The Expression Of Extracellular Matrix And Its Relationship With Apoptosis And Chemoresistance In Non-small Cell Lung Cancer

PartⅠThe expression of ECM and its relationship with apoptosis and chemoresistance in non-small cell lung cancer cell linesBackground and purpose: Lung cancer is the major reason of cancer-ralated death, patients cannot be cured with current therapeutic modalities. Drug resistance is one of the major causes in treatment failure of non-small cell lung cancer(NSCLC). Classical drug resistant mechanisms have been elucidated upon monocellular models. Recently, interactions between the tumor cells and extracellular matrix (ECM)have been shown to be one of the mechanisms of drug resistance. We investigated effects of ECM on cisplatin-induced apoptosis of the cell line A549 and cisplatin-resistant cell line A549/DDP , and the role of phosphatidylinositol 3'-kinase(PI3-K) in ECM's signal pathway.Methods: A549 and A549/DDP cells were cultured in RPMI 1640 supplemented with 10% fetal calf serum in vitro, 6μmol/L cisplatin (DDP) was added to maintain the drug resistant phenotype of the A549/DDP cells. The inhibitory effects of DDP and/or LY294002 (PI3-K inhibitor) on cellular proliferation were tested by methabenzthiazuron (MTT) assay. Immunocytochemistry was used to detect the expression of extracellular matrix and integrinβ1 of these two cell lines, respectively. Cell adhesion experiments were used to test cell adhesion rates. Flow cytometry was applied to study the influence of collegenⅣ(CⅣ) and LY294002 on A549 cells'apoptosis and cell cycle distribution before and after DDP treatment.Results: Expression of ECM proteins in cisplatin-sensitive and cisplatin-resistance lung cancer cells was different. Extracellular matrix–mediated cell adhesion promotes survival of A549 and A549/DDP cells from cisplatin insults. Pre-coating with collagenⅣprotects non-small cell lung cancer cells from drug-induced growth inhibition, which is PI 3-kinase/Akt-dependent. Combining cisplatin with LY294002 caused greater increases in apoptosis compared with that caused by the inhibitor or cisplatin alone. CollagenⅣchanged A549 cells cell cycle distribution, with an increase in G1 phase,a reduction in S and G2/M phases, as treated with cisplatin or combined with LY294002.Conclusions: ECM may provide protection against cisplatin-induced apoptosis of A549 and A549/DDP cells, while LY294002 increases the growth inhibitory effect of cisplatin. Integrin ?1 signaling and PI3-Kinase pathway may be important survival pathways of drug-induced apoptosis in lung cancer cells and activation of this pathway may contribute to the generation of drug resistance.Part II Expression of dominant-negative forms of integrinβ1 prevent ECM-induced protection against chemotherapeutic attackBackground and purpose: Chemotherapy is one of the most important therapeutic strategy in non-small cell lung cancer except IA patients. New drug resistance mechanism needs to be found to enhance the response rates of tumor cells to chemotherapy. We use RNA interference technic to understand the role of integrin ?1 in this pathway.Methods: A549 and A549/DDP cells were transiently transfected using the Invitrogen reagent lipofectamine 2000 following manufacturer's instructions(V-gene Biotechnology limited). We constructed three plasmids of integrin ?1 siRNA. encoding target sequence (17-1TGCACGATGTGATGATTTA;17-2GGATTCTGACAGCTTTAAA;17-3GACTGTTCTTTGGATACTA). Then these plasmids and a control one were synthesized by Invitrogen (V-gene Biotechnology limited). Cells were grown to 80%-90% confluens on 6-well plates and washed twice, resuspended in antibiotic free medium DMEM (5×105 cells/mL) and transfected with 16μg/mL of total DNA/106 using Lipofectamine 2000 (1 : 2.5-3.0 ratio). Transfection medium was removed after 6h, and medium containing 2% fatal bovine serum was added to induce transgene expression. Twenty-four hours later, the cells were washed with PBS and used for subsequent experiments. (V-gene Biotechnology limited). At least three independent experiments were performed in each case.Results: When transfected with dominant-negative forms of integrinβ1, cells pre-cultured with collagenⅣbecame more sensitive to cisplatin, especially when in combination with LY294002.Conclusions: Integrin ? 1 signaling and PI3-Kinase pathway may be important survival pathways of drug-induced apoptosis in lung cancer cells and activation of this pathway may contribute to the generation of drug resistance.Part III Extracellular matrix expression in non-small cell lung cancer and the relationship with neo-adjuvant chemotherapyBackgrounds and objectives: Chemoresponse rates are always influenced by drug resistance of tumor cells. Extracellular matrix are found to be a new drug resistant mechanism. This study was about to investigate the expression of extracellular matrix in neo-adjuvant chemotherapy and control group, and its relationship with clinical significance in non-small cell lung cancer.Methods: Expression of extracellular matrix in lung cancer tissues was detected in 73 patients by using immunohistochemistry Envision method.Results: The median survival of 14 patients with low expression of fibronetin was 67 month, 59 patients with positive expression was 38 month. A significant difference existed between the two groups (P=0.048). High expression of laminin in neo-adjuvant chemotherapy group was negatively related to clinical response. When analyzed only patients randomized to neo-adjuvant chemotherapy using cox step-wise regression analysis, TNM stage is the only prognosis factor to survival.Conclusion: Overexpression of some extracellular matrix may be involved in the negative chemoresponse rate. The survival time of laminin negative patients may be longer than positive patients.