The Relationship Of FasL And Caspase-3 Expression On Non-small Cell Lung Cancer With Tumor Infiltrating T Lymphocytes

Background and objective:Malignant tumor development or degeneration, to some extent, is dependent upon the interaction between the tumor and the host immunity system. Therefore, to improve the host immune ability, and induce tumor cells to apoptosis is an important objective for tumor therapy. Up to date, it is found that there are two message pathways to regulate cell apoptosis: mitochondria C pathway and death receptor pathway. FasL is an important apoptosis molecule, which can couple with its death receptor Fas to induce apoptosis of Fas positive cells. Fas was found expressing almost all on the somatic cells. While FasL was orignally discovered on cells of the lymphoid/myeloid lineage, including activated T cells, natural killer (NK) cells, and immune privilege regions, such as eye, testis and placenta. Caspase is the main enzymes to execute apoptosis, and Caspase-3 is a specific cysteine proteinase which plays critical role in the series of caspases cascade reaction. It was presented that there was infiltration of immune cells in malignant tumor matrix named tumor infiltrating lymphocytes (TILs), which is a group of heterogenicity lymphocytes mainly composed of T cells playing specific and non-specific actions in killing tumor cells. It is believed that the TILs perfom its tumor-killing activity by binding the FasL on the lymphocytes to Fas on the tumor cell surface to induce the tumor cell for apoptosis. However, both tumor cells and tumor infiltrating T lymphocytes express FasL on their surface. What is the relationship between TILs and the expresion of FasL and Caspase-3 on non-small cell lung cancer? We presumed that high expression of FasL on non-small cell lung cancer might counterattack the TILs, and escape the host immune surveilence. And the same time, we were wondering if the FasL expression on the tumor cells is the result while receiving stimulation from some factors produced by the TILs, and then tumor cells might possess the ability to counterattack host immune system for its own survival and development. We believe that deep study on the relationship of FasL and Caspase-3 expression on non-small cell lung cancer with tumor infiltrating T lymphocytes seems to have an important biological significance.Methods: The following methods have been taken in the study:(1) To start with this study, 45 samples of surgically resected non-small cell lung cancer were used as research object. Immune histochemical stain was employed to detect the expression of FasL and Caspase-3 protein on the tumors.(2) With laser capture micro disection technique, cells in peripheral and non-peripheral region in NSCLC were obtained, examed for 10-case NSCLC samples, which were freezed freshly surgically resected, and then RT-PCR was utilized to assay the expression level of FasL and Caspase-3 mRNA on these tumor cells.(3) The tumor infiltrating lymphocytes were labeled with immune histochemical stain, and then TUNEL in situ in the same section of the tumor was used to detect apoptosis cells in TILs. Finally, the tumor infiltrating lympocyte apoptosis rates was calculated.(4) Four portions of HLA-A2+ PBL were obtained from 12 health blood donors. A549 cells were transinfected with plasmid to steadily express HLA-A2. Then the lymphocytes expressing HLA-A2+ were becoming CTLs to A549 with evaluation trial. After 12 days culture, the lymphocytes were accounted to 3×105/ml in concentration, and the total volume was 246ml. And 96 mls of supernatant was acquired from the medium. Then 50μl, 100μl, 150μl, 200μl, 250μl and 500μl of the TILs supernatant was added to A549 cells for co-culture for 30min, 60min, 120min, 180min and 240min respectively. Negative control was also set up. RT-PCR and Western blot technique were utilized to assay the expression level of FasL mRNA and protein, and Caspase-3 mRNA and protein for each group respectively.(5) Statistical analysis was performed by using the software SPSS 10.0.Results:The major results in this study were as follows:1. The assay result for non-small cell lung cancer FasL protein expression: (1) The level of FasL protein expression in adenocarcinoma and squamous cell lung cancer was 73.3%±15.8% and 60.4 %±15.8 % respectively, and the expression level of FasL protein in adenocarcinoma was higher than that in squamous cell lung cancer (p<0.01); (2) The expression of FasL protein in mid- and high differentiated lung cancer and low differentiated lung cancer was 60.6%±15.8 % and 70.1 %±16.9 % respectively, and the rate of FasL protein expression in mid- and high differentiated lung cancer was significantly lower than that in low-differentiated lung cancer (p<0.05). (3) The expression of FasL protein in two groups of NSCLC with and without local lymph node metastasis was quite different (p<0.05). In the former, FasL expression positive rate was 62.4 %±15.6 %, and in the latter, FasL expression was 55.8 %±11.9 %. (4) There was no significant FasL expression difference among the group of NSCLC with different pTNM. However, the FasL expression rate in stageⅠ+Ⅱwas 62.4%±15.6%, and in stageⅢA+ⅢB, 72.8 %±18.0%. The FasL protein expression rate in group of NSCLC of stageⅢA+ⅢB was significantly higher than that in group of stageⅠ+Ⅱ(p<0.05). (5) In the peripheral region of NSCLC, the FasL protein expression rate was 74.4%±18.4%, while in the non-peripheral region of NSCLC, the rate was 64.4%±13.1%; and the FasL protein expression rate in NSCLC peripheral region was signifigcantly higher than that in the non-peripheral region ( p<0.05 ).2. The results of FasL and Caspase-3 mRNA expression