| Data have showed that pulmonary carcinoma has been one of the major tumor types resulting in death, and at present there is an upward tendency. Pulmonary carcinoma is often divided into two types: small cell lung cancer (SCLC) and non-small cell lung cancer (non-small cell lung cancer, NSCLC). NSCLC is the common type of lung cancer, which is about 80% of lung cancer. Furthermore, pulmonary carcinoma can not be perceived until in its advanced stage. Along with the development of chemotherapy drugs, paclitaxel has been proved to be the significant anti-tumor activity against the advanced non-small cell lung cancer. However, the emergence of obtained drug resistance has impeded the effective treatment of NSCLC greatly. Paclitaxel is a new type of anti-tubule drugs which is extracted from yew barks, and its broad spectrum of anti-tumor effect has been generally recognized. At present, paclitaxel has been used for the treatment of ovarian cancer, breast cancer, non-small cell lung cancer, bladder cancer, head and neck tumors. The mechanism of resistance of paclitaxel is more complex and it is not clear, it may be have a variety of mechanisms.DNA polymerase beta (DNA polβ) is an important repair gene for DNA damage in eukaryote cells. As a housekeeping gene, the DNA sequence of DNA polβgene is highly conservative. The main function of DNA polβis to excise and repair DNA base mutation, and the expression level of DNA polβis relatively stable in the whole cell cycle without being regulated by proliferation of cell cycle. It has been reported that DNA polβgene overexpressions have been found in many tumor tissues. The overexpressions of DNA polβgene which lead to the increase of genomic instability and the acquisition of mutation phenotype may be an important molecular mechanism for mediating the formation of multidrug resistance.However, at present the studies on the expression of DNA polβgene in the human lung adenocarcinoma resistance cells, and its relation to the occurrence of MDR have not been reported yet both at home and at abroad.The multidrug resistance (MDR) is one of the major obstacles in successful chemotherapy of cancer. The investigation of resistant mechanism developed by tumor and the search for new and potent reversal agents of MDR are the focus of the development of new chemotherapeutic agents. To establish MDR cell line in vitro is an important method to study the MDR mechanism and search the new reversal agents of MDR.In this study, at first, human lung adenocarcinoma MDR cell lines (A549/TXL20) were established in vitro by exposure to stepwise increased concentrations of the paclitaxel in a cell culture medium for nearly 180d, and biological characteristics of the drug-resistance cell line were valued preliminarily. Then, the mRNA expressions of DNA polβand some drug-resistance related gene were measured. Meanwhile, on account of the overexpression of DNA polβin human lung adenocarcinoma MDR cell line A549/TXL20, we utilized RNA interference (RNAi) technique to construct DNA polβtargeted siRNA eukaryotic expression vectors, and then transfected the vectors into A549/TXL20 cell lines to observe the silencing effects of DNA polβtargeted siRNA on the expressions of DNA polβ, and to research the effect of DNA polβtargeted siRNA eukaryotic expression vectors on its MDR reversal and reversal mechanism after transfected into A549/TXL20.Partâ… Establishment and biological characteristics of human lung adenocarcinoma multidrug resistance cell lines induced by paclitaxel Methods1. A human lung adenocarcinoma multidrug-resistant cell lines (A549/TXL20) was established in vitro by exposure to stepwise increased concentrations of the paclitaxel in a cell culture medium.2. Methyl tetrazolium assay was performed to value the chemoresistance indexes of cell lines and to measure the cross-resistance of the cell lines to cisplatin, mitomycin, 5-fluouracil (5-FU) and vincristin respectively.3. Drug sensitivity of cell lines to paclitaxel was evaluated by colonyforming assay4. Drug concentration in cultured cells of paclitaxel was analyzed by solid-phase of extraction high performance liquid chromatographyResults1. Compared with A549 cell lines, the decrease of cellular size, and the increase of N/C ratio were found in the