Apoptotic signaling in lung carcinoma cells : With focus on mechanisms of radioresistance

In this thesis, IR-induced apoptotic signaling was studied in a panel of lung carcinoma cell lines. The goal was not only to achieve a better understanding of the process, but also to identify possible targets for pharmacological modulation to enhance the efficiency of radiotherapy. Lung cancer is the most common malignant disease and the leading cause of cancer mortality in the western world. Radiotherapy is a cornerstone in the treatment of lung cancer. However, inherent or acquired radioresistance is a common clinical problem. Many types of anti-cancer treatment, including radiotherapy, work via the induction of apoptosis. The absence of IR-induced apoptosis in NSCLC (non-small cell lung carcinoma) cells was previously described by our group. The expression of pro-caspases, μ-calpain and the Bcl-2 family proteins Bcl2, Bcl-XL and Bax, was analyzed in a panel of lung carcinoma cell lines. No correlation was observed between the expression of the investigated proteins and intrinsic radiosensitivity. Moreover, SCLC (small cell lung carcinoma) cells were characterized by a high Bcl-2/Bax ratio and the lack of pro-caspase-8, -10, -4 and –1 expression. The functionality of the apoptosis signaling pathways was therefore studied in more detail. Mitochondrial release of cytochrome c and subsequent caspase activation was observed in both SCLC and NSCLC cells after IR-treatment. However, only SCLC cells had nuclear localization of active caspase-3 and underwent apoptosis. This led us to analyze the importance of possible inhibitors of apoptotic signaling at this level. Neither the expression level nor the intracellular localization of IAPs (inhibitor of apoptosis proteins) correlated with radiosensitivity. Moreover, although all cell lines had a strong expression of HSP72 (heat shock protein), known to have versatile and potent anti-apoptotic properties, knockdown of this protein by RNAi did not sensitize NSCLC cells to IR-induced apoptosis. The role of the pro-apoptotic mitochondrial regulators Bax and Bak in IRinduced apoptosis was investigated in U1810 (NSCLC) and U1285 (SCLC) cells. IR-treatment induced pro-apoptotic conformational changes in Bak, followed by mitochondrial depolarization, caspase-3 activation and appearance of apoptotic nuclear morphology changes, only in the radiosensitive U1285 cells. Moreover, in response to IR, an early and sustained activation of stress-activated protein kinases p38MAPK and JNK was detected in U1285, but not in U1810 cells. Blocking of SAPK activation in U1285 cells by chemical inhibitors totally abrogated IR-induced apoptosis. The data presented here demonstrate that IR-induced apoptotic signaling in radioresistant NSCLC cells is abrogated at several levels. Interestingly, in response to other agents, initiation and execution of apoptosis can be activated also in radioresistant cells. This suggests that the apoptotic machinery is functional in NSCLC cells, but is not activated vigorously enough to trigger apoptosis in response to IR. LIST OF PUBLICATIONS I. Joseph B*, Ekedahl J*, Sirzen F, Lewensohn R and Zhivotovsky B Differences in expression of pro-caspases in small cell and non-small cell lung carcinoma Biochemical and Biophysical Research Communications. 1999. 262(2): 381-387. II. Joseph B, Ekedahl J, Lewensohn R, Marchetti P, Formstecher P and Zhivotovsky B Defective caspase-3 relocalization in non-small cell lung carcinoma Oncogene. 2001. 20(23): 2877-88. III. Ekedahl J, Joseph B, Grigoriev M Y, Muller M, Magnusson C, Lewensohn R and Zhivotovsky B Expression of Inhibitor of Apoptosis Proteins in small and non-small cell lung carcinoma cells Experimental Cell Research. 2002. 279(2): 277-290. IV. Ekedahl J, Joseph B, Marchetti P, Fauvel H, Formstecher P, Lewensohn R and Zhivotovsky B The role for Heat Shock Protein 72 in the resistance of non-small cell lung carcinoma to ionizing radiation (submitted for publication) V. Viktorsson K*, Ekedahl J*, Lindebro M.C, Lewensohn R, Zhivotovsky B, Linder S and Shoshan M C Defective stress kinase and Bak activation in response to ionizing radiation but not cisplatin in a non-small lung carcinoma cell line (submitted for publication) *Authors contributed equally to this work Previously published papers were reproduced with permission from the respective copyright holders Additional papers Zhang A, Zheng C, Lindvall C, Hou M, Ekedahl J, Lewensohn R, Yan Z, Yang X, Henriksson M, Blennow E, Nordenskjold M, Zetterberg A, Bjorkholm M, Gruber A and Xu D Frequent amplification of the telomerase reverse transcriptase gene in human tumors Cancer Research. 2000. 60(22): 6230-5. Meijer A E, Ekedahl J, Joseph B, Castro J, Harms-Ringdahl M, Zhivotovsky B and Lewensohn R High-LET radiation induces apoptosis in lymfoblastoid cell lines derived from ataxiatelangiectasia patients International Journal of Radiation Biology. 2001. 77(3): 309-17.