| ObjectiveDevelopement of multidrug resistance(MDR) in malignancy cell to chemotherapy drugs is the most important cell prevent mechanism avoid malignancy cell lesion and might be particularly involved in the consequent chemotherapy failure.To elucidate the mechanisms of drug resistance and research reversal agents,most investigators have utilized cell lines that have acquired drug resistance in vitro or MDR cell line-bearing nude mice.However,it is hard to represent the real process of MDR when malignancy cell were exposed to chemotherapy drugs in vivo.With respect to controlling drug resistance,effective reversal agent such as verapamil and ciclosporin A have been reported.However,their use in the clinical setting has been limited because of the high plasma levels required for reversing drug resistance and its resulting serious toxicity.Therefore the development of more potent and less toxicity agents is anticipated urgently.Cepharanthine hydrochloride(CH) have diverse biological effects.Its reversing effect on multidrug resistance has been proven by many investigations.Our study was to establish acquired ascitic hepatocellular carcinoma(Hca/FAP) MDR mouse model induced by FAP combination chemotherapy regimen and investigate the molecular mechanism based on cell and molecular biological technology.Meanwhile,another purpose of our studies was to research the reversing effect and molecular mechanism of CH to Hca/FAP MDR model for supply basic data in future clinical liver cancer therapy.MethodsThe aims of our reseach was to developed acquired Hca/FAP MDR mouse model. We have adopted FAP combination chemotherapy regimen(5-Flurouracil+ Adriamycin+Cisplatin) that commonly used to treat hepatocellular carcinoma in clinic and induced Hca-bearing KM mouse with continuous exposure to increasing concentrations of these drugs to acquire MDR.Drug resistant spectrum and resistance index of seven chemotherapy drugs were detected by MTT assay in Hca/FAP tumor cells.Accumulation and efflux ability of Rhodamine123(Rh123) in Hca and Hca/FAP tumor cells was determined by measuring Rh123 mean fluorescene intensity(MFI) on flow cytometry;Multidrug resistance gene 1(mdr1) and multidrug resistant-associated protein gene 1(mrp1) mRNA expression level in Hca and Hca/FAP tumor cells was measured using RT-PCR technology.For study the model stability after drug treatment was stopped,mdr1 and mrp1 mRNA expression was also detected by RT-PCR.Moreover,we further examined the reversing effect of CH on Hca/FAP tumor cells.Based on flow cytometry technology,the effect of CH or VER on the accumulation and efflux function of Rh123 were detected by measuring MFI of Rh123.CH or VER combined with FAP regimen were treated with Hca/FAP-bearing mouse,prolonging life rate was calculated to evaluate its curative effect,cell apoptosis ratio detected by PI dyeing on flow cytometry,mdr1 and mrp1 mRNA expression level determined by RT-PCR technology.The purpose of this study are to elucidate the generate mechanism of Hca/FAP MDR model and reversing effect of CH on Hca/FAP tumor cells.Results1.Drug resistant spectrum and resistance index of seven chemotherapy drugs in Hca/FAP tumor cellsThe values of IC50 of seven drugs by MTT assay in Hca and Hca/FAP tumor cells have a significant difference compared with each other at P＜0.05.Drug resistance index of induced drugs:Adriamycin(ADR),Cisplatin(CDDP) and 5-Flurouracil(5-Fu) was higher and reached 21.45,19.80,8.15 times in Hca/FAP tumor cells,respectively;As for Daunomycin(DNR),Etoposide(VP-16), Mitomycin(MMC),Vincristine(VCR),Hca/FAP tumor cells also has drug resistance to some degree,reached 3.83,2.85,3.48,1.60 times,respectively.2.Accumulation and efflux ability of Rhodamine123(Rh123) in Hca and Hca/FAP tumor cellsThe results of Rh123 accumulation test by flow cytometry indicate that MFI of Hca and Hca/FAP tumor cells was 204.3±11.2 and 158.5±6.7,with significantly differences at P＜0.01;The curve of Histogram in Hca/FAP cells moved to left evidently;About Rh123 efflux test,histogram shows the curve of Hca/FAP cells lower and flater than Hca cells and moved to left obviously;MFI of Hca/FAP cells decreased sharply and efflux ratio was 68.2±0.6%,but Hca tumor cells was 37.2±0.5 %,with significant differences from each other at P＜0.01.3.Mdr1 and mrp1 mRNA expression level in Hca and Hca/FAP tumor cellsThe results of electropherogram shows the gray level ratio of mdr1 and mrp1 mRNA in Hca/FAP tumor cells was 0.4192±0.0468 and 0.7882±0.3171,compared with 0.0480±0.0076 and 0.1953±0.0568 in Hca tumor cells,respectively,mdr1 mRNA expression in Hca/FAP tumor cells increased obviously with significantly differences at P＜0.01 by statistical treatment and mrp1 expression has also increased and statistics indicated with significant difference at P＜0.05.4.Mdr1 and mrp1 mRNA expression level after withdraw