A549/TXL20 cell lines.2. The growth property of A549/TXL20 didn't change significantly compared with A549.3. The resistant cells, A549/TXL20, were 19.3 folds more resistant to paclitaxel and 67.4 folds more resistant to cisplatin than the parent cells. The resistant cells also demonstrated cross-resistance to mitomycin, vinblastine, and 5-fluouracil (5-FU).4. Compared with the A549 cell lines, an unreasonably higher level of drug-resistance and lower drug concentration was detected in A549/TXL20 cells after exposure to the drug in a culture medium.Partâ…¡The expression of DNA polymeraseβand multidrug resistance-related genes (mdrl, mrpl,GST-Ï€, lrp, and topoâ…¡)in a human lung adenocarcinoma MDR cell lines A549/TXL20 and a human lung adenocarcinoma cell lines A549 Methods1. Total RNA was extracted from human lung adenocarcinoma multidrug-resistant cell lines A549/TXL20 and human lung adenocarcinoma cell lines A549, and was reversely transcribed to cDNA.2. DNA polβ, mdrl, GST-Ï€, mrp1, lrp and topoâ…¡genes were amplified by semi-quantitative PCR whileβ-actin was performed as inner control. The mRNA expression levels of these genes in human lung adenocarcinoma multidrug-resistant cell lines A549/TXL20 and human lung adenocarcinoma cell lines A549 were analyzed.3. Western blot assay was preformed to measure the expression of DNA polβprotein in human lung adenocarcinoma multidrug-resistant cell lines A549/TXL20 and human lung adenocarcinoma cell lines A549.4. Statistical analysis SPSS 12.0 was used to perform t test, analysis of variance P value less 0.05 was considered as statistically significant.ResultsThe expressions of mRNA of DNA polβ, mdrl, GST-Ï€, mrpl, lrp and topoâ…¡genes by RT-PCR assay indicated: 1. The mRNA expression levels of DNA polβgene in human lung adenocarcinoma MDR cell lines A549/TXL20 were 0.9862±0.1768, whereas the expressions of DNA polβin human lung adenocarcinoma cell lines A549 were 0.2531±0.048. The results showed that DNA polβgene mRNA expressions in human lung adenocarcinoma MDR cell lines A549/TXL20 was significantly higher than those in human lung adenocarcinoma cell lines A549(P＜0.05).2. The mRNA expression level of mdr1 gene in human lung adenocarcinoma MDR cell lines A549/TXL20 were 1.3563±0.4953, whereas the expressions of mdrl in human lung adenocarcinoma cell lines A549 were 0.4032±0.2506. The results showed that mdrl gene mRNA expressions in human lung adenocarcinoma MDR cell lines A549/TXL20 were significantly higher than those in human lung adenocarcinoma cell lines A549(P＜0.05).3. The mRNA expression level of GST-Ï€gene in human lung adenocarcinoma MDR cell lines A549/TXL20 were 1.6577±0.1892, whereas the expressions of GST-Ï€in human lung adenocarcinoma cell lines A549 were 0.5223±0.1769. The results showed that GST-Ï€gene mRNA expressions in human lung adenocarcinoma MDR cell lines A549/TXL20 were significantly higher than those in human lung adenocarcinoma cell lines A549(P＜0.05).4. The mRNA expression level of mrp1 gene in human lung adenocarcinoma MDR cell lines A549/TXL20 were 1.2109±0.4361, whereas the expressions of mrpl in human lung adenocarcinoma cell lines A549 were 0.5219±0.2646. The results showed that mrpl gene mRNA expressions in human lung adenocarcinoma MDR cell lines A549/TXL20 were significantly higher than those in human lung adenocarcinoma cell lines A549(P＜0.05).5. The mRNA expressions level of 1rp gene in human lung adenocarcinoma MDR cell lines A549/TXL20 were 1.5783±0.1402, whereas the expressions of 1rp in human lung adenocarcinoma cell lines A549 were 0.6479±0.2642. The results showed that 1rp gene mRNA expressions in human lung adenocarcinoma MDR cell lines A549/TXL20 were significantly higher than those in human lung adenocarcinoma cell lines A549(P＜0.05).6. The mRNA expressions level of topoâ…¡gene in human lung adenocarcinoma MDR cell lines A549/TXL20 were 0.5247±0.0174, and the expressions of topoâ…¡in human lung adenocarcinoma cell lines A549 were 0.4925±0.3345. The results showed that topoâ…¡gene mRNA expressions in human lung adenocarcinoma MDR cell lines A549/TXL20 were similar to those in human lung adenocarcinoma cell lines A549, and there was no significant difference of topoâ…¡gene mRNA between the A549/TXL20 cell lines and A549 cell lines (P＞0.05).7. Western blot showed that the expressions of DNA polβprotein in the A549/TXL20 celllines were 94.3±0.36, and significantly higher than those in A549 cell lines (56.82±0.53), (P＜0.05).Partâ…¢The construction and identification of DNA polβtargeted siRNA expression vectorMethods1. Single strand DNA templates encoding short hairpin siRNA against DNA polβwere designed and synthesized.2. Both single strand DNA templates encoding short hairpin siRNA against DNA polβwere annealed and purified;3. Target DNA templates encoding short hairpin siRNA against DNA polβwere added to adenine (A), and T-A clone were constructed, and then the recombinant was screened and identified.4. Target DNA templates encoding short hairpin siRNA against DNA polβwere subcloned into expression vector pslience2.0-GFP and then the recombinant was screened and identified.Results1. According to DNA polβsequence, two pairs of single strand DNA template encoding small interference RNA (siRNA) were designed and synthesized, and DNA polβtargeted siRNA eukaryotic expression vectors pslience2.0-GFP-sipolbl were successfully constructed.2. According to DNA polβsequence, two pairs of single strand DNA template encoding small interference RNA (siRNA) were designed and synthesized, and DNA polβtargeted siRNA eukaryotic expression vectors pslience2.0-GFP-sipolb2 were successfully constructed.3. According to DNA polβsequence, two pairs of single strand DNA template encoding small interference RNA (siRNA) were designed and synthesized, and DNA polβcontrol siRNA expression vector pslience2.0-GFP-SC were successfully constructed.Partâ…£The effect of DNA polβtargeted siRNA eukaryotic expression vectors on its MDR reversal and reversal mechanism after transfected into A549/TXL20Methods1. DNA polβtargeted siRNA eukaryotic expression vectors were transfected into human lung adenocarcinoma MDR cell lines A549/TXL20 by lipofectin reagent. After selection of G418, A549/TXL20 cell lines with stable overexpressions of polβtargeted siRNA were obtained.2. Methyl tetrazolium assay was performed to value the MDR reversal by polβtargeted siRNA to A549/TXL20 cell lines on paclitaxel and cisplatin respectively.3. The naked mouse models of grafting tumor were established by injecting subcutaneously transfected A549/TXL20 cells and untreated A549/TXL20 cells in axillas, and observed the effects of polβtargeted siRNA on grafting tumor.4. Semi-quantitative RT PCR was performed to detect the expressions level of mRNA of DNA polβ, mdr1, GST-Ï€and mrp1 genes in transfected cells.5. Western blot assay was preformed to measure the expression of DNA polβprotein in transfected A549/TXL20 cells and untreated A549/TXL20 cells.6. Gene spontaneous mutation rate in transfected cells was assayed by HGPRT gene test.7. The statistical analysis SPSS 12.0 software was used to perform t test, the analysis of variance. P value less than 0.05 was considered as statistically significant.Results1. The A549/TXL20 cell lines with stable overexpressions of polβtargeted siRNA were obtained.2. Compared with A549/TXL20 cell lines, the IC50 value of A549/TXL20 cells transfected with sipolbl and in A549/TXL20 cells transfected with sipolb2 both to paclitaxel and to cisplatin were significantly decreased. Meanwhile, there was no significant difference of IC50 value to paclitaxel and cisplatin in A549/TXL20 cells transfected with SC and A549/TXL20 cells transfected with empty vectors compared with A549/TXL20 cell lines.3. In the naked mouse models of human lung adenocarcinoma MDR cell line A549/TXL20, the tumor growth rate of sibolbl group and sibolb2 group were 20.26±0.25 and 20.87±0.39 respectively, which were significantly lower than that in A549/TXL20 group. Compared with A549/TXL20 group, the tumor weight of sibolb1 group and sibolb2 group were 2310±131mg and 2305±163mg respectively, which decreased significantly, (P＜0.05); The tumor inhibitory rate of sibolbl group and sibolb2 group were 25.6% and 25.8% respectively.4. RT-PCR indicated that there existed mRNA overexpression of DNA polβand mdr1 genes in untreated A549/TXL20 cells and in A549/TXL20 cells transfected with control siRNA vectors (pslience2.0-GFP-SC) and empty vectors (pslience2.0-GFP) respectively, but there was no significant difference among