assay in different region of NSCLC: (1) FasL mRNA in peripheral region of non-small cell lung cancer (with the average of 0.926 + 0.41) was significantly higher than that in non-peripheral district of NSCLC (with mean of 0.892 + 0.26), p<0.05. (2) However, the Caspase-3 mRNA expression was no significantly different between peripheral and non-peripheral region of NSCLC (p>0.05), the former being 0.912 + 0.07 on average, and the latter being 0.923 + 0.04 on average.3. The Caspase-3 protein examination result in nonsmall cell lung cancer: (1) The expression level of Caspase-3 protein in squamous cell lung cancer was significantly higher than that in adenocarcinoma (p<0.05). The expression rate in the former was 76.9 %, and in the latter was 47.4%. (2) The Caspase-3 protein expression rate in mid- and high differentiated lung cancer and low differentiated lung cancer was 70.0% and 64.0% respectively. Caspase-3 protein expression in mid- and high differentiated NSCLC was higher than that in low differentiated NSCLC (p<0.05). (3) Caspase-3 expression was related with local lymph node metastasis. The expression rate of Caspase-3 protien in group of nonsmall cell lung cancer with and without local lymph node metastasis was 61.8% and 81.8% respectively. The expression rate of Caspase-3 in the group with local lymph node metastasis was significantly higher than that without lymph node metastasis (p<0.05). (4) There was no significant diference in Caspase-3 protein expression between the two groups of peripheral and non-peripheral region of NSCLC (p>0.05).4. The nonsmall cell lung cancer TILs apoptosis examination result: (1) The apoptosis index (AI) in adenocarcinoma was 5.51±1.56, and in sqamous cell lung cancer was 4.11±2.03. AI in adenocarcinoma was higher than that in squamous cell lung cancer (p<0.01). (2) AI in mid- and high differentiated nonsmall lung cancer and low differentiated NSCLC was 3.84±1.71 and 5.38±1.90 respectively. And the AI in low differentiated NSCLC was significantly higher than that in high differentiated NSCLC, with p<0.01. (3) AI was found no significant difference in the NSCLC with different pTNM (P>0.05). (4) AI of TIL had relationship with the phathological type and differntiation degree of the tumor. With increasing FasL expression in NSCLC, AI was growing with significant and positive corelationship ( r = 0.707, p < 0.01).5. FasL and Caspase-3 mRNA and protein assay result for the co-culture of A549 with different amount of TILs supernatan fluid: (1) When the A549 cells cocultured with 250μl of tumor infiltrating cell supernatant fluid for 60min, FasL mRNA expression was significanttly higher than the control group, (p<0.05). (2) In two goups of A549 cells co-cultured with 200μl and 250μl of TILs supernatant fluid for 90min, the FasL mRNA was higher than the control group respectively (p<0.05). However, there was no significant diference in FasL mRNA expression between any other two groups (p>0.05). (3) In any other group of A549 cells co-culture with more than or less than 250μl of the TIL supernatan liquid for 30min, 120min, 180min, 240min, no significant change was found in FasL mRNA expression on A549 cells, compared with the control group (p>0.05). (4) No sigificant change of FasL protein expresstion was found in all groups for co-culture with 50μl to 500μl of TIL supernatant liquid for 30min to 240min (p>0.05). (5) And the expression of Caspase-3 mRNA and protein in all experimental groups were found to be without any significant change for co-culture with 50μl to 500μl TIL supernatant liquid for 30min to 240min, compared with the control one ( p>0.05).Conclusions: The conclusion was as follows from the study:1. FasL protein expression on non-small cell lung cancer was closely related with its biological charateristic. High FasL protein expression on NSCLC might be liable to metastasize, and the prognosis of the patients with upregulation of FasL could be poorer.2. FasL protein distribution on the non-small cell lung cancer was of charateristic with that FasL protein in the peripheral area was obviously higher than that in non-peripheral area. Addtionatinally, FasL mRNA expression in peripheral area was also significantly higher than that in the non-peripheral area. Therefore, it might be implied that the microenvironment factors might impact the expression of FasL on the tumor cells. Peripheral NSCLC cells might receive the stimulation from the factors produced by tumor infiltrating T lymphocytes or other humor factors.3. Apotosis of TILs in NSCLC was closely related with its FasL expression. And the higher FasL expression, the higher apoptosis rate of tumor infiltrating T lymphocyte in the tumor. Upregulation of FasL expression on non-small cell lung cancer might be more liable to counterattack human tumor infiltrating T lymphocytes and immune system through FasL expression.4. FasL expression on tumor cells might be influenced or regulated by some cytokine produced from tumor infiltrating lymphocytes. The tumor cells make quick reaction only after the TIL supernatant liquid was added for coculture for 60min and 90min; however, for longer than 90min co-culture of the tumor cell TIL supernatant liquid, no upregulation expression of FasL was found. What's more, more than 250μl of tumor infiltrating T cell supernatant liqid was added for tumor cell co-culture, still there was no significant change on the FasL expression without any obviously understood mechanism. While, there are so many factors involved apoptosis, and the regulation mechanism is very complicated; therefore, it need get futher research that the cytokines produced by tumor infiltrating lymphocytes regulate FasL expression on NSCLC.