| BackgroundLung cancer is one of the highest incidence of malignant tumors in worldwide. In China, the incidence of lung cancer increased year by year, and has become the leading cause of cancer deaths. Most lung cancer patients have been diagnosed in an advanced stage, especially lung cancer ccounted for 80% of non-small cell lung cancer(Non-small cell lung cancer, NSCLC), and lost the chance of surgical treatment. Therefore, chemotherapy has become one of the main means of treatment of lung cancer. Even though the treatment has made some progresses, in the past 25 years, the 5-year survival rate yet to see significant increase of only 15%. In lung cancer chemotherapy, cisplatin(DDP) based combination chemotherapy is NSCLC first-line chemotherapy drugs, but because of the problem of drug resistance, the effect of chemotherapy significantly reduced, which has become plagued by lung cancer treatmentis a major problem. Therefore, searching more specific platinum drug resistance-associated molecular targets, changing lung cancer platinum drug resistance and resistance reversal mechanism has become one of the hotspots of clinical research at present.Multidrug resistance (MDR) is one of the main failure of chemotherapy in lung cancer. MDR refers to the phenomen on that tumor cells have intrinsic or acquired cross resistance to multiple chemotherapeutic agents, thus impeding the effective treatment of cancer. Research showed that multidrug resistance mechanism is very complex, which is the combined effects of multi-gene and multiple steps. Multidrug Resistance (MDR) is defined as insensitivity to administered medicines that are structurally unrelated and have different molecular targets. Cancers possess numerous mechanisms of drug resistance, involving various aspects of cell biology. A pivotal role in this phenomenon is played by proteins-enzymatic or structural parts of the cell.The mechanisms of resistance to chemotherapeutic drugs is extremely complex, oncogenes, tumor suppressor genes as well as several cytokines may be involved in the formation of drug-resistant process, but the exact mechanism is not fully clear. Study of the cells drug efflux transporters has become one of the most the most widely and effectively studied in the mechanisms of MDR. The molecular research shows that leading to the MDR gene encoded by membrane transporters, including the main members of ABC protein family:P-gp, MRP1 and BCRP, as well as LRP, which builds structure of vaults, determine the multidrug-resistant phenotype by decreasing drug concentration within the cell or modifying its distribution to intracellular compartments. At present, using a variety of MDR inhibitors interfere with this transport protein efflux pumps strategy in the different research phase. Human multidrug resistance gene (MDR gene) have two main subtypes:MDR1 and MDR2. Multidrug resistance are mainly mediated by MDR1 encoded gene product. Among them, the most extensively study is the P-gp. P-gp is a 170kD transmembraneglyco protein encoded by the MDR1 gene on chromosome 7. The expression of P-gp was positively correlated with cell resistance. P-gp expression in non-small cell lung cancer (NSCLC) is higher than small cell lung cancer (SCLC), in adenocarcinoma is higher than squamous cell carcinoma, is especially higher in the-well-differentiated adenocarcinoma, which is consistent with their insensitivity to chemotherapy.There was an significant correlation between P-gp expression level and cancer drug resistance, the choice drug of drug, prognosis, but irrelevant with the staging of lung cancer. One of the main reasons of MDR is chemotherapeutic drug-induced apoptosis istolerated by tumor cells. Apoptosis tolerance was mainly caused by the apoptosis suppressor gene, such as overexpression of Bcl-2, Survivn, and mutant P53.Bcl-2 is the most intensively studied and most important inhibitor of apoptosis genes. Bcl-2 gene is located on chromosome 18q21, most tumors can occur inpatients with chromosome (14; 18) translocation, so that Bcl-2 gene is located on chromosome 14q32, close to the immunoglobul in heavychain transcriptional enhancer, resulting in high expression of Bcl-2. Overexpression of Bcl-2 gene may promote cellsurvival, it also suppressed apoptosis induced by chemotherapeutic drugs. Cell with Bcl-2 overexpression, chemotherapy drugs can still enter the cell and induce cell injury, but the damage can not be efficiently converted into apoptosis signal, so that apoptosis is inhibited and the expression of Bcl-2 in cancer cells during chemotherapytore-grow at a faster rate, finally resulting in drug resistance.Recent studies have shown that RhoE gene also affects the tumor resistance. RhoE geneis closely related with tumor development, invasion, metastasis and prognosis. RhoE is an atypical RhoGTPase family members, and it has been in the activated GTP-bound state. Recently, some studies have found that the expression of RhoE downregulated in some lung cancer cell lines and cancer tissues was downregulated, which can promote the invasion and metastasis of tumor cells. RhoE has many important functions, which can regulate cytoskeletal reorganization and cell morphology, cell proliferation, apoptosis and differentiation.Inhibitors of differentiation/DNA binding (Id) proteins, which belong to the group of helix-loop-helix (HLH) transcription factor family members, play a negative regulatory role due to lack the basic DNA binding domain. There are four members of Id family (Idl, Id2, Id3 and Id4). In recent years, studies have shown that Id protein family not only inhibit cell differentiation, promote cell proliferation, but also have a broader biological effects, such as regulate cell fate, orientation differentiate and promote embryogenesis and organ formation, induce apoptosis, maintain cell survival, angiogenesis, promote tumor invasion, oncogene. Id protein especially Idl not only involved in normal cell growth and development, also overexpression in many tumors. It has been confirmed that Idl expression levels related with invasion and metastasis of tumor cell. Lwatsuki found that expression of Idl in bone marrow and peripheral blood are higher than without distant metastasis of gastric cancer in patients with gastric cancer lymph node metastasis and peritoneal metastasis. And proposes Id1 is the "true indicator" of gastric cancer with lymph node metastasis and peritoneal. Many studies have confirmed Idl expression in tumor cells associated with differentiation and invasiveness of tumor cells. Id1 are considered to be "quasi-oncogene" by researchers, which is widely studied. It has been widely recognized that biological effects of Idl in tumors. However, the complexity regulation mechanism of Id3 determine the diversity of its functions. Compared with Id1, Id3 in cell cycle regulation and its mechanism still need to be further studied.Id3 gene was firstly identified as a serum inducible immediate early gene in an established murine fibroblastic cell line. The expression of Id3 is broad and specific cell types, its regulation mechanism is very complex. Id3 expression pattern and regulation mechanism is not identical with Id1. Id3 has different expression patterns in tumor tissues and cell lines. Id3 expression in primary colorectal cancer and small cell lung cancer are high expression level, but in thyroid cancer, ovarian cancer and prostate cancer are low expression level. Id3 gene knockout ((Id3-/-) mice can lead to occurrence of γδT cell lymphoma similar to humans, suggesting that γδT cell lymphoma patients may exist Id3 gene mutation. In the previous study, we found that: ①There is not Id3 expression in PBMC, but the degree of expression in tumor cells has great difference. In human prostate cancer cell line PC-3M, Id3 expression level is the highest, significantly higher than lung adenocarcinoma cells (A549), ovarian cancer cells (COC2), Burkitt’s B lymphomacell (Daudi), T lymphocytic cell leukemia (Jurkat), and so on. ②Indirect immunofluorescence assay (IFA) showed that, Id3 protein expression in A549 cells was significantly lower than that in MCF7 and PC-3M cells. The above results suggest that the expression of Id3 is different according to the level of the cell types and different stages of growth and development, so as to perform a variety of special function of cell biology.According to the literature reports,.. in addition to Id3 can promote cell cycle progression, inhibit cell differentiation, Id3 also plays an important role in cell apoptosis. In early 2001, Kee found Id3 can lead to BLPs growth arrest and apoptosis when Id3 was imported in BLPs by retroviral vector, overexpression of exogenous Id3 in the BLPs can be simulated TGF beta induced BLPs apoptosis. Recently, Lee has found that X ray irradiated mice epidermis and HaCaT keratinocytes formed cells can lead to intracellular Id3 up-regulated and Id3 recombinant adenovirus in HaCaT cells can induce cell apoptosis, and enhanced X-ray induced apoptosis sensitivity. Our previous studies revealed that Id3 gene is down-regulated in human lung adenocarcinoma epithelial cells (A549), and the exogenous expression of Id3 gene in A549 cells can induce cell proliferation inhibition and apoptosis. In the previous study we found that: ①Id3 gene is down-regulated in human lung adenocarcinoma epithelial cells(A549), and MTT, Annexin V/7AAD-FCM and Hoechst 33258 nuclear staining results showed that the exogenous expression of Id3 gene in A549 cells can induce cell proliferation inhibition and apoptosis.②iRNA interference vector was successfully constructed by RNA interference targeting Id3 gene (pcDNA/miRId3), which can inhibit the expression of A549 Id3 in cells in vitro and reversed the founction of cell proliferation and apoptosis by exogenous expression of Id3.