| ObjectiveTo study alteration of radiosensitivity of nasopharyngeal carcinoma cell lines in vitro before and after inducing multidrug resistance, and alteration of radiosensitivity before and after different reversal methods to multidrug resistance cells. To study alteration of expression of multidrug resistance of nasopharyngeal carcinoma cell lines before and after radiotherapy, and alteration of expression of multidrug resistance before and after reversal therapy during or after radiotherapy. And analysis the relationship between P- gp, GST-π, TOPⅡ, MRP1 protein expression in nasopharyngeal carcinoma and it's pathology, clinical stage, therapeutic effect in the near future.Methods1 . Inducing and reversion multidrug resistance of CNE1 and CNE2 cell lines.Comparsion effect of different reversal methods on multidrug resistance ofnasopharyngeal carcinoma cell lines. Using the IC50 value of DDP for CNE1 andCNE2 as initial concentration respectively, and 3.0 and 1.5μmol/L as ultimumconcentration respectively, and induce multidrug resistance of nasopharyngealcarcinoma cell lines. The cell lines were stimulated 12 weeks with gradually increasing concentration of DDP, and then cultivatived 2 weeks with non-durg nutrient medium, and renamed as CNE1/DDP and CNE2/DDP cell line respectively. The survival rate of CNE1/DDP and CNE2/DDP cell lines were detected by MTT colorimetric assay. Three reversion methods, which were 3.0umol/L cyclosporine A, 500U/ml interferon, and 3.0umol/L cyclosporine A united with 500U/ml interferon, were studied in this research. And the survival rate of CNE1/DDP-C and CNE2/DDP-C cell lines, which were reversed witn the three methods respectively, were detected by MTT colorimetric assay too. Calculate and compare the IC50 value and conversion multiple of each group. Cloning efficiency of CNE1/DDP, CNE2/DDP, and reversion groups of two cell lines were detected by clone formation method, and cytotoxicity of cyclosporine A and interferon to each group were assayed.2. Detct and compare the effect on radiosensibility of multidrug resistance cell lines reversed with cyclosporine A and interferon with clone formation experiment.CNE1 cell line was divided to three groups as CNE1, CNE1/DDP and CNE1/DDP-C. CNE2 cell line was divided to three groups as CNE2, CNE2/DDP and CNE2/DDP-C too. CNE1/DDP-C and CNE2/DDP-C were CNE1/DDP and CNE2/DDP cell lines reversed with cyclosporine A and interferon respectively. Tumour cell was digested to monoplast cell suspension with trypsase during exponential phase of growth, and each group was divided to 7 subsets again. Then, each 7 subsets were cultivated in 6cm diameter culture dish, and irradiated with X-ray in 0, 0.5, 1.0, 2.0, 4.0, 6.0, 8.0 Gy buy turns. After irradiation, each subset was cultivated for 12 days to clone formation. All the cultivated cells were stained with Gentian violet, and the clone number was counted. Calculate the cloning efficiency and survival rate of each group. Survive curve was fitted by one-hit multitarget model, and calculated Dq^ Do^ NU SF2 of each group. And analysis the radiotherapysensibility of each group with these results.3. Expression of multidrug resistance related gene on CNE1 and CNE2 cell lines before and after radiotherapy.CNE1 and CNE2 cell lines were renamed as CNE1/RT and CNE2/RT after 40 Gy radiation in 2.5 Gy every time and twice per week lasted for 8 weeks. Then renamed as CNEl/RT-Cl and CNE2/RT-C1 dealed with 3.0umol/L cyclosporine A and 500U/ml interferon after radiotherapy. Another two cell lines were renamed as CNE1/RT-C2 and CNE2/RT-C2 administerated with 3.0umol/L cyclosporine A and 500U/ml interferon twice per week during radiotherapy. Protein expression of P-gp, MRP, TOP II and GST-tt of each group were detected by flow cytometry, and gene expression of mdrl, MRP, TOP II and GST-7t of each group were detected by RT-PCR. Detect the survival rate of CNE1/RT, CNE2/RT, CNEl/RT-Cl and CNEl/RT-Cl with MTT colorimetric assay, and calculate the ICsoof each group.4. Dependablity study between expression of P- gp, GST-sr, TOP II