drugs in Hca/FAP tumor cellsElectropherogram indicate the gray level ratio of mdr1 mRNA by RT-PCR in Hca/FAP tumor cells at 0,4,8 weeks for drugs withdrawal was 0.6874±0.0427, 0.5615±0.0837,0.4097±0.0841 and mrp1 mRNA was 1.1276±0.3256,0.7639±0.1612, 0.1232±0.0253.Statistic data showed mdr1 and mrp1 mRNA expression after drugs absence for 4 weeks without significantly differences at P＞0.05 from 0 week,but 8 weeks and 0 week has significant difference at P＜0.01.5.Effects of CH or VER on accumulation and efflux of Rh123 in Hca/FAP tumor cells The results of Rh123 accumulation test by flow cytometry indicate that verapamil(VER) in 4μmol·L-1 and CH in 8μmol·L-1,4μmol·L-1,2μmol·L-1 could increased obviously Rh123 accumulation of each observation time point in Hca/FAP tumor cells with significantly difference at P＜0.01 compared with MFI in control group.Moreover,statistics showed the dose of CH in 8μmol·L-1 and 4μmol·L-1 have significant difference at P＜0.01 compared with VER in 4μmol·L-1.As for Rh123 efflux test,MFI of CH 8μmol·L-1 and 4μmol·L-1 during each observation time point have significant difference compared with control group at P＜0.01.At 60min and 120min observation time point,VER 4μmol·L-1 and CH 2μmol·L-1 have also signification difference vs control group at P＜0.01.But CH 8μmol·L-1 and VER 4μmol·L-1have no statistic difference at P＞0.05.6.Prolonging life rate in Hca/FAP-bearing mouse treated with CH combined with FAP regimenCH or VER combined with FAP regimen has some inhibitory effect on Hca/FAP tumor cells.VER 2.5mg·kg-1 or CH 5mg·kg-1,CH2.5mg·kg-1 combined with FAP regimen group significantly increased average live span of Hca/FAP-bearing mouse with 26.38±7.31,26.44±9.37,27.25±8.53 days respectively and prolonging life rate reached 45.18%,45.51%,49.97%with significant difference compared with normal saline(NS) group for 18.17±5.42 days at P＜0.05 and these three group without significant difference compared with each other.If Hca/FAP-bearing mouse treated with FAP regimen or CH5mg·kg-1,2.5mg·kg-1 alone,all of the three groups have no significant difference compared with NS group at P＞0.05.7.Mdr1 and mrp1 mRNA expression level in Hca/FAP-bearing mouse treated with CH combined with FAP regimenAll coadmistration groups include VER 2.5mg·kg-1,CH5mg·kg-1 or CH2.5mg·kg-1 combined with FAP regimen could downregulate mdr1 mRNA expression in Hca/FAP tumor cells and Gray level ratio of the three groups was 0.4093±0.1570,0.3733±0.2317,0.4318±0.5324,respectively with significant statistic difference compared with NS group for 0.7835±0.3027 at P＜0.05 and without significantly difference each other at P＞0.05;Mdr1 mRNA expression of groups treated with FAP regimen,CH5mg·kg-1 and CH 2.5mg·kg-1 alone have no significant difference with NS group at P＞0.05.As for mrp1 mRNA expression,all groups have no significant difference with NS group at P＞0.05.8.Apoptosis ratio in Hca/FAP-bearing mouse treated with CH combined with FAP regimenThe figures of cell cycle indicates that sub-G1 peak appeared on the histogram by PI dyeing.Natural apoptosis ratio of NS group in Hca/FAP tumor cells was 3.8% and cell apoptosis ratio of FAP,CH5mg·kg-1 and CH 2.5mg·kg-1 alone treated groups was 6.13±0.56%,4.65±0.90%,3.75±0.75%,respectively,there is no significant difference compared with NS group at P＞0.05.But the three treated groups of VER 2.5mg·kg-1,CH 5mg·kg-1 and CH 2.5mg·kg-1 combined with FAP regimen raise the cell apoptosis ratio with 17.91±2.81%,20.41±1.69%,15.05±1.05%,respectively. These coadministration groups have significantly statistic difference with NS group at P＜0.05.Conclusions1.After long term treated with FAP chemotherapy regimen,the Hca tumor cells are able to develop cross-resistance to many other cytotoxic agents to which they are exposed or not such as anthracycline antibiotics,vinca alkaloids,etoposide,MMC and platin antitumor drugs.2.The mechanism of multidrug resistance of Hca/FAP tumor cells correlate with mdr1 and mrp1 mRNA overexpression and decrease intracellular drug concentration by adjusting the function of P-glycoprotein.3.Maintenance of Hca/FAP MDR model depend on drugs administration.Mdr1 and mrp1 mRNA expression depress and acquired MDR probably eliminate with absence of induced drugs.4.CH show a strong potential of reversing drug resistance in Hca/FAP tumor cells by increasing intracellular drug concentrations.This beneficial effect seems to be connected to the modulation of the P-gp function.The effect depend on concentration of CH and reversing effect of CH 8μmol·L-1 is stronger than VER.5.Coadministration of CH significantly enhance cytotoxicity of antitumor drugs and reverse drug-resistance.CH combined with FAP chemotherapy regimen downregulate mdr1 mRNA expression,induce Hca/FAP tumor cells apoptosis and prolong live span of mouse. |
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