③The Id3 gene was cloned into the adenovirus expression vector pAxCAwtit. Prepared the recombinant adenovirus Ad-Id3 after transfected into 293 cells. The results showed that adenovirus mediated transgene expression of Id3 can inhibit A549 cells proliferation. These results suggested that Id3 transgenic expression in A549 cells can induce apoptosis and inhibit cell proliferation.Id as a negative regulator which inhibit apoptosis by anticancer drugs, is expected to become a new therapeutic target for enhanced sensitivity to chemotherapy. In cisplatin induced apoptosis of sarcoma MG63 experiments, overexpression of Id3 may lead to increased sensitivity of cells to cisplatin induced apoptosis. Studies have shown that Idl not only as a molecular marker of lung cancer prognosis, and downregulate the expression of Idl can increase the sensitivity of lung cancer chemotherapy, but its mechanism is still not clear. Li found that by down regulating the expression of Idl can enhance the sensitivity of gastric cancer MGC803 cells and AGS cisplatin resistance; at the same time, Li also found that through down-regulation of p53 expression in negative regulation of PTEN protein at the level of transcription, lead to the activation of PI3K/Akt/Wnt signaling pathway, resulting in cell proliferation occurs, so that the PI3K/Akt signaling pathway may be involved in the biological role of Idl, such as multiple biological effects of cell proliferation, anti apoptosis, invasion and metastasis. Recent studies also found that Id1 and Id3 can serve as a marker for the prognosis of chemotherapy for patients with non small cell lung cancer stage III N2. The two proteins may play a different role in the different treatments of tumors, especially in the treatment of tumor chemotherapy.Cisplatin is a nonspecific cell cycle cytotoxic drug, its main target is DNA. The main mechanism is the role in the DNA chain and chain cross chain, formed DDP-DNA complexes, interfering with DNA replication or combined with nuclear proteins and cytosolic proteins, causing cancer cell DNA replication barriers and arrest the rapidly proliferating cells in the G2/M phase of the cell cycle and induced cell apoptosis, with strong broad-spectrum anti-cancer effect. At present, it is believed that inhibition of cancer cell apoptosis, increase of shear in the process of repairing DNA, and changes of expression of regulatory protein related with signaling pathway and drug intake reduce lead to intracellular DDP concentration decrease may be associated with cisplatin resistance. However, drug resistance mechanism in lung cancer cells has not been fully elucidated. The study on mechanism of cisplatin resistance in lung cancer is mainly in the following aspects:1, drug iptake disorder or elimination of obstacles resulting in increased intracellular drug concentration decreased; 2, enhance cell detoxification function; 3, DNA damage and repair dysfunction; 4, inhibition of cell apoptosis. In addition, angiogenesis, cytoskeletal abnormalities, abnormal extracellular matrix surrounding abnormalities are also involved in cisplatin resistance in lung cancer. Therefore, further study cisplatin resistance mechanism and find new targets for reversing resistance to improve the effect of chemotherapy of lung cancer to improve the survival rate of 5 years has important significance.The main mechanism of the anti-tumor effect of cisplatin is induced tumor cells apoptosis. In vivo/vitro study found that the change of apoptosis of tumor cells mainly due to the apoptosis signal transduction pathway abnormalities, and the abnormal expression of apoptosis related factors.Our previous studies show that overexpression of Id3 can induce A549 and A549/DDP cells apoptosis. A recent study found that the PI3K/Akt pathway is the most important anti-apoptotic pathway, also found that PI3K/Akt signaling pathway is closely related with tumor resistance. We hypothesize that Id3 may through the PI3K/Akt signaling pathway to regulate lung adenocarcinoma cisplatin resistance. Therefore, this study is intended to non-small cell lung cancer cells and cisplatin-resistant cells as the main object of study, observed the change of biological behavior and resistance to cisplatin before and after adding cisplatin in case of overexpression of Id3. To investigate whether Id3 related with cisplatin resistance in lung cancer. Study the effect of Id3 overexpression on cell sensitivity to cisplatin. Clarify the molecular mechanism of regulation of Id3 lung cancer cells cisplatin resistance, and provide a theoretical basis and experimental evidence for further analysis Id3 can become resistant to cisplatin reversal lung molecular targets.This study was conducted according to the following three parts:Part 1:Analysis of drug resistance of A549/DDP cells and study on the expression level of Id3 gene and protein in cellObjective:To analyze the drug resistance of A549/DDP cells to cisplatin, as well as the expression level of Id3 gene and protein in the cells.Method:1. CCK-8 method was used to determine the inhibitory effect of different concentration of DDP on A549/DDP and A549 cells, and calculate the half inhibitory concentration (IC50), calculated the resistance index of cells;2. RT-PCR, Western Blot detect the expression level of Id3 mRNA and protein in A549/DDP cell.Result:1. Cisplatin can significantly inhibit the proliferation of human lung adenocarcinoma A549, cisplatin has certain inhibitory proliferation effect on A549/DDP cells, but there are obvious tolerance phenomenon, at higher concentrations (20μg/ml) showed certain inhibition. The inhibition was concentration dependent, according to