Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition

Abstract

Metabolic viability based high throughput assays like MTT and MTS are widely used in assessing the cell viability. However, alteration in both mitochondrial content and metabolism can influence the metabolic viability of cells and radiation is a potential mitochondrial biogenesis inducer. Therefore, we tested if MTT assay is a true measure of radiation induced cell death in widely used cell lines. Radiation induced cellular growth inhibition was performed by enumerating cell numbers and metabolic viability using MTT assay at 24 and 48 hours (hrs) after exposure. The extent of radiation induced reduction in cell number was found to be larger than the decrease in MTT reduction in all the cell lines tested. We demonstrated that radiation induces PGC-1α and TFAM to stimulate mitochondrial biogenesis leading to increased levels of SDH-A and enhanced metabolic viability. Radiation induced disturbance in calcium (Ca2+) homeostasis also plays a crucial role by making the mitochondria hyperactive. These findings suggest that radiation induces mitochondrial biogenesis and hyperactivation leading to increased metabolic viability and MTT reduction. Therefore, conclusions drawn on radiation induced growth inhibition based on metabolic viability assays are likely to be erroneous as it may not correlate with growth inhibition and/or loss of clonogenic survival.

Sirt3 Exerts Its Tumor-Suppressive Role by Increasing p53 and Attenuating Response to Estrogen in MCF-7 Cells

“…Several studies have shown that E2 has the ability to increase mitochondrial function and biogenesis (reviewed in [42]), therefore, we explored whethe Sirt3 plays a role in E2-induced metabolic fitness of MCF-7 cells. As observed in Figure 3A,C, Sirt3 potentiated this inducing effect of E2 leading to increased metabolic activity, primarily as a result of the induced rise in SDH-A expression ( Figure 3B) [26]. This is not surprising, considering that ERα is an essential estrogen receptor for most E2-mediated increases in respiratory chain proteins and antioxidant enzymes [43,44].…”

“…First, we tested the effect of E2 and Sirt3 on metabolic activity of MCF-7 cells using MTT assay. The MTT salt is reduced to formazan in the metabolically active cells predominantly by mitochondrial complex-II subunit succinate dehydrogenase A (SDH-A) and is considered to be a marker of metabolic potential of the cell [26]. The Sirt3-overexpressing cells had significantly higher basal metabