Expression of Novel Anti-Apoptotic Factors Associated with Clinical Outcome in Lung Adenocarcinoma
Date Issued
2011
Date
2011
Author(s)
Fang, Hsin-Yuan
Abstract
Lung cancer is one of the most frequent malignant disease in Taiwan as well as in the world. The mortality of lung cancer in Taiwan increases nearly 12 times within recent 40 years. In 2009, lung cancer occupied the first position of cancer death in both male and female in Taiwan. The incidence rate of lung cancer in Taiwan is increasing rapidly and probably is the highest in the world. The male and female ratio in lung cancer maintains approximately about 2:1.The histological type of lung cancer can be divided into the small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) which includes squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. According to the annual statistical report of Department of Health in Taiwan, the number of lung adenocarcinoma continuously increases and has already surmounted the number of squamous cell carcinoma, especially in female. The behavior of lung adenocarcinoma in Taiwan is different from that in other countries. According to the studies from National Health Research Institutes in Taiwan, the mutation rate in epidermis growth factor receptor gene in lung adenocarcinoma patients is 55%, much higher than the rate in western countries. The clinical response rates of chemotherapy and target therapy in lung adenocarcinoma are also different. These evidences demonstrate that the clinical behavior of lung adenocarcinoma in Taiwan is obviously different from that in western countries. To investigate the mechanism of survival, proliferation, apoptosis, local invasion, and distant metastases in lung adenocarcinoma cells becomes quite important. By doing the research, we hope we can help lung adenocarcinoma patients and improve their survival rate.
Apoptosis is one of the cell death methods. It plays an important part in cellular growth, differentiation, and the death in cancer cells. In order to increase the survival rate, adapt to the micro-environment, prevent being killed by immunological cells, cancer cells develop many anti-apoptosis methods to prevent apoptosis. Mitochondrion is an important organelle for energy supply and also determines the cells to die or live. When the cell apoptosis is induced by mitochondrial pathway, the apoptosis induced factor (AIF) and cytochrome C are released to cytoplasm and nucleus from the inner membrane of mitochondria. AIF and cytochrome C will dissect the deoxyribonucleic acid in the nuclease with other associated enzymes which cause the cell to die. Therefore the cancer cells develop a method for stabilizing the mitochondria to prevent the apoptosis exposing to worse environment. This makes the cancer cells to survive. According to our previous research and literature review, AIF acts quite an important key role in the apoptosis of lung adenocarcinoma cells.
The ATPase family, AAA domain containing 3A (ATAD3A) protein belongs to AAA family. It contains the enzyme activity of adenosine triphosphatase. ATAD3A in cells distributes over endoplasmic reticulum and membrane portion of mitochondria. It is transported from endoplasmic reticulum to mitochondria through small vesicles which associate with dynamin-related protein 1 (DRP1), mitofusin-2, and optic atrophy type 1(OPA1). These proteins associate with mitochondrial fusion and fission. We consider that ATAD3A prevents cellular apoptosis through stabling mitochondria and stopping the release of AIF and cytochrome C. OPA1 is one of dynamin families protein involved in mitochondrial fusion. It also associates with cellular apoptosis via mitochondria through caspase dependent pathway. Caspase-14 belongs to the cysteine protease family members. It correlates with the epithelial cell differentiation. However, it is still not so clear in apoptosis pathway. In cells, caspase-14 is distributed in cytoplasm and nucleus, not in mitochondria. From the analysis of protein interaction, we know that caspase-14 interacts with AIF in cytoplasm when AIF released from mitochondria. Caspase-14 combines with AIF and blocks its function. It might stop the process of apoptosis through caspase independent pathway. By veiwing above phenomenon, we consider that ATAD3A, OPA1 and caspase-14 affect the activity of AIF and cytochrome C in cytoplasm and mitochondria. Moreover, ATAD3A is the key factor in controlling mitochondrial morphology and stability with other associated proteins. In this research, we focus on several parts as follows: (1) ATAD3A, OPA1 and caspase-14 involving the cellular apoptosis; (2) the distribution and location of ATAD3A, OPA1 and caspase-14 in cells; (3) the function and phosphorylation of these proteins; (4) the change of mitochondria and organelle morphology by ATAD3A, OPA1 and caspase-14; (5) the suppression of these proteins activity in cells affecting the response to chemotherapy; and (6) the association in the expression of these proteins and clinical outcome in lung adenocarcinoma patients.
In this research, we used lung adenocarcinoma cell lines and lung tissues after surgical resection. The messenger RNA expression in lung adenocarcinoma cells and tissue were detected by reverse transcription polymerase chain reaction method. We also prepared the monoclonal antibodies of ATAD3A, OPA1 and caspase-14 from mice. Using Western blotting, the protein expression of ATAD3A, OPA1 and caspase-14 were detected in lung adenocarcinoma cells and tissue. Confocal microscope and immunohistochemistry were used to localizate the position of ATAD3A, OPA1 and caspase-14 inside lung adenocarcinoma cells. We found the expression of ATAD3A in cells were in endoplasmic reticulum and mitochondria. The OPA1 was in the inner membrane of mitochondria. The caspase-14 was in cytoplasm and nucleus. Using electric microscope, we observed the morphologic change in endoplasmic reticulum and mitochondria after knocking down the ATAD3A and OPA1 expression. Clinically, the high expression of ATAD3A, OPA1 and caspase-14 in tumor tissue is related to local invasion and poor outcome. We used the small interfering RNA to decrease the expression of these proteins in cancer cells and examined the growth rate of lung adenocarcinoma cells with cisplatin. Moreover, we found that phosphorylation of ATAD3A, OPA1 and caspase-14 improved stability and function. Finally, we found the interaction among ATAD3A, mitofusion 1, mitofusion 2, AIF, caspase-14 and OPA1.
In conclusion, this research evidences ATAD3A, OPA1 and caspase-14 as new anti-apoptosis factors in lung adenocarcinoma. They have high correlation with the clinical prognosis. By these discoveries, we believed that they can affect and improve the clinical outcome and prognosis in lung adenocarcinoma in the future.
Subjects
Lung cancer
Lung adenocarcinoma
Apoptosis
Mitochondria
ATAD3A
OPA1
Caspase-14
SDGs
Type
thesis
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