An Experimental Study On T Lymphocyte Killing By Non-small-cell Lung Cancer Cells

Despite an immunologic surveillance system in the body, tumors form and develop. Tumor cells can escape the surveillance of the human immune system through various mechanisms. For instance, because of absence of tumor surface antigens, immune cells fail to recognize tumor cells, and multiple immunosuppressant factors can be produced to impair the host's immunity. In the elderly patients, in particular, tumor cells are more likely to escape immunologic surveillance and result in tumorigenesis.It has recently been demonstrated that the Fas/FasL system, consisting of Fas and FasL, either as membrane-bound proteins or as soluble molecules, and many other factors, plays an important role in development, immune regulation and homeostasis, as well as cell immunity; hence, the Fas/FasL system is closely associated with tumorigenesis.To bind to FasL is the only means for Fas to function in the body. It has been found recently that FasL is also expressed by some non-lymphatic system tumor cells. For instance, FasL expression was detected in malignant tumor cells in the human colon, stomach, and pancreas. Hence, it is thought that FasL on the tumor cell surface binds to Fas on the surface of activated T lymphocytes, resulting in T cell apoptosis. In this way, tumor cells escape surveillance of the immune system.To investigate the possible mechanism by which non-small-cell lung cancer cells kill T lymphocytes, we performed an experimental study in three parts.Part 1: Fas/FasL specific siRNA target sequence was designed and synthesized using the siRNA target sequence design software and the Angela principles of siRNA design. The target sequence was then inserted into the pGCsi-U6 plasmid to construct siRNA plasmid. Fas/FasL expression was detected by immunoblotting in human lung cancer A549 cell strain transfected with siRNA plasmid, so as to determine the plasmids with the optimal interference effect. The determined Fas/FasL siRNA plasmids were used to transfect A549 cells, and the transfected cells were screened with 800μg/ml G418. The screened cell clones were subcultured, and the two clones that grew best were screened by Western blotting. Finally A549 cells with optimal interference effect were used in the following experiments.Part 2: A549 cells were transfected with Fas/FasL specific siRNA plasmid (Fas/FasL silencing) and psiNEGative RNAi plasmid (independent control), and then mRNA and protein expression of Fas, FasL, Caspase-3, Caspase-8 in A549 cells was determined by RT-PCR and Western blotting, respectively. Meanwhile, cell viability and apoptosis were assessed by MTT and flow cytometry, respectively. The aim was to investigate the effect of Fas/FasL expressed by tumor cells on tumor cell viability, i.e., to determine whether Fas/FasL expressed by tumor cells causes tumor cell suicide. In addition, T cells may commit apoptosis by an autocrine mechanism, i.e., Fas/FasL expressed by T cells may cause T cell apoptosis. Hence, in the present study, anti-FasL antibody (NOK-1) was used to block FasL expression by tumor cells, and the changes in tumor cell viability and apoptosis after FasL expression blockage were determined by MTT and flow cytometry, respectively.Part 3: Human umbilical vein endothelial cells (HUVECs) were cultured and identified by immunohistochemistry using VIII factor-related antigen, and Fas expression by HUVECs was detected immunohistochemically. Jurkat cells were used to activate T lymphocytes: the A549 cell groups that were transfected with pSi-FasL and psiNEGative RNAi plasmids were co-cultured with HUVECs and Jurkat cells, respectively. Meanwhile, the effect/target ratio, FasL expression after NOK-1 blockage, and apoptosis of HUVECs and Jurkat cells were determined by flow cytometry.Main results are as fellows:1. The siRNA plasmid interference efficiency differed between groups. Western blotting results demonstrated that in the Fas-siRNA design, pSi-Fas1DNA sequence complied with six Angela principles, and the sequences of the other two DNAs complied with five Angela principles. pSi-Fas1 interference was efficient. In the FasL-siRNA design, pSi-FasL 2 DNA sequence complied with six Angela principles, and the sequences of the other two DNAs complied with five Angela principles. pSi-FasL 2 interference was efficient. A549 cells with specific Fas/FasL silencing were obtained by transfecting these cells with pSi-Fas1 and pSi-FasL 2 plasmids.2. A549 cells lowly expressed Fas, Caspase-3, Caspase-8 mRNA and protein, and highly expressed FasL mRNA and protein. A549 cells transfected with pSi-Fas plasmid lowly expressed Fas, Caspase-3, Caspase-8 mRNA and protein, and highly expressed FasL mRNA and protein. A549 cells transfected with pSi-FasL plasmid lowly expressed Fas, FasL, Caspase-3, Caspase-8 mRNA and protein; A549 cells transfected with psiNEGative RNAi plasmid lowly expressed Fas, Caspase-3, Caspase-8 mRNA and protein, and highly expressed FasL mRNA and protein. MTT and flow cytometry did not show significant differences in cell viability and apoptosis between the three groups.3. MTT and flow cytometry showed that A549 cells and A549 cells transfected with psiNEGative RNAi did not show changes in the cell viability and apoptosis with or without NOK-1 blockage of FasL expression. In contrast, FasL expression blockage decreases Jurkat cell viability, but did not increase apoptosis significantly. After FasL expression blockage by NOK-1, the viability and apoptosis of Jurkat cells were normal.4. HUVECs were successfully cultured and identified immunohistochemically. HUVECs were also shown immunohistochemically not to express Fas.5. Flow cytometry (Annexin+PI) showed apoptosis of Jurkat cells transfected with psiNEGative RNAi, with the apoptotic rate increasing with the effect/target ratio (1: 30>1: 10>1: 3), but no apoptosis of Jurkat cells transfected with FasL siRNA. After culture of HUVECs with A549 cells transfected with pSi-FasL plasmid and A549 cells transfected with psiNEGative RNAi, HUVECs apoptosis did not increase at various effect/target ratios.6. FasL expression was blocked by anti-FasL antibody (NOK-1) in A549 cells and A549 cells transfected with psiNEGative RNAi, and then these cells were co-cultured woth HUVECs and Jurkat cells. Flow cytometry (Annexin+PI) indicated no apoptosis of Jurkat cells and HUVECs which were co-cultured with A549 cells or A549 cells transfected with psiNEGative RNAi.Conclusions are as fellows:1. When screening siRNA targets, Angela criteria should be considered to increase the interference efficiency. Western blotting can be used to screen siRNA recombinant plasmids.2. The Fas/FasL apoptosis dynamic signal chain remains unactivated in A549 cells in the absence of external stimuli, and apoptosis of non-small-cell lung cancer cells will not result from Fas/FasL expression by cancer cells. 3. Fas/FasL expression by T cells causes T cell apoptosis, and blockage of FasL expression by T cells abolishes T cell apoptosis.4. HUVECs do not express Fas; hence, lung cancer cells cannot kill HUVECs through the Fas/FasL pathway, and penetration of vascular basement membrane by lung cancer cells may be mediated by other pathways.5. A549 cells kill T lymphocytes through the Fas/FasL pathway.6. The present study investigated the killing of T lymphocytes by non-small-cell lung cancer cells, helping the establishment of tumor specific CTL models and providing a basis for studying the biological behaviors of tumors, such as immune escape and immune tolerance.