M.W. Roomi, T. Kalinovsky, A. Niedzwiecki and M. Rath
Dr. Rath Research Institute, 1260 Memorex Drive, Santa Clara, CA 95050
Open Journal of Apoptosis 5:2-8, 2016
Fanconi anemia (FA) is a fatal heterogeneous autosomal recessive disorder, characterized by progressive bone marrow failure, congenital defect and cancer predisposition. Cell culture from FA fibroblast (FAF) displays certain abnormalities as compared to normal human dermal fibroblast (NHDF). This prompted us to investigate the effect of a novel nutrient mixture (NM) containing ascorbic acid, lysine, proline and green tea extract, which has demonstrated a broad spectrum of pharmacological activities, on FAF compared to NHDF.
We investigated the in vitro effect of NM on FAF and NHDF cell proliferation by MTT assay, MMPs secretion by zymography, morphology by H&E staining and apoptosis by green caspase assay. FAF (FA-A:PD20, FA-A:PD220) and NHDF were cultured in modified Dulbecco Eagle media. At near confluence, the cells were treated with different concentrations of NM (0, 50, 100, 250, 500 and 1000 µg/ml) in triplicate. The cells were also treated with PMA to induce MMP-9 activity. NM had no effect on FAF cell viability in both cell lines compared to control. In contrast NM exhibited 20% at 50 and 100, 50% at 250, 60% at 500 and 70% toxicity at 1000 µg/ml on NHDF cells. Zymography demonstrated MMP-2 and MMP-9 on PMA stimulation in FAF and NM inhibited the activity of both MMP-2 and MMP-9 in a dose response fashion with total block at 500 µg/ml. In contrast, NHDF exhibited only MMP-2, both active and inactive forms, and NM inhibited their activities in a dose- dependent manner with total block at 1000 µg/ml. H&E staining did not indicate any morphological changes in FAF nor induced apoptosis at higher concentrations, as seen by caspases assay. However, although no morphological changes in NHDF were noted up to NM 100 µg/ml, progressive changes in cell shrinkage, rounding and nuclear condensation, pertaining to apoptosis, were observed at higher concentrations. These changes were consistent with the results from the green caspases apoptosis assay. Our data demonstrate that NM exhibited different responses toward FAF and NHDF. This may in part be due to elevated chromosomal break, deletion and hypersensitivity to cross linking agents, a DNA repair disorder in FAF that is lacking in NHDF.
Running title: Nutrients exhibit different responses in FA fibroblasts than NHDF
Key words: Fanconi anemia fibroblasts, normal human dermal fibroblasts, nutrients, cell viability, MMP-2 and -9, apoptosis