| Part I The mechanism of intrinsic resistance to TRAIL in lung cancer cellsObjective To verify the differences in TRAIL-sensitivity of A549and H460lung cancer cell lines. To analyze the relationship of the expression level of death receptors and the sensitivity to TRAIL in A549and H460cells. To explore the effect on TRAIL sensitivity of DR4gene expression suppressed A549cells. To detect the differences in the level of death receptors redistribution onto membrane lipid rafts induced by TRAIL in A549and H460cells. To investigate the correlation between TRAIL sensitivity and the death receptors translocation onto cell membrane lipid rafts in lung cancer cells.Methods TRAIL-induced cytotoxicity of A549and H460cells were detected by MTT assay. TRAIL-induced cell apoptosis was analysed by the flow cytometry. The expression levels of DR4/DR5of A549and H460cells were evaluated by RT-PCR and western blot. A549cells were transfected by siRNA targeted DR4, then confirmed suppressed expression of DR4gene through RT-PCR and western blot, and the TRAIL sensitivity of A549cells with DR4gene silence were analysed by MTT assay and flow cytometry. TRAIL-induced membrane aggregation levels of death receptors were detected by immunofluorescence combined flow cytometry. Lipid rafts and non-lipid raft were separated by discontinuous sucrose gradient centrifugation, where the proteins were analysed by western blot, to investigate the differences in location of DR4, DR5, and pro-caspase8of A549and H460cells. At last, the correlation between TRAIL sensitivity and the death receptors translocation onto cell membrane lipid rafts was reverse verified by TRAIL-sensitive H460cells with nystatin pretreatment.Results It was indicated that H460cells were sensitive to TRAIL and A549cells were intrinsic resistant to TRAIL by MTT assay and flow cytometric analysis. TRAIL-sensitive H460cell and TRAIL-resistant A549cell showed the similar mRNA and protein level of DR5, whereas A549cells showed a higher expression level of DR4than H460cells. Under DR4-siRNA transfection, the expression of DR4was restrained and DR5was not changed in A549cells, but there was almost no change in its apoptosis induced by TRAIL. With TRAIL pretreatment, H460cells showed a elevated formation of TRAIL contained immunocomplex in a time-dependent manner. However, the uptake of TRAIL in A549cells is almost no change. After lipid rafts and non-lipid rafts were separated by discontinuous sucrose gradient centrifugation, it was identified fractions4and5as lipid rafts by the presence of the lipid raft marker caveolin-1, and just H460cells not A549cells treated by TRAIL resulted in the redistribution of DR4, DR5and procaspase-8from the nonraft to the lipid raft fractions. At last, H460cells with nystatin pretreatment showed the failure of DR4, DR5and procaspase-8migrate into lipid rafts. As a result, the nystatin treatment significantly down-regulated the TRAIL-induced apotosis of H460cells.Conclusion H460cells were sensitive to TRAIL, and A549cells were intrinsic resistant to TRAIL. There is no positive correlation between the expression levels of death receptors and sensitivity to TRAIL, and DR4expression levels couldn’t affect the capacity of TRAIL in inducing apoptosis in A549cells. TRAIL-induced redistribution of DR4and DR5in lipid rafts contributed to the sensitivity to TRAIL in TRAIL-sensitive H460cell line, and the intrinsic resistance to TRAIL of A549cells is conferred by incapacity of death receptors redistribution in lipid rafts. Part II The establishment and identification of acquired TRAIL-resistant cell line H460RObjective To establish an isogenic aquired TRAIL-resistant cell line H460R from the TRAIL-sensitive human non-small cell lung cancer cell line H460, and confirm the differences in sensitivity to TRAIL of both H460and H460R cells, and assess whether H460R cells were cross-resistant to cisplatin.Methods Aquired resistant cell line model H460R was established on the basis of TRAIL-sensitive H460cell line, through the method of infinite dilution cloning experiments and low concentration induced by TRAIL. To confirm H460R cells of acquired resistance to TRAIL, H460and H460R cells were pretreated by various concentrations of TRAIL, then cellular morphology was directly observed through a optical microscope; cellular toxicity was detected by the CCK-8assay; TRAIL-induced cell apoptosis was analysed by the flow cytometry. At last, cisplatin dose survival rate of both cell lines were detected by the CCK-8assay.Results Acquired TRAIL-resistant cell line H460R was successfully established. The results of direct observation through an optical microscope:without any pretreatment, the morphology of H460R cells were the same as the parental H460cells; with the incubation of various concentrations of TRAIL, H460cells showed sensitive to TRAIL, whereas H460R cells showed resistant to TRAIL. The TRAIL-induced