| Chapter One Reversal of the taxol-resistant phenotype by cisplatin in nasopharyngeal carcinomaObject:To compare the difference of cell growth inhibition between taxol-resistance NPC cells and its parental cells treated by cisplatin, and observe if cisplatin reverse the taxol-resistance phenotype in NPC.Method:A taxol-resistant NPC cell line(CNE-1/taxol) and its parental cell line (CNE-1) were cultured in normal condition. The sensitivity of both CNE-1 and CNE-1/taxol to cisplatin was detected using the colony formation assay. Apoptotic death was measured by flow cytometry. The CI-isobologram was used to analyze the drug combination assays of taxol and cisplatin.Result:The IC50 value of CNE-1 and CNE-1/taxol was respectively 1.33±0.05nmol/L,11.24±0.51nmol/L when both cell lines treated by taxol. The index of CNE-1/taxol was 8.43. The IC50 value of CNE-1/taxol and CNE-1 was respectively 591.1±23.2 nmol/L and 1269.6±52.2 nmol/L when both cell lines treated by different doses of cisplatin (300nmol/L,600nmol/L,900nmol/L,1200nmol/L and 1500nmol/L). The sensitivity to cisplatin in CNE-1/taxol was significantly higher than in CNE-1 (p<0.01). When the cells were pretreated with low-dose cisplatin (IC20), the growth inhibition rates of taxol in CNE-1 cells remained unchanged, however, the rate significantly increased in CNE-1/taxol cells(p<0.01), its IC50 value was changed to 5.34±0.29nmol/L. The coadministration of taxol and cisplatin produced the synergistic effect in taxol-resistant NPC cells, but only additive effects in parental NPC cells. The apoptotic rate was significantly higher in CNE-1/taxol than in CNE-1 cells when both cell lines were treated by different doses of cisplatin (P＜0.01)Conclusion:CNE-1/taxol cells were more sensitive to treatment with cisplatin than its parental cells, CNE-1.Cisplatin reversed the taxol-resistant phenotype in NPC cells. Chapter Two Screening the differential expression of taxol-resistance related genes of nasopharyngeal carcinoma by cDNA microarrayObject:To screen the profiling of gene expression related to taxol resistance and reversal of taxol resistance, and search for genes related to taxol-resistance phenotype.Method:To detect the differential expression of CNE-1/taxol and CNE-1 treated by taxol and cisplatin using cDNA microarray.Result:297 genes was traped in our designed criteria through multiple steps. Some of these genes should be taxol-resistant genes.133 genes were down-expressed in CNE-1/taxol, and its expression level could be restored by cisplatin.164 genes were overexpressed in CNE-1/taxol, and its expression level could also be recovered by cisplatin. These genes involved in angiogenesis, cell growth, cell metabolism, apoptosis, etc.17 genes were screened out when the fold change must be more than 5, including TSP1,LAMC2,PSG7,NRG1,KRT14,AKAP12,G0S2,TMEM40,MYEOV,SERPINA3,CFI,MAOA,MYOM2,METTL7A,FOS,NOV,ABP1.1.Through Analyzing documented drug-resistant genes, MDR1 expression was not detected in all NPC samples. Eight ATP-binding cassette transporters that highly expressed in paclitaxel-resistance nasopharyngeal carcinoma cells were detected. However, the expression of these genes were not increased in CNE-1/taxol cells after treated by IC50 taxol.2.CYP1A1 of P450 family members was not expressed in CNE-1, but significantly increased expression was found in CNE-1/taxol and these increased expression could be restored by cisplatin.3.The expression level of 4 genes and 3 receptor genes of tumor necrosis factor family members were decresed in CNE-1/taxol, and restored by cisplatin. These genes included TNFAIP1,3,6,8 and TNFRSF10B,12A,21。4.The expression of CASP4 was decreased in CNE-1/taxol, but not changed by cisplatin. The other genes of Caspase family members were not significantly changed. 5.The expression ofβ-tubulin I and IV were not detected in all samples. No differential expression was found in P-tubulin III and VI when both CNE/taxol and CNE-1 cells treated or untreated by taxol and cisplatin(p>0.05). The expression ofβ-tubulin II were down-regulated in CNE-1/taxol.6.The expression of TXR1 was higher in CNE-1/taxol than in CNE-1. TSP1 was obviously down-regulated in CNE-1/taxol compared with CNE-1, and a more significant down-regulation of TSP1 was found when CNE-1/taxol treated by taxol. However, it was greatly up-regulation after treated by cisplatin in CNE-1/taxol.Conclusion:17 Significant changed genes in CNE-1/taxol may relate to taxol resistance and reversal of taxol resistance in NPC cells, such as TSP1, LAMC2, etc. ATP-binding cassette transporters (including MDR1) were not associated with taxol resistance of NPC, and P-tubulin isotype were not key genes in drug resistance. Caspase family members of apoptotic-related genes were nothing to do with CNE-1/taxol resistance. CYP1A1 may play a role of taxol resistance and reversal of taxol resistance in NPC cells.Chapter Three The role of TXR1/TSP1 in taxol resistance and reversal of taxol resistance in NPC cellsObject:To understand the role of TXR1/TSP1 in taxol resistance and reversal of taxol resistance in NPC cells.Method:Both CNE-1/taxol and CNE-1 cells were treated by different doses of taxol and cisplatin. The mRNA expression of genes was determined by RT-PCR, protein western of genes was detected by immunofluorescence and western blot.Result:Approximate 7-fold increase of TXR1 mRNA expression and 8.9-fold decrease of TSP1 mRNA expression were observed in taxol-resistant cells compared to their parental cells. An 8.7-fold increase in TSP1 mRNA expression was observed in CNE-1/taxol cells exposed to 590 nM cisplatin for 24 hours. An increase in TSP1 protein expression was obtained in a dose-dependent manner after CNE-1/taxol cells were exposed to cisplatin. However, there was no change in TXR1 mRNA expression after both CNE-1 and CNE-1/taxol cells were exposed to cisplatin.Conclusion:The TXR1/TSP1 regulation pathway may relate to taxol resistance in NPC cells. It was determined that cisplatin reverses drug resistance through the up-regulatin of TSP1 downstream of TXR1. |
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