However, the intracellular MFI in the c-fos WT group was approximately 60% lower than that for the NC group (p?0.05; Fig.?5a). The expressions of the c-fos and mdr1genes were significantly higher in the HEp-2/VCR cells than in control cells. C-fos overexpression in HEp-2 cells (c-fos WT) resulted in increased P-gp expression and increased the IC50 for 5-FU. C-fos knockdown in the HEp-2/VCR cells (c-fos shRNA) resulted in decreased P-gp expression and decreased IC50 for 5-FU. An intracellular Rh-123 accumulation assay showed that the mean intracellular fluorescence intensity (MFI) Finafloxacin hydrochloride was lower in the HEp-2/VCR cells than in HEp-2 cells. C-fos WT cells also showed lower MFI. By contrast, c-fos shRNA cells exhibited a higher MFI than the control group. Klf2 Conclusion C-fos increased the expression of P-gp and mdr1 in the HEp-2/VCR cells, and enhanced the efflux function of the cells, thereby contributing to the development of MDR. values less than 0.05 were considered statistically significant. Results Drug resistance of HEp-2/VCR cells We established a drug-resistant human laryngeal carcinoma cell line, named HEp-2/VCR, by selection against an increasing drug concentration gradient. The IC50 of VCR was increased from 0.04??0.01?mol/l in the normal HEp-2 cells to 1 1.7??0.19?mol/l in the HEp-2/VCR cells (Table?2). The 42.5-fold increase in IC50 indicates successful establishment of the drug-resistant HEp-2/VCR cell line. Table 2 Comparison of the IC50 values for HEp-2 and HEp-2/VCR cells exposed to 4 chemotherapeutics
VCR0.04??0.011.7??0.1942.5MTX1.2??0.358.3??0.236.90DDP0.5??0.251.9??0.163.85-FU61.1??4.35332??5.215.44 Open in a separate window Data are shown as the means SD The IC50 values for other common chemotherapeutic drugs were also assessed (Table?2). HEp-2/VCR cells were respectively 6.90, 3.8 and 5.44 times as resistant as HEp-2 cells to MTX, DDP and 5-FU. The results indicate that HEp-2/VCR is a multidrug-resistant cell line. Expression of c-fos and mdr1 in HEp-2/VCR cells Real-time PCR results showed that the expression of the proto-oncogene c-fos was low in HEp-2 cells, but increased 4.66-fold in the drug-resistant HEp-2/VCR cells (p?0.05; Fig.?1a). The drug resistance gene mdr1 was expressed at low levels in HEp-2 cells but increased 9.57-fold in HEp-2/VCR cells (p?0.05; Fig.?1b). The protein levels of c-fos and P-gp (which is encoded by mdr1) were also significantly elevated in HEp-2/VCR cells (Fig.?1cCf). This indicates a relationship between c-fos and mdr1 (P-gp). Open in a separate window Fig. 1 Differences in the expressions of c-fos and mdr1 (p-gp) in HEp-2 and HEp-2/VCR cells. a Expression of c-fos mRNA in HEp-2 and HEp-2/VCR cells. b Expression of mdr1 in Finafloxacin hydrochloride HEp-2 and HEp-2/VCR cells. c, d Western blot analysis of the expression of c-fos and p-gp. e, f The statistical quantification analyses of c-fos and p-gp protein levels in Finafloxacin hydrochloride HEp-2 and HEp-2/VCR cells. Data are shown as the means SD.*p?0.05, **p?0.01 Expression of mdr1 and P-gp in HEp-2 cells overexpressing c-fos To confirm the correlation between c-fos and the drug resistance gene mdr1 and its corresponding protein P-gp, we overexpressed c-fos in HEp-2 cells and determined the expressions of mdr1 and P-gp. At 48?h after transfection, the transfection efficiency exceeded 90% (Fig.?2a), and the c-fos expression had significantly increased in the transfected HEp-2 cells, which were named the c-fos WT group (Fig. ?(Fig.2b,2b, Finafloxacin hydrochloride ?,d,d, ?,e).e). More importantly, both the expression of the mdr1 gene (Fig.?2c) and P-gp (Fig.?2d and f) was significantly higher in the c-fos WT group than in the HEp-2 cells and NC group. Open in a separate window Fig. 2 Expression of mdr1 and p-gp in HEp-2 cells after overexpression of c-fos. a Fluorescence microscopy examination of the efficiency of transfecting the c-fos overexpression plasmid into HEp-2 cells (named c-fos WT). The negative control (NC) was HEp-2 cells transfected with an empty vector. The scale bar represents 100?m. b, c Real-time PCR results showing the expression of c-fos and mdr1 in HEp-2 cells after the overexpression of c-fos. d Western blot results showing the expression level of c-fos and p-gp in HEp-2 cells after the overexpression of c-fos. e, f The statistical quantification analyses of c-fos and p-gp protein levels in c-fos overexpression HEp-2cells.Data are shown as the means SD. **p?0.01, ***p?0.001 Expression of mdr1 and P-gp in HEp-2/VCR cells with c-fos knockdown We further examined the expressions of mdr1 and P-gp in HEp-2/VCR cells after c-fos downregulation. HEp-2/VCR cells with c-fos knockdown, named the c-fos shRNA group, showed a very low expression of.