and MRPl protein in nasopharyngeal carcinoma and it's pathology, clinical stage and therapeutic effect in the near future.Detect P- gp, GST-ir, TOP II and MRPl protein expression in nasopharyngeal carcinoma of 45 patients with immunohistochemistry stain, and dectect it's pathology type with HE stain, and definite it's clinical stage. Then, analysis the relationship between P- gp, GST-sr, TOP II and MRPl protein expression in nasopharyngeal carcinoma and pathology or clinical stage. Same chemotherapy ang radiotherapy were given to these patients, and clinical effect in the near future were evaluated 3 monthes after therapy. Then, analysis the relationship between P- gp, GST-jr, TOP II and MRPl protein expression in nasopharyngeal carcinoma patients and it's clinical effect in the near future.Results1 ? The IC50 of DDP for CNE1, CNE2, CNEl/DDP and CNE2/DDP were 0.35, 0.23, 6.72, and 4.16 umol/L respectively. Reversal multiple of cyclosporine A to CNEl/DDP and CNE2/DDP were 5.01 and 9.67 fold respectively, and reversal multiple of interferon were 1.95 and 2.34 fold respectively, and reversal multiple of cyclosporine A united with interferon were 6.34 and 11.89 fold respectively. So, 3.0umol/L cyclosporine A and 500U/ml interferon can reverse the multidrug resistance of CNEl/DDP and CNE2/DDP no matter applied alone or united with another. The reversal activity of cyclosporine A to CNEl/DDP and CNE2/DDP was stronger than that of interferon obviously (P<0.01), and the reverseon activeity of cyclosporine A united with interferon was stronger obviously than that of cyclosporine A or interferon applied alone (P<0.01). Clone formation experiment showed the clone formation rate of CNEl/DDP, CNEl/DDP -tcyclosporine A, CNEl/DDP + interferon and CNEl/DDP + cyclosporine A+ interferon cell lines were 73.67±2.08, 73.00±2.65, 74.00±2.65 and 73.33±1.53 respectively, and analysis of variance showed no differentiation between each group. So, cyclosporine A and interferon in reverseon concentration showed no cytotoxicity to CNEl/DDP no matter applied alone or together. And the clone formation rate of CNE2/DDP, CNE2/DDP +cyclosporine A, CNE2/DDP + interferon and CNE2/DDP +cyclosporine A+ interferon cell lines were 69.33±2.52, 68.00±2.65, 68.67±3.06 and 68.33±3.79 respectively, and analysis of variance showed no differentiation between each group. So, cyclosporine A and interferon in reverseon concentration showed no cytotoxicity to CNE2/DDP no matter applied alone or together.2. Survival curve and radiation biological parameter were fitted by one-hit multitarget model. SF2 value of CNE1, CNEl/DDP and CNE1/DDP-C were 0.641, 0.686 and 0.619 respectively. SF2 value of CNE2, CNE2/DDP and CNE2/DDP-Cwere 0.187, 0.355 and 0.277 respectively. The radiosensibility of CNE1 showed no variance before and after DDP induction, even reversed with cyclosporine A and interferon; And the radiosensibility of CNE2 was down-regulated after inducted with DDP, and up-regulated again reversed with cyclosporine A and interferon.3. Flow cytometry showed protein P-gp, MRP and GST-u expression of CNE1 were 33.76±3.98, 41.92±3.89 and 53.60±6.28 respectively. And protein P-gp, MRP and GST-rc expression of CNEl/RT were up-regulated obviously afer radiotherapy (p<0.01), which were 61.12±5.92, 58.27±3.81 and 86.00±5.62 respectively. And protein P-gp and MRP expression of CNEl/RT-Cl were down-regulated again reversed with cyclosporine A and interferon after radiotherapy (p<0.01), which were 38.69±3.86 and 45.79±3.04 respectively, and showed no difference compared with CNE1 (p>0.05). Protein P-gp and MRP expression of CNE1/RT-C2 showed no fifference compared with CNEl/RT (p>0.05), which were 59.02±2.31 and 57.43±2.97 respectively. Protein GST-rcexpression of CNEl/RT-Cl and CNE1/RT-C2 showed no fifference compared with CNEl/RT (p>0.05). Protein TOPII of CNE1, CNEl/RT, CNEl/RT-Cl and CNE1/RT-C2 showed no fifference compared with eachother (p>0.05), which were 2.91+1.10, 1.44±0.75, 2.28±1.64 and 2.45±0.75 respectively. Flow cytometry showed protein P-gp, MRP and GST-rc expression