a linear regression calculated in two cell lines to cisplatin half inhibitory concentration(IC50). DDP on A549 cells with an IC50was 5.32μg/ml, was 19.38μg/ml on A549/DDP cells, so the resistance index of A549/DDP cells to DDP is 3.64;2. RT-PCR and Western blot results showed that Id3 mRNA and protein in the resistant strains A549/DDP and parental A549 cells expression level is low, two cell lines without significant difference.Conclusion:1. Effect of DDP on the parent cell line A549 and multidrug resistant cell line A549/DDP in a concentration dependent manner; the resistance index of A549/DDP cells to DDP is 3.64;2. Id3 expression level in multidrug resistant human adenocarcinoma cells is low.Part 2 Overexpression of Id3 gene in A549/DDP cells and its role in reversing cisplatin resistanceObjective:Explore the reversal effect of apoptosis inducing factor Id3 resistance to DDP.Method:1. human lung adenocarcinoma cells (A549 and A549/DDP) and human lung adenocarcinoma cell line (SPC-A-1) were transfected into by lipofectamineTM2000, fluorescence microscopy observe the EGFP-Id3 fusion protein expression within three groups of cells, inverted microscope observe Id3 morphological changes in human lung adenocarcinoma cells;2. Fluorescence quantitative RT-qPCR detect the expression of Id3 in A549 cells and A549/DDP cells;3. CCK-8 method was used to detect the reversal effect of overexpression of Id3 in drug resistance of A549/DDP cells;4. A549/DDP cells were transfected with Id3 and added different concentrations of DDP (0.0μg/ml, 1.0μg/ml,2.0μg/ml) after 24h, using Annexin V-FITC and PI double staining combined with flow cytometry flow cytometry to detect the apoptosis rate.Results:1. Under the fluorescence microscope observation, cells untransfected with the plasmid were not green fluorescence, cells itransfected with pEGFP/Id3 that green fluorescent was mainly expressed in the nucleus, cells transfected with empty vector pEGFP that green fluorescence expressed throughout the cell, and the fluorescence was diffuse, uniform distribution, there was no significant difference in the cytoplasm and nucleus;2. Fluorescence quantitative qRT-PCR results showed that, compared with control cells and transfected with empty vector pEGFP group, Id3 mRNA expression were significantly increased in A549/DDP cells transfected with pEGFP/Id3 group (P< 0.05).3. Compared with the control group, Id3 transfected A549/DDP cells can significantly increase the sensitivity of cells to cisplatin resistance, the reversal index increases 1.66 times;4. Compared with the untreated group and pEGFP/control group, Id3 after transfection into A549/DDP cells, the apoptosis rate was significantly increased. And the apoptotic rate increased with the increase of DDP concentration, which indicated that the inhibitory effect of Id3 gene and DDP on cell growth was synergistic.Conclusion:Id3 gene and DDP have a synergistic effect on the inhibition effect of A549/DDP cells. The sensitivity of cells to DDP was significantly increased after the overexpression of the Id3 gene, the cell apoptosis increased, and the drug resistance was partly reversed.Part 3 Analysis of the mechanism of Id3 reversing the drug resistance of A549/DDP in cisplatinObjective:To investigate the reversal effect and the molecular mechanism of apoptosis inducing factor Id3 on multidrug resistance in DDP.Method:1. Western blot and Real time PCR to detect the expression of multidrug resistance MDR protein, RhoE protein, Bcl-2 protein, and the changes of Akt phosphorylation.2. Flow cytometry to detect the apoptosis of A549/DDP cells and the expression of multidrug resistance MDR protein.Results:1. Real time PCR detection found that compared with the control group, the Id3 transfected A549/DDP cells in tumor drug resistance related gene MDR and Bcl-2 mRNA expression levels were significantly reduced;2. Western blot detection found that compared with the control group, the Id3 transfected A549/DDP cells in tumor drug resistance related gene MDR and Bcl-2 protein expression levels were significantly reduced, and RhoE protein expression level was significantly increased (P<0.05);3. At the same time, Western blot analysis also found that compared with the control group, the expression of p-Akt expression was significantly decreased in Id3 transfected A549/DDP cells (P<0.05), suggesting that Id3 may inhibit the activity of PI3K/Akt signaling pathway, thereby inhibiting cell proliferation, promote apoptosis and reverse the drug resistance. Id3 has negative moderating effect in this pathway;4. Flow cytometry detection found that, compared with the control group, in drug resistance related gene MDR protein expression levels were significantly reduced in the Id3 transfected A549/DDP cells, the difference was statistically significant (P<0.05), and consistent with Western blot detection.Conclusion:Id3 can reverse the drug resistance to cisplatin A549/DDP, its mechanism may be related to the inhibition of drug efflux, the expression of negative regulation of tumor multidrug resistance associated protein, promote cell apoptosis. |
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