| Objective:Cytological experiments was used to investigate whether rh-Apo2L can reverse cisplatin resistance; to investigate whether the reversal of cisplatin resistance is associated with PARP1, ERCC1; immunohistochemistry (IHC) method was used to detect ERCC1 and PARP1, we aimed to find the prognostic value of ERCC1 and PARP1.Methods:1. Totaling 81 Cases are from Fuzhou General Hospital, with the diagnosed as lung cancer patients, by retrospective analysis. Detect the ERCC1 and PARP1 by using immunohistochemical expression PV9000. Collect the data for statistical analysis with SPSS 16.0.2. A549/DDP cells were treated with various concentration of rh-Apo2L or Cisplatin for 24～48 hours. Proliferation inhibition rate of A549/DDP cells were measured by MTT assay, and half inhibitory concentration (IC50) were analyzed.3. A549/DDP cells were divided into four groups:control, rh-Apo2L, Cisplatin, rh-Apo2L combined with Cisplatin, every group was treated with associated conditions and administrations for 24 hours. The apoptosis rates of different groups were detected by FCM.4. Immunofluorescence was used to quantify the position and the relative expression quantity distribution of ERCC1 and PARP1 in A549/DDP.5. RT-PCR was used to quantify the change of the Cisplatin resistance related mRNA ERCC1、PARP1.6. Western blot was used to quantify the change of the Cisplatin resistance related protein ERCC1、PARP1.Results:1. We found no difference in ERCC1/PARP1 expression level with regarding to sex age,smoking status,pathology type,TNM stage,lymphnode metastasis in all 81 NSCLC patients.2.The survival curve of ERCC1 negative group is better than ERCC1 positive group(48.8mVS44.7m P=0.291), no difference was found on overall survival time, and no difference was found on disease free survival time between ERCC1 negative and positive group (34.1mVS25.1m,p=0.016). The survival curve of PARP1 negative group is better than PARP1 positive group(49.6mVS44m P=0.088),no difference was found on overall survival time, while difference was found on disease free survival time between PARP1 negative and positive group (36.2mVS23.5m, P<0.001).3.rh-Apo2L could induce lung adenocarcinoma cell line A549/DDP apoptosis, presenting obvious dose-dependent cytotoxicity, and IC50 of 24h was 397.1 μg/ml, while IC50 of 48h was 287μg/ml. Cisplatin also could induce lung adenocarcinoma cell line A549/DDP apoptosis, presenting Cisplatin obvious dose-dependent cytotoxicity, and IC50 of 24h was 139.17μg/ml, while IC50 of 48h was 99.06μg/ml. rh-Apo2L combined with Cisplatin can induce more lung adenocarcinoma cell line A549/DDP apoptosis, which has a significant difference between different groups (P<0.05). And the co-efficient of drug interaction (CDI) was 0.611.The apoptosis rate of the lung adenocarcinoma cell line A549/DDP was obviously increased by intervention of rh-Apo2L combined with Cisplatin (54.13±7.02%),compared to the group of rh-Apo2L (12.8±1.82%) or Cisplatin (13.03±8.38%) treatment, which has a significant difference(P<0.05).4. ERCC1 and PARP1 was expressed in A549/DDP and mainly located in the nucleus.5.The expression level of the related mRNA ERCC1、PARP1 was downregulated in group of rh-Apo2L combined with Cisplatin, and there was a significant difference between different groups (P<0.05).6.The expression level of the related protein ERCC1、PARP1 was downregulated in group of rh-Apo2L combined with Cisplatin, and there was a significant difference between different groups (P<0.05).Conclusions: 1.PARP1 is related to DFS in patients of non-small cell lung cancer.2.rh-Apo2L combined with cisplatin can apparently increase the apoptosis rate of the lung adenocarcinoma cell line A549/DDP and reverse the cisplatin resistance.3.The synergistic effect mechanisms of rh-Apo2L combined with cisplatin may be associated with the down regulation of ERCC1,PARP1 mRNA and protein level. |
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