| Objective:To evaluate the reversal effect of Xiaochaihu Tang (XCHT) on multi-drug resistance (MDR) of hepatocellular carcinoma (HCC) in vitro and in vivo, and investigate the underlying molecular mechanisms and target.Methods:1. In vitro study:Cell viability of the parental cell or the MDR cell was deternmined by MTT, and used the SPSS package for Windows (Version 11.5) to calculate the resisitance index (RI) and the reversal fold (RF) of XCHT. The cell viability was detected by MTT assay, Microscopy assay were used to evaluate the cell morphology, the colony formation assay was used to evaluated the survival capability, Flow cytometry (FCM) was used to analysed the cell cycle, those were used to evaluated the effect of XCHT on the proliferation of BEL-7402/5-FU. The cell apoptosis was analysed by Hoechst 33258 staining or FACS analysis with Annexin V/PI, respectively. The content of ATP was detect by luciferase assay. The intracellular retention of 5-fluorouracil (5-FU), adriamycin (ADM) or rhodamine was detected by high performance liquidchromatography (HPLC) or FCM analysis. The mRNA and protein expression of Bax, Bcl-2, CyclinGl, ABCB1 (p-gp), ABCC1 (MRP1) and ABCG2 (BCRP) were detected by RT-PCR and western blot assay.2. In vivo study:Nude mice were implanted with human hepatocellular carcinoma parental cell BEL-7402 or its resistant cell BEL-7402/5-FU to establish the xenograft model in vivo and intraperitoneal injection of 5-FU(20 mg/kg) for 21 days, the body weight was used to evaluated the side-effect of 5- FU, the tumor volumn and tumor weight were used to evaluated the MDR of the xenograft model. The MDR model were established and randomly devided to four group:Control,5-FU (20 mg/kg), XCHT (14.2g/kg) and combination (5-FU+XCHT), and fed with 5-FU (20 mg/kg) or XCHT (14.2 g/kg) for 21 days, the tumor volumn and tumor weight were used to assess the effect of 5-FU and XCHT, Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick-End Labeling (TUNEL) assay was used to determine the effect of XCHT on apoptosis. Immunohistochemistry (IHC) was performed to examine the positive expression of Ki-67. The mRNA and protein expression of Bax, Bcl-2, CyclinG1, ABCB1 (p-gp), ABCC1 (MRP1) and ABCG2 (BCRP) were determined by RT-PCR and Western-blot assay.3. Target validation:To establish the growth curve of BEL-7402 or BEL-7402/5-FU and calculated the double time, respectively. The expression of miR-122 in tumor cells or tissues was determined by qPCR. BEL-7402 or BEL-7402/5-FU cells were transtcted with inhibitor-miR-122(ASO-miR-122) or mimics-miR-122, and detected the expression of miR-122 and the resistance to 5-FU. The cell morphology and cell number was evaluated after BEL-7402/5-FU cells were transtcted with mimics-miR-122. The apoptosis was determined by DAPI staining and FCM analysis, respectively. Colony formation and cell cycle were used to ssesed the proliferation. FCM was used to analysed the intracellular accumulation of Rho123 and ADM. qPCR and western blot were used to determined the expression of target genes of miR-122.Result:1. In vitro study:(1)The IC50 of BEL-7402/5-FU to 5-FU, ADM or DDP were significantly higher than that on BEL-7402 cells after treated 48 h, and the Resistant Inedx (RI) of BEL-7402/5-FU to 5-FU, ADM or DDP was 6501.99,31.73 and 13.82, respectively(that was resisitant to chemotherapeutic drug when the RI was more than 1.5). (2) The combination of XCHT (0.5 or 1.0 mg/mL) and 5-FU or ADM significantly decreased the IC50 of BEL-7402/5-FU cells to 5-FU and ADM, and the Reversal Fold (RF) of 0.5 mg/mL or 1.0 mg/mL to 5-FU or ADM were 1.66,3.44 and 1.50,5.58, the relative reversal rate (RRR)was 39.88%,71.34% and 