Evaluation of effects of α-lactalbumin and sulindac on primaryand metastatic human colon cancer cell lines via mitochondrialapoptotic pathway by immunohistochemistry and electronmicroscopy assays

Colon cancer is the second reason of death which is caused by cancer. Colorectal cancer prevalence is fourth order in women whereas it is third in men. Although surgery is applied to treatment the cancer drug treatments are applied and appropriate protocols are tried to build. HAMLET (human α-lactalbumin made lethal to tumor cells) and Sulindac are active ingredients which are used to treatment of cancers. HAMLET consists of Ca+2 binding protein α-lactalbumin, which is obtained from female human milk, and fatty acid (oleic acid). In in vivo and in vitro conditions, it attaches to surface of tumor cells and get in inside of the cells. It connects to the chromatin by histone proteins in nucleus. It damages the chromatin irreversibly, and activates the caspases by effecting the mitochondria and that induces apoptosis. Sulindac is one of nonsteroidal anti-inflammatory drugs (NSAIDs). It is inhibitor of growth and induces apoptosis. It cures some cancers (intestine, head and neck) by inducing apoptosis of tumor cells. Apoptosis is triggered by two ways in mammalian cells depending on the source of death signals: Intrinsic and extrinsic pathway. Intrinsic pathway is triggered by various signals (activation of oncogenes, DNA damages) which comes from cytosol and the mitochondria plays major role whereas extrinsic pathway is activated by binding the signal molecules to the receptors where are on cell membrane. In this study, COLO-320 Primary and COLO-741 Metastatic human colon carcinoma cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 1% L-glutamine and 1% penicillin-streptomicin at 37°C and 5% CO2 in air condition. Each cell lines were divided in four groups. The first groups are control groups and there were not treated with any drugs. The second groups were treated with α-Lactalbumin, the thirds with Sulindac and the fourth groups were exposed to αLactalbumin and Sulindac. Effects of drugs were determined at 24., 48. and 72. hours of culture. For cytotoxic analyses MTT and level of lactic dehyrogenase, for ultrastructure of the cells electron microscopy technique, for distribution of apoptotic cells TUNEL and indirect immunuperoxidase assays for intensity of caspase-3, caspase-9 and cytochrom-c were analyzed. After electron microscopic analyses, it was observed that contours of cell and nucleus were normal in control groups of each cell lines, in treated groups apoptotic blebs were detected, but the number of apoptotic blebs were more in group which were treated with both drugs . The number of TUNEL positive cells were increased in all treated groups especially in COLO-320 cell line. Immunoreactivities of caspase-3, caspase-9 and cytocrom-C were detected in all treated groups, these immunoreactivities were increased in groups which were treated with both drugs. After our study, we suggest that α-Lactalbumin and Sulindac may be trigggered mechanisms of apoptosis in both primary and metastatic colon carcinoma cell lines and primary colon carcinoma cell line was affected much more than metastatic colon carcinoma cell line. We suppose that these results will lead the in vivo experiments. Key words: Cancer, Apoptosis, α-Lactalbumin, Sulindac.