Role Of Fas Gene In Cis-dichlorodiamine Platinum-induced Multidrug Resistance Of Human Small Cell Lung Cancer

Background:SCLCs are well known for their initial sensitivity to chemotherapeutic agents and thereafter frequently recur at which time the tumour is drug resistant. Cisplatin, formally known as cis-diamminedichloroplatinum (Ⅱ) (CDDP) is a metal-base oncolitic agent which binds to the nucleophilic sites of DNA resulting in changes in DNA synthesis and cell death . For this reason, Cisplatin is recommended in chemotherapy of SCLC, however for many patients with SCLC, cisplatin resistance hampers cisplatin treatment. It was well known that a number of cellular adaptations, including: 1.Exoeytosis is enhanced by transport protein in cell membrane such as P-gP, MRP, BCRP and LRP; 2.The tumour cell increase its resistance to the anti-tumour drug (e.g. GST); 3.Alternation in target enzyme (e.g. TopoⅡ) lead to a decrease in the drug susceptibility; 4.DNA repair function is enhanced (e.g. ERCC1); 5.The correlation factor in signal transduction is abnormally expressed. Drug resistance can also be caused by the dysfunction of apoptotic signal transduction pathway of the malignant cell. The downregulation of pro-apoptotic genes such as Bax, Fas/FasL and upregulation of anti-apoptotic gene such as Bcl-2 can cause drug resistance.Fas, a 45 kDa typeⅠtransmembrane protein, is expressed on cell membranes of varieties of normal cells and malignant cells including lung cancer cells . Its ligand, FasL, is expressed on the cell membrane of activated T lymphocytes and some malignant cells . After trimerization of Fas on the cell membrane by extracellular FasL, Fas-associated death domain (FADD) and caspase 8 bind to the intracellular death domains of Fas and induce a death signal in the cells, leading to the activation of a cascade of caspases and eventually to cell death. Since FasL can induce apoptosis in Fas-expressing malignant cells, the Fas/FasL system plays an important role in T cell-mediated cytotoxic reaction and malignant cell-mediated autocrine suicide or paracrine death against malignant cells. On the other hand, malignant cells can avoid being killed by downregulating Fas expression. It has been demonstrated that cisplatin-resistant lung cancer cells express low level of Fas, and correspondingly, their apoptosis decreases significantly. Some reports have correlated multidrug resistance (MDR) with the decreased Fas expression and resistance to Fas-mediated apoptosis. They found that Fas-resistant cells were resistant to chemotherapeutic drug treatment, which presumably due to the disruption of pathways responsible for the induction of cell death by chemotherapeutic drugs . But it is still obscure whether upregulation of Fas expression can reverse cisplatin resistance in human SCLC cells and cisplatin-induced apoptosis and alter the expression of any drug-resistant or cell signal transduction gene.Objective:Fas/FasL system is a major regulator of apoptosis. The mechanisms by which Fas mediates cisplatin resistance remain unclear. The aim of this study is to explore the effect of Fas overexpression on cisplatin resistance of small cell lung cancer cells and its possible mechanisms.Methods:1. H446/CDDP obtained resistance to cisplatin, but also to a wide range of other chemotherapeutic agents, by stepwise increasing concentrations of cisplatin.2. The Fas-containing adenoviruses Vector were constructed.3. Fas gene was over-expressed in H446/CDDP cells by infection with Fas-containing adenoviruses. Sensitivity of Fas-overexpressed H446/CDDP cells to cisplatin and other chemotherapeutants was evaluated using CCK assay.4. Expressions of Fas, P170, MRP, TOPOⅡ, Bcl-2, Bax, GST-π, ERCC1 and some cell signal transduction gene were detected by gene chips, RT-PCR and Western blot analysis.Apoptosis rate were examined by FACS.Results:1. H446/CDDP line was obtained which was resistance not only to cisplatin, but also to a wide range of other chemotherapeutic agents.2. In H446/CDDP line, the expression of MRP, P170, GST-π, ERCC1, TOPOⅡand Bcl-2 were significantly upregulated, and the expression of Fas were significantly downregulated (P<0.01, compared with H446 line). 3. In H446/CDDP line, the PI3K, PKC, JAK-STAT, ERK and p38MAPK cell signal transduction were activated, and JNK was inhibited.4. Clone open reading frame sequence of gene Fas, and successfully construct the Fas-containing adenoviruses Vector. The Fas gene was stablely upregulated after the Fas-containing adenoviruses was transferred into cell strain H446/CDDP.5. Up-regulation of Fas significantly decreased the tolerance of H446/CDDP cells to cisplatin and other chemotherapeutants.6. Over-expression of Fas in H446/CDDP cells led to more cells in apoptotic status (P<0.01).7. Over-expression of Fas in H446/CDDP cells significantly decreased Bax, TOPOⅡ, MRP, P170, GST-πand ERCC1 mRNA and protein levels (P<0.01), and increased the expression of Fas and bcl-2 (P<0.01).8. Up-regulation of Fas, the PI3K, PKC, JAK-STAT cell signal transduction were inhibited, and the JNK, p38MAPK cell signal transduction were activated.Conclusion:1. H446/CDDP obtains resistance not only to cisplatin, but also to a wide range of other chemotherapeutic agents, which may be due to 1)the downregulation of Fas, and upregulation of MRP, P170, GST-π, ERCC1, TOPOⅡ; 2)the activation of PI3K, PKC, JAK-STAT, ERK and p38MAPK pathway, and the inhibition of JNK pathway.2. Fas-containing adenoviruses Vector which can stably up-regulate the expression of Fas gene is successfully constructed. The mRNA and protein expression of Fas are significantly downregulated after Fas-containing adenoviruses are transfected with cell strain H446/CDDP.3. Fas over-expression reverses drug resistance of H446/CDDP cells, possibly due to 1) the upregulation of Fas and the decreased MRP, P170, TOPOⅡ, GST-πand ERCC1 expression; 2) the inhibition of PI3K, PKC, JAK-STAT and the activation of JNK, p38MAPK pathway.