34.11%,84.51%,respectively. (3) XCHT suppressed the proliferation of BEL-7402/5-FU:We observed that XCHT treatment significantly decreased the cell viability of BEL-7402/5-FU cells in a dose-and time-dependent manner. Colony formation assay showed that XCHT could inhibit the cell proliferation in a dose-manner. XCHT could block the cell cycle on G2/M phase and down-regulate the expression of cyclinGl. (4) XCHT induced the apoptosis of BEL-7402/5-FU:We observed that XCHT could induced cell apoptosis by hoechst 33258 staining. The FCM assay showed that XCHT treatment induce the apoptosis in a dose- dependent manner. XCHT treatment also upregulated the expression and downregulated the expression of Bcl-2. (5) XCHT inhibited the chemotherapeutic drug efflux outside BEL-7402/5-FU:XCHT increased the intracellular accumulation of 5-FU, ADM and Rho123 in BEL-7402/5-FU. XCHT treatment could significantly reduces the content of ATP. XCHT downregulated all the expression of ABCB1 (p-gp), ABCC1 (MRP1) and ABCG2 (BCRP).2. In vivo:(1) The tumor volumn and tumor weight showed that the xenograft tumor of BEL-7402/5-FU was more resisitant to chemotherapeutic drug and XCHT could suppress the growth of xenograft, together these, we thought that XCHT could reverse the MDR in vivo. 5-FU treatment have no effect on the tumor volumn and tumor weight. (2) We observed that XCHT and combination both could significantly induce the cell apoptosis of BEL-7402/5-FU xenogrft by TUNEL assay. The treatment of 5-FU had no effect on apoptosis. (3) XCHT and combination treatment both significantly inhibited the positive expression of Ki-67 though IHC assay. The treatment of 5-Fu had no change (4) XCHT or combination treatment both significantly down-regulated the expression of Bcl-2, cyclinG1, ABCB1 (p-gp)、ABCC1 (MRP1)、ABCG2 (BCRP) and upregulated the expression of bax by RT-PCR and Western-blotassayt. Treatment of 5-FU did not show significant change on these genes.3. Target validation:Compared the BEL-7402, the double time (TD) was relonged 17.04 h and more resistant to 5-FU. The expression of miR-122 both were downregulated, the decreased fold change was 0.57- or 0.61-fold in resisitant cells or tissues, XCHT increased the expression of miR-122, the fold chagen was 2.36-or 2.82-fold, respectively. The BEL-7402 cells was transfected with the inhibitors of miR-122, that is a antisese oligonucleotides (ASO), the fold change of miR-122 reduced to 0.71-fold, and it had no significant effect on 5-FU. The BEL-7402/5-FU cells were transfected with mimics of miR-122, the expression of miR-122 was increased to 2056-fold, and those cells were more sensitive to 5-FU within this range of 0-1200μM. MiR-122 could inhibited the grouth of BEL-7402/5-FU cells, inhanced the sensitivity of BEL-7402/5-FU cells to 5-FU. MiR-122 also promoted the apoptosis of BEL-7402/5-FU cells. XCHT treatment inhibited the proliferation of BEL-7402/5-FU cells. MiR-122 could increase the intracellylar accumulation of ADM and Rho123 in BEL-7402/5-FU cells. MiR-122 regulated the expression of its target genes,such as Bax, CDK4, CyclinGl and ABCB1.Conclusion:1. The results in vitro and in vivo showed that XCHT treatment could reverse the MDR of HCC2. The mechanisms by which XCHT reversed the MDR of HCC maybe:(1) XCHT inhibited the proliferation of BEL-7402/5-FU cells.(2) XCHT promoted the apoptosis of BEL-7402/5-FU cells.(3) XCHT inhibited the drug efflux activity of drug pump of BEL-7402/5-FU cells.(4) XCHT regulated the apoptosis, proliferation and drug pump function ofBEL-7402/5-FU cells via regulating the expression of miR-122 and its target genes. |
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