The Expression Of ABCG2 In Nasopharyngeal Carcinoma And Its Function In Nasopharyngeal Carcinoma Chemotherapy

Objective:This study investigated the expression of ABCG2 in nasopharyngeal carcinoma biopsy samples and CNE-2 cell line and its relationship with the multidrug resistance in nasopharyngeal carcinoma chemotherapy.Material and Methods:The tumor biopsy samples were from 30 patients with nasopharyngeal carcinoma, and the tissues in control group were chronic inflammatory biopsy tissues from 20 patients with chronic nasopharyngitis. ABCG2 mRNA expression was detected by Real-time polymerase chain reaction (Real-time PCR) and ABCG2 protein expression was evaluated by immunohistochemical analysis.Results:It was found that the expression levels of ABCG2 mRNA and protein were higher in nasopharyngeal carcinoma tissues than in the chronic inflammatory tissues (t=5.4060, P=0.0124), and it was markedly increased in patients with lymph node metastasis (t=5.4547, P= 0.014). And then we chose low concentrating of Topotecan (0.75μg/ml) and Mitoxantone (0.6μg/ml) to treat CNE-2 cells for 24,48 and 72 h respectively. The results of real-time PCR showed that the ABCG2 mRNA expression was increased in a time-dependent manner after treatment of CNE-2 cells with Topotecan or Mitoxantone (r= 0.9857, P= 0.0143).Conclusion:The expression of ABCG2 is higher in nasopharyngeal carcinoma, which is related with the N staging. ABCG2 may be a marker of lymph node metastasis in nasopharyngeal carcinoma. And ABCG2 is potentially involved in Topotecan and Mitoxantrone resistance in nasopharyngeal carcinoma chemotherapy. Objective:This study investigated the function of ABCG2 in Mitoxantrone treatment in CNE-2 cells.Methods:ABCG2 function was determined by measuring the IC50s of CNE-2 cells for different concentrations of Mitoxantrone (0,10 nM,20 nM,40 nM,60 nM,80 nM) in the presence and absence of the specific ABCG2 inhibitor fumitremorgin C (FTC) for 24h,48h and 72h. The expression of ABCG2 before and after Mitoxantrone treatment was detected by the real-time polymerase chain reaction (Real-time PCR) and western blot at both mRNA and protein levels in CNE-2 cells.Results:We found that the IC50s of CNE-2 cells for Mitoxantrone were reduced by approximately one-third of that without FTC. Mitoxantrone could induce ABCG2 mRNA expression, which was dose-dependent at 24h,48h and72h (r2=0.9520, P=0.0009; r2=0.8826, P=0.0054 and r2=0.9351, P=0.0016) and was also exposure time-dependent at 10 nM,20 nM,40 nM,60 nM and 80 nM(r2=0.9505, P=0.0251; r2=0.9650, P=0.0177; r2=0.9923, P=0.0039; r2=0.9671, P=0.0166; r2=0.9672, P=0.0155). The protein expression of ABCG2 was determined by Western Blot and found to consistent with the Real-time PCR data.Conclusion:We speculated that ABCG2 would transport mitoxantrone out to protect the cells in Mitoxantrone treatment in CNE-2 cells and ABCG2 is potentially involved in Mitoxantrone resistance in nasopharyngeal carcinoma chemotherapy. Objective; To construct three short hairpin RNA (shRNA) interference expression plasmid vectors of human ABCG2 gene, to assay the expression of ABCG2 in a human nasopharyngeal carcinoma (NPC) cell line, CEN-2 cell line, after transfected with recombinant plasmids, and to detect the RNAi effect of shRNA.Methods:Targeting ABCG2 gene sequence, three plasmid expression vectors coding for shRNA and a control vector containing random DNA fragment were constructed. The recombinant plasmids were amplified in Ecoli. DH5 and then identified by restriction digestion, PCR and sequencing. The vectors were transfected into CEN-2 cells. ABCG2 expression was assayed by real-time quantitative PCR and western blot.Results:The successful construction of pGC-silencer-U6/Neo/GFP/ABCG2 was confirmed by DNA sequencing. Transfection of shRNA plasmids significantly down-regulated ABCG2 expression in CEN-2 cells at both mRNA level and protein level. Recombinant plasmid 1 had the strongest effect, with an inhibition ratio of 75% at the mRNA level and 68% at the protein level, which showed a significant difference from plasmids 2 and 3 (P<0.05).Conclusion:pGC-silencer-U6/Neo/GFP/ABCG2 has been successfully constructed and it can down-regulate ABCG2 expression after transfected into CEN-2 cells, which could facilitate further studies of ABCG2 functions CEN-2 cell line and its application in NPC gene therapy. Objective:To investigate the mechanism of multidrug resistaance in nasopharyngeal carcinoma by detecting the sensitivity to Mitoxantrone of CNE-2/siRNA-ABCG2 cells after inhibition of ABCG2 by RNAi technology.Methods:pGC-silencer-U6/Neo/GFP/ABCG2 and control plasmids were transfected into CNE-2 cells separately and then stable clones were isolated named as CNE-2/ siRNA-ABCG2 cells and CNE-2/control cells, respectively. The sensitivity to Mitoxantrone of CNE-2, CNE-2/siRNA-ABCG2 and CNE-2/control cells were evaluated by measuring the IC50s of these cells for different concentrations of Mitoxantrone (0,10 nM,20 nM,40 nM,60 nM,80 nM). Then CNE-2, CNE-2/ siRNA-ABCG2 and CNE-2/control cells were exposed to 35.3 nM Mitoxantrone, which is the IC50 of CNE-2 cells at 24h, and their growth and cell cycle were detected by CCK-8 and PI staining.Results:For all three time points, the IC50s of CNE-2/siRNA-ABCG2 for Mitoxantrone were significantly lower than that of untransfected CNE-2 cells (t=11.9137, P=0.0003). We examined the growth rate of these cells for 10 days, and found that the proliferation of CNE-2/siRNA-ABCG2 cells was significantly slower than that of CNE-2 cells (t=2.2614, P=0.0364) with Mitoxantrone treatment. After treated with Mitoxantrone, proportion of apoptosis cells was increased from 8.70% to 20.91%, proportion of G1 stage cells were increased from 37.22% to 40.43%, whereas G2/M stages cells were reduced from 33.76% to 17.54% in CNE-2 cells, however in CNE-2/siRNA-ABCG2 cells, proportion of apoptosis cells was increased from 8.32% to 29.73%, proportion of G1 stage cells were increased from 39.94% to 41.15%, whereas G2/M stages cells were reduced from 30.47% to 6.70%. Conclusion:The sensitivity of CNE-2 cells to Mitoxantrone was increased after transfected with pGC-silencer-U6/Neo/GFP/ABCG2 plasmid Objective:To investigate the sensitivity to Mitoxantrone of CNE-2 cells after inhibition of ABCG2 by RNAi technology in nude miceMethods:Created the model of human nasopharyngeal carcinoma xnograft in nude mice. The base of the tumors and the tumors themselves CNE-2, CNE-2/ siRNA-ABCG2 or CNE-2/control cells formed were micro-multi-point injected of Mitoxantrone (10n M,20μl) per week for 4 weeks. An equal amount of PBS (20μ1) was injected as control. The growth curves of tumors were recorded. Then tumors were isolated from nude mice after 4weeks. The apoptotic indexes of these tumors were detected using the in situ TUNEL method.Results:No one died in experimental nude mice, and their body weight continued to grow. Tumor formation rate reached 100%. the increase of volume in control groups was faster than that in experimental groups in all of CNE-2 group, CNE-2/ siRNA-ABCG2 group and CNE-2/control group (as described in Materials and methods); (2)between the group injected with CNE-2/control cells and the group injected with untreated CNE-2 cells, there was no significant difference in tumor growth in their experimental groups as well as their control groups(t=0.9678,P=0.3460); (3)in these three groups, the growth of tumors was no significant difference among their control groups (p>0.05); (4) injected with Mitoxantrone, the growth of CNE-2/siRNA-ABCG2 cells formed tumors was significantly suppressed compared with that of untreated CNE-2 cells formed tumors (t=2.6255,P=0.0172). Using the in situ TUNEL method, we found that treated with Mitoxantrone, the apoptotic index was 29.45±5.29% for the tumors CNE-2/ siRNA-ABCG2 cells formed,18.51±4.33% for the tumors untreated CNE-2 cells formed, and 12.13±6.04% for the tumors CNE-2/control cells formed, respectively. Injected with PBS, the apoptotic indexes for the three groups were no significant difference.Conclusion:The sensitivity to Mitoxantrone of CNE-2 cells after inhibition of ABCG2 by RNAi technology in nude mice was increased.