core position in the mechanism of ovarian cancer.NF-κB get through the regulation of downstream target protein Bcl-x L、Bcl-2、Fas/ FasL、XIAP、survivin、cIAP1/2、CDK2、VEGF、COX-2 and other antiapoptosis effect,increased the survival ability of tumor cells,eventually leading to resistance to chemotherapy.NF-κB signaling pathway is closely related to the occurrence and development of tumors.Based on the previous research,our experiment use SKOV3、SKOV3/DDP cells as the research object,to explore the effects of naringin on ovarian cancer cells and related mechanisms,and the reversal of cisplatin resistance related signal pathway regulating mechanism,so as to further reveal the mechanism of naringin,provide a theoretical basis for clinical reversal of ovarian cancer resistance to chemotherapy.Part One: The effect of naringin on proliferation of ovarian cancer cells and the Drug resistance of SKOV3/DDP in vitroObjective: To observe the effect of naringin on the proliferation of human ovarian cancer cell line SKOV3 cells and human ovarian cancer cell resistant to cisplatin SKOV3/DDP cells in vitro,and to observe the effect of naringin on the SKOV3/DDP cells in vitro.Methods: 1.Cell culture: SKOV3 cells grow in RPMI l640 complete medium that containing 10% fetal bovine serum,penicillin and streptomycin 100U/ml 100U/ml,at 37℃,5%C02,saturated humidity condition culture,medium was changed every other day,2、3 days a passage.2.MTT assay The sensitivity of SKOV3 and SKOV3/DDP cells on cisplatin: cells in the logarithmic growth phase、suitable concentration(5×103)was inoculated into 96 well plates,two kinds of cells in experimental group were added with different concentrations of cisplatin(1μg/ml、 2μg/ml、 4μg/ml、 8μg/ml、 16μg/ml、 32μg/ml),the control group with drug containing 1640 culture medium,cultured 48 h,MTT method was used to detect the OD value of each hole,calculation of each concentration group cell proliferation inhibition rate and IC50,the drug resistance and calculation of SKOV3/DDP cells by publicity.3.Detecting the effect of naringin on the proliferation of SKOV3/DDP cells by MTT:selecting the logarithmic growth phase cells,conventional trypsin digested with 10% fetal bovine serum RPMI 1640 medium,then made into single cell suspension and cultured in a 96 well culture plate,each containing about 100μl(5 x 103cells),after the cells adhered to the culture medium,adding different concentrations of naringin(10μmol/L,20μmol/L,40μmol/L,80μmol/L),with 5 wells in each group,were cultured for 24 h,48h and 72 h,set up the control group.According to the measured absorbance(OD)value,the growth inhibition rate of two groups of cells at different time was calculated.It is concluded that the drug concentration of the growth rate is more than 90%,which is the non cytotoxic dose of the drug.4.Detecting the reversal effect of naringin on multidrug resistance in SKOV3/DDP cells by MTT : Take the logarithmic growth phase cells,the appropriate concentration(5×103)concentration was inoculated into 96 well plates,the control group with different concentrations of cisplatin(1μg/ml,2μg/ml,4μg/ml,8μg/ml,16μg/ml,32μg/ml),each with 5 wells,the experimental group add the naringin,which final concentration of 20μmol/L,after 48 h,calculate each cell proliferation inhibition rate、IC50、The drug resistance reversal fold,to determine whether naringin can reverse the drug resistance of ovarian cancer cell lines.Results: 1.The MTT results showed that different concentrations of DDP(1μg/ml,2μg/ml,4μg/ml,8μg/ml,16μg/ml,32μg/ml)in two groups,SKOV3 cells’ IC50 was 8.618,SKOV3/DDP cells IC50 was 18.876,the resistance ratio was 2.190,shows SKOV3 / DDPcellswere DDP resistant resistant cells.2.MTT results showed that different concentrations of naringin could inhibit the proliferation of SKOV3/DDP cells,the inhibition increased with the drug concentration(10μmol/L,20μmol/L,40μmol/L,80μmol/L)and the extension of action time(24h,48 h,72h),presents the concentration-time dependence.after 24 h,the proliferation inhibition rate was 3.05 + 0.15%,3.89 + 0.44%,11.67 + 0.53% and 22.38 + 0.63% respectively,There wereno statistically significant differences between the 20μmol/L and 10μmol/L groups(P>0.05);after 48 h,the proliferation