of CNE2 were 16.56±3.17, 10.71±0.90 and 29.80±2.22 respectively. And protein P-gp, MRP and GST-n: expression of CNE2/RT were up-regulated obviously afer radiotherapy (p<0.01), which were 47.99±4.51, 45.60±4.32 and 59.75±4.12 respectively. And protein P-gp and MRP expression of CNE2/RT-C1 were down-regulated again reversed with cyclosporine A and interferon after radiotherapy (p<0.01), which were 19.97±4.57 and 15.52±4.68 respectively, and showed no difference compared with CNE2 (p>0.05). Protein P-gp and MRP expression of CNE2/RT-C2 showed no fifference compared with CNE2/RT (p>0.05), which were 45.25±1.43 and 42.99±4.93respectively. Protein GST-rcexpression of CNE2/RT-C1 and CNE2/RT-C2 showed no fifference compared with CNE2/RT (p>0.05). Protein TOP II of CNE2, CNE2/RT, CNE2/RT-C1 and CNE2/RT-C2 showed no fifference compared with eachother (p>0.05), which were 1.61±0.87, 1.89±0.79, 2.49±1.34 and 1.91±0.71 respectively. PT-PCR showed mRNA mdrj, MRP and GST-7t expression of CNE1 and CNE2 were upregulated after radiotheraoy, and cyclosporine A and interferon showed no reversal effect on mRNA level used no matter after or during radiotherapy. And mRNA TOP II expression of CNE1 and CNE2 were all very low, and showed no alteration reversed with cyclosporine A and interferon. The IC50 value of CNE1/RT, CNE2/RT, CNE1/RT-C1 and CNE2/RT-C1 was 7.24, 6.35, 1.08 and 0.44umol/L respectively determined by MTT colorimetric assay, cyclosporine A and interferon showed reversal effect applied after radiotrerapy, and the reversal multiple of cyclosporine A and interferon to CNE1/RT and CNE2/RT was 6.70 and 14.43 fold respectively.4. Dependablity study between expression of P- gp, GST-*, TOP II and MRP1 protein in nasopharyngeal carcinoma and it's pathology, clinical stage and therapeutic effect in the near future.Expression rate of P- gp protein in KSCC, NK-DIFF and NK-UD were 60.0%, 30.8% and 11.1% respectively, and that of GST-jf were 60.0%, 69.2% and 66.7% respectively, and that of TOP II were 20.0%, 30.8% and 48.2% respectively, and that of MRP were 40.0%, 46.2% and 48.2% respectively. Statistical analysis showed that expression rate of P- gp was correlation with pathology, and expression rate of GST-*r, TOP II and MRP1 had nothing to do with pathology. Expression rate of P- gp protein in clinical stage I, II, III and IV patients were 33.3%, 18.8%, 23.5% and 22.2% respectively, and that of GST-ir were 66.7%, 62.5%, 70.6% and 66.7% respectively, and that of TOP II were 33.3%, 43.8%, 35.3% and 44.4% respectively, and that of MRP1 were 0%, 43.8%, 52.9% and 55.6% respectively. Statisticalanalysis showed independence between P- gp, GST-*, TOP II and MRP protein expression and clinical stage. Statistical analysis showed independence between P- gp, GST-ir, TOP II and MRP protein expression and therapeutic effect in the near future.ConclusionMultidrug resistance was correlated with radiotherapy of nasopharyngeal carcinoma closely. The alteration of radiosensitivity was different between different nasopharyngeal carcinoma cell lines after chemotherapy, and it was also different after reversial therapy between different nasopharyngeal carcinoma cell lines. Cyclosporine A and interferon showed no cytotoxity in reversial concentration no matter applied alone or combination. Radiotherapy can induce multidrug resistance to nasopharyngeal carcinoma. P-gp, MRP and GST-tc were correlated with multidrug resistance of nasopharyngeal carcinoma induced by radiotherapy, and TOP II was independent with multidrug resistance of nasopharyngeal carcinoma induced by radiotherapy. Cyclosporine A and interferon can reverse multidrug resistance of nasopharyngeal carcinoma induced by radiotherapy partly in protein level when applied after radiotherapy. Expression of P- gp was correlation with pathology, and expression rate of GST-a-, TOP II and MRP had nothing to do with pathology. Expression of P- gp, GST-a-, TOP II and MRP protein were independent with clinical stage and therapeutic effect in the near future. |
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