cell toxicity by measuring the percentage of cells with a CCK-8assay showed that the concentrations of TRAIL-induced50%cell death in H460cells was50ng/ml; In contrast, exposure of H460R cells at concentrations of TRAIL up to250ng/ml resulted in50%cell death, and the TRAIL-induced cell toxicity in both cell lines was in a dose-dependent manner, with a higher slope of dose-survival curve in H460cells. The cell apoptosis by flow cytometric analysis showed that H460cells exhibited a significant higher apoptotic rate than H460R cells after50ng/ml TRAIL pretreatment for4hours, which suggested that the acquired TRAIL-resistant cell model H460R was successfully established. At last, H460R cells and the parental H460cells were exposed to increasing concentrations of ciplatin and cell survival was determined by CKK-8. The result indicated a similar susceptibility to ciplatin of both cell lines.Conclusion Acquired TRAIL-resistant cell line H460R was successfully established. The morphology H460R cells was similar with the parental H460cells. H460R cells were detected to be acquired TRAIL-resistant and remained sensitive to ciplatin. Part III The mechanism of acquired resistance to TRAIL in lung cancer cellsObjective To detect the expression levels of death receptors in H460R cells and parental H460cells, and further investigate the relationship of the expression levels of death receptors and the sensitivity to TRAIL. To detect the differences in the levels of death receptors redistribution onto membrane lipid rafts induced by TRAIL in H460and H460R cells. To confirm the correlation between acquired TRAIL-resistance and the redistribution of death receptors in membrane lipid rafts of lung cancer cells.Methods The expression levels of DR4/DR5in H460and H460R cells were evaluated by RT-PCR and western blot. After DR4/DR5overexpression plasmid/siRNA transfection, the expression of DR4/DR5in H460and H460R cells was detected by RT-PCR to confirm the transfection efficiency, and the TRAIL sensitivity of which was analysed by CCK-8assay and flow cytometry. TRAIL-induced membrane aggregation levels of death receptors were detected by immunofluorescence combined flow cytometry. lipid rafts and non-lipid rafts were separated by discontinuous sucrose gradient centrifugation, where the proteins were analysed by western blot, to investigate the differences in location of DR4, DR5, and pro-caspase8in H460and H460R cells. We further examined the status of DR4and DR5localization in H460and H460R cells by confocal microscopy observation of immunofluorescence. At last, the correlation between acquired TRAIL-resistance and the death receptors translocation onto membrane lipid rafts was reverse verified by TRAIL-sensitive H460cells with nystatin pretreatment.Results Compared with the parental H460cells, the mRNA and protein expression levels of DR4/DR5were not altered in acquired TRAIL-resistant H460R cell, and the mRNA and protein expression levels of DR4/DR5were not altered in TRAIL-treated both cell lines. After DR4/DR5overexpression plasmid transfection, the TRAIL sensitivity of H460and H460R cells was significantly elevated; After DR4/DR5siRNA transfection, the TRAIL sensitivity of H460and H460R cells was significantly decreased. With TRAIL pretreatment, the parental H460cells showed a progressive increase of DR4and DR5on cell membrane in a time dependent manner, compared with the acquired TRAIL-resistant H460R cells. After lipid rafts and non-lipid rafts were separated by discontinuous sucrose gradient centrifugation, it was identified fractions4and5as lipid rafts by the presence of the lipid raft marker caveolin-1, and H460R cells showed a decrease of DR4, DR5and procaspase-8from the nonraft into the lipid raft fractions induced by TRAIL, compared with parental TRAIL-sensitive H460cells. Confocal microscopy observation of DR4and DR5localization in lipid rafts of H460and H460R cells was consistent with the previous results. At last, H460cells with nystatin pretreatment showed the failure of DR4, DR5and procaspase-8migrate into lipid rafts. As a result, the nystatin treatment significantly down-regulated the TRAIL-induced apotosis of H460cells.Conclusion The mRNA and protein expression levels of DR4/DR5in acquired TRAIL-resistant H460R cells are same as the parental H460cells, and the expression levels of DR4/DR5are not altered in TRAIL-treated both cell lines. Nevertheless, tests on both cell lines with DR4/DR5overexpression or silence suggest that death receptors expression on cell membrane surface is necessary but not sufficient for TRAIL-induced apoptosis. Our foundings indicate that death receptors redistribution mediated assembly of the DISC in lipid rafts is a critical upstream event in TRAIL induced apoptosis and its incapacity lead to the development of acquired resistance to TRAIL in H460R cells. |
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