rate was 4.14 + 0.21%,7.49 + 0.62%,28.81 + 1.22% and 36.49 + 1.14% respectively,there were statistically significant differences in groups of different concentrations of naringin(P<0.05),after 72 h,there were statistically significant differences in groups of different concentrations of naringin(P< 0.05)And,in 40μmol/L naringin groups there was no significant difference between 72h、48h(P> 0.05),in order to avoid too many cells died during the experiment,we chosen 20μmol/L naringin 48 h as the most suitable concentration and action time in the following research.3.With the increase of DDP concentration,20μmol/L naringin after 48 h action,the proliferation inhibition rate of SKOV3/DDP cell was also gradually increased,8.09 + 0.47%,17.49 + 1.30%,25.40 + 1.10%,41.31 + 2.34%,71.06 + 2.12%,83.90 + 1.15%,respectively,the experimental group of SKOV3/DDP cells IC50 was 9.112 g/ml,IC50 of the control group was 18.876 g/ml,the difference was statistically significant(P< 0.05);The drug resistance reversal fold was:2.072.Conclusion: 1.Different concentrations of naringin alone or in combination with DDP can inhibit the proliferation of SKOV3 and SKOV3/DDP cells in vitro,and have a concentration-time dependence.2.The combination of non cytotoxic naringin and DDP can improve the anti ovarian cancer activity in vitro and partly reverse the drug resistance of SKOV3/DDP cells.Part Two: The mechanism of naringin in reversing SKOV3/DDP resistance in vitroObjective: To investigate the molecular mechanism of naringin reversing SKOV3/DDP resistance in vitro.Methods: Detecting the expression of P-gp in SKOV3 and SKOV3/DDP cells: selecting the appropriate density of cells seeded in 6 well plates,cultured 48 h,observe differential expression of multidrug resistance protein P-gp in SKOV3 、SKOV3/DDP cells.1.The expression of P-gp in SKOV3 and SKOV3/DDP cells in ovarian cancer: selecting the appropriate density(5×103)of cells seeded in 6 well plates,cultured 48 h,Detecting expression of multidrug resistance protein P-gp in SKOV3 and SKOV3/DDP cells.2.The expression of different concentrations of naringin on P-gp protein of SKOV3/DDP cells: different concentrations(10μmol/L,20μmol/L,40μmol/L,80μmol/L)of naringin cultured in SKOV3/DDP cells,after 48 h,Detecting the expression of P-gp protein by using single naringin3.Selecting the cells in logarithmic growth phase,cultured with appropriate concentration(5 × 103)and the best time of naringin,SKOV3/DDP cells were divided into following groups:group with single naringin(20μmol/L)、 cisplatin group(5μg/ml)、naringin(20μmol/L)combined with cisplatin(5μg/ml)group、control group,after 48 h,the expression of P-gp was detected to determine the mechanism of naringin reversal of cisplatin resistance;Detecting the expression of cyclinD1、 C-myc、caspase3、caspase 7、bcl-2、survivin、MMP2、MMP9、VEGF、β-catenin gene and protein.In order to understandthe way of inhibiting proliferation、 inducing apoptosis and inhibiting the invasion and metastasis of ovarian cancer cells.To get the underlying mechanism ofresistance reversal.Results:The expression of esistance related protein in SKOV3 and SKOV3 /DDP cells.1.The two groups of cells were cultured for 48 hours,RT-PCR and Western blotting showed that the P-gp expression existed in two groups,the expression of P-gp in SKOV3 was lower than SKOV3/DDPcells,the difference was statistically significant(P < 0.05).2.Different concentrations of naringin(10μmol/L、20μmol/L、40μmol/L、80μmol/L)cultured in SKOV3/DDP cells after 48 h,the expression of P-gp protein decreased gradually.(4.920 + 0.142,4.750 + 0.123,3.123 + 0.146,1.883 + 0.114),80μmol/L、 40μmol/L、20μmol/L 10μmol/L groupcompared with control group,the difference was statistically significant(P< 0.05).3.Naringin can reverse drug resistance in SKOV3/DDP cells by down regulating the expression of P-gp alone: naringin group(20μmol/L),naringin(20μmol/L)combined with DDP(5μg/ml)group were compared with control group,the expression of P-gp protein was significantly decreased(P< 0.05).To compare the expression of P-gp protein between naringincombined with DDP group and DDP alone group,the protein expression was decreased,the difference was statistically significant(P < 0.05).4.Effect of