them, P＞0.05. However, compared with A549/TXL20 cells, the mRNA expression levels of in DNA polβand mdrl genes in A549/TXL20 cells transfected with pslience2.0-GFP-sipolb1 and pslience2.0-GFP-sipolb2 respectively, were significantly decreased, and these results showed that DNA target siRNA may decrease mRNA expression levels of DNA polβand mdrl mRNA in A549/TXL20 cells. There existed mRNA overexpressions of GST-Ï€, mrp1, and 1rp genes in untreated A549/TXL20 cells and in A549/TXL20 cells transfected with pslience2.0-GFP-sipolb1, pslience2.0-GFP-sipolb2, control siRNA vectors (pslience2.0-GFP-SC) and empty vectors (pslience2.0-GFP) respectively. The results showed that DNA target siRNA had little effect on mRNA expressions of GST-Ï€, mrp1 and lrp genes.5. Western blot showed that there existed mRNA overexpression of DNA polβprotein in untreated A549/TXL20 cells and in A549/TXL20 cells transfected with control siRNA vectors (pslience2.0-GFP-SC) and empty vectors (pslience2.0-GFP) respectively, but there was no significant difference among them, P＞0.05. However, compared with A549/TXL20 cells, the expression levels of in DNA polβprotein in A549/TXL20 cells transfected with pslience2.0-GFP-sipolb1 and pslience2.0-GFP-sipolb2 respectively, were significantly decreased, and these results showed that DNA target siRNA may decrease expression levels of DNA polβprotein in A549/TXL20 cells.(P＜0.05).6. Compared with A549/TXL20 cells transfected with control siRNA vectors (pslience2.0-GFP-SC) and empty vector (pslience2.0-GFP) and untreated A549/TXL20 cells, the spontaneous mutation rate of HGPRT gene in A549/TXL20 cells transfected with pslience2.0-GFP-sipolb1, pslience2.0-GFP-sipolb2 significantly decreased. Conclusions1. The paclitaxel-induced human lung adenocarcinoma MDR cell lines A549/TXL20 are successfully established which are cross-resistance to cisplatin, mitomycin, 5-FU and vincristin, and may be used as an experimental system for the search of a means to overcome drug resistance and elucidate possible mechanisms of acquired MDR involved in human lung adenocarcinoma.2. It is the first time to reveal that the mRNA and protein expression levels of DNA polβgene in human lung adenocarcinoma MDR cell lines A549/TXL20 are significantly higher than those in human lung adenocarcinoma cell lines A549, which indicates that overexpressions of DNA polβmight confer the development of drug resistance in A549/TXL20 cell lines.3. The overexpressions of mdr 1, GST-Ï€, mrp1 and 1rp genes existed in human lung adenocarcinoma MDR cell lines A549/TXL20, and there is no significant difference of expressions of topoâ…¡gene between A549/TXL20 cells and A549 cells. The results indicate that the overexpressions of mdr 1, GST-Ï€, mrp1 and lrp genes might be related to the formation of A549/TXL20 MDR.4. DNA polβtargeted siRNA eukaryotic expression vectors (pslience2.0-GFP-sipolb1, pslience2.0-GFP-sipolb2) are successfully constructed and successfully transfected into human lung adenocarcinoma MDR cell lines A549/TXL20, which can significantly inhibit the mRNA expressions of DNA polβgene.5. DNA polβtargeted siRNA may result in reversal resistance to paclitaxel and cisplatin of A549/TXL20 in vitro study, and may decrease tumor growth rate of human lung adenocarcinoma grafting tumor of naked mouse significantly in vivo study.6. DNA pol targeted siRNA may decrease the mRNA expression levels of DNA polβand mdr1 genes in A549/TXL20 cell lines, decrease the expressions of DNA polβprotein, and decrease spontaneous mutation rate of HGPRT gene significantly.7. The results of comprehensive studies show that human lung adenocarcinoma multidrug-resistant cell lines (A549/TXL20) are established in vitro by exposure to stepwise increased concentrations of the paclitaxel in a cell culture medium, whose multidrug resistant mechanism may have relation to mRNA overexpressions of DNA polβ, mdr 1, GST-Ï€, mrpl and lrp genes; DNA polβtargeted siRNA may reverse the drug resistant characteristics of A549/TXL20 cell lines to some extent. The overexpressions of DNA polβgene may result in formation of A549/TXL20 MDR by inducing the mRNA overexpressions of mdr 1 gene and increasing the spontaneous mutation rate of HGPRT gene. |
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