Acceleration of tumorigenesis is observed when apop tosis is suppressed by overexpression of anti apoptotic proteins such as Bcl2. When anti apoptotic Bcl 2 family members are overexpressed, the ratio of pro and anti apoptotic Bcl 2 family members is disturbed and apoptotic cell death can be prevented. Targeting the anti apoptotic Bcl 2 family of proteins can improve apoptosis. Apoptosis induction is arguably the most potent defence against cancer growth. Evidence suggests that certain chemopreventive agents can trigger apoptosis in transformed cells in vivo and in vitro, which appears to be associated with their effectiveness in modulating the process of carcinogenesis. In this study, we analyzed the effect of CF on 12 differ ent cell lines showing that the nutraceutical has anti cancer activity.
Among all, colon cancer and mesothelioma cell L-Mimosine c-Met inhibitor lines were the most sensitive and were selected to study the action of CF on cancer. The nutraceutical treatment induced death by apoptosis, upregulation of p53 and downregulation of c myc, pAkt, and Bcl 2. Given the central role of these molecular targets in cell proliferation and death, the potential preventive benefits of CF in human cancers are self evident. Methods Cell culture Breast, colorectal, lung, melanoma, mesothelioma cancer cell lines, and fibroblast and mesothelio cell lines were gradually conditioned in DMEM F12 Glutamax supplemented with 10% FBS and antibiotics and maintained at 37 C and 5% CO2. Cellfood CF was kindly provided by Eurodream srl and stored at room temperature. CF was diluted in phosphate buffered saline and sterilized using a 0.
45 um syringe filter before a replacement use. Cell growth assays For cell growth experiments, cells were plated in quintu plicates in 96 well culture plates at a density of 3 × 103 cells well. 24 h later, the medium was replaced with fresh growth medium containing 1,200, 1,400, 1,800, 1,1600 dilutions of CF. At 24 and 48 h of treatment, XTT labelling reagent was added to each well, and the sam ples were incubated for an additional 4 h at 37 C. The XTT assay, Roche Molecular Biochemicals, Indianapolis, IN is based on the cleavage of the yellow tetrazolium salt XTT to form an orange forma zan dye by metabolic active cells. Absorbance was mea sured at 492 nm with a reference wavelength at 650 nm and the absorbance values of treated cells were presented as a percentage of the absorbance versus non treated cells. All experiments were repeated three times. The anti proliferative CF activity was assessed in mono layer cell culture conditions by plating cell lines in a T25 flask. After 24 h, CF was added for the time indicated in the experiments.
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