naringin on apoptosis and invasion related protein of SKOV3/DDP cells: Western blotting results showed that: compared with the blank control group、 single naringin group、naringin combined with DDP group,the expression of inhibitor of apoptosis protein bcl-2 and Survivin were decreased and the pro apoptotic protein caspase3 、caspase7 expression increased,the difference was statistically significant(P< 0.05),The cell cycle regulated protein cyclinD1、C-myc expression decreased in the combination group、single drug group,compared with the control group,the difference was statistically significant(P<0.05),the expression of the combination group was lower than in single DDP group,the difference was statistically significant(P<0.05);The expression of tumor invasion related protein MMP2,MMP9,VEGF,β-catenin reduced in the combination group and single drug group(naringin alone and DDP alone),compared with the control group,the difference was statistically significant(P<0.05).Conclusion: Naringin can downregulating the expression of resistant protein P-gpin SKOV3/DDP cells by downregulating of inhibitor of apoptosis protein bcl-2 and survivin,upregulating of proapoptotic protein caspase3 and caspase7 expression,downregulating of cell cycle regulatory proteins cyclinD1,C-myc,downregulating of tumor invasion related protein MMP2、MMP9、VEGF、β-catenin expression etc,thus reverse of drug resistance of SKOV3/DDP,which may be part of the molecular mechanism of naringin reverses cisplatin resistance.Part Three: The effect of naringin on reversing cisplatin resistance in SKOV3/DDP cells by regulating NF-κB signaling pathwayObjective: To investigate the effect of naringin on reversing cisplatin resistance in SKOV3/DDP cells by regulatingNF-κB signaling pathway.Methods: 1.RT-PCR and Western blotting assay were used to detect the expression of NF-κB in the cultured SKOV3/DDP cells;2.Designning and synthesising small interfering RNA targeting NF-κB(siRNA),targeting silence NF-κB;construct overexpression of NF-κB recombinant plasmid and transfected into cells,cell groups as follows: blank control group(not transfected plasmid and siRNA),empty plasmid control group,overexpression of NF-κB,siRNA control group,NF-κB siRNA group.The expression of P-gp mRNA and protein was detected by RT-PCR and Western blotting after transfection of NF-κBsiRNA and over expression plasmid 48 h,respectively;3.SKOV3/DDP cells were transfected with NF-κB overexpression plasmid and were treated with naringin at the same time.The groups were as follows: naringin 20μmol/L + NF-κB overexpression plasmid was chosen as the experimental group,naringin 20μmol/L + empty plasmid as control group,After cultured 48 h,the expression of P-gp.mRNA and protein was detected.Results: 1.RT-PCR and Westem blotting showed that,with the increase of the concentration of naringin,mRNA and protein expression of NF-κB gradually decreased,compared with the blank control group,the difference was statistically significant(P< 0.05),80μmol/L,40μmol/L,20μmol/L compared with 10μmol/L of naringin,the difference was statistically significant(P< 0.05),but in 40 μmol/L and 20μmol/L group,there was no statistical significance(P>0.05).2.RT-PCR and Westem blotting showed that P-gp mRNA and protein expression was higher in NF-κB overexpression group,meanwhile,in siNF-κB group,the expression was low,and had significant difference when compared with the blank control group 、 empty plasmid control group and siRNA control group(P<0.05).3.In the experimental group,overexpression of NF-κB plasmid was transfected into the experimental group,empty plasmid was transfected into the control group.The Westem blotting results showed that the expression of NF-κB protein in the experimental group was significantly higher than that in the control group,indicate that transfection was successed.After overexpression of NF-κB,the expression of P-gp mRNA and protein was higher than that in control group,and the difference was statistically significant(P<0.05).Conclusion: Naringin can reverse the cisplatin resistance in ovarian cancer cells by inhibiting the expression of drug resistance protein P-gp in SKOV3/DDP cells,which is related to the regulation of NF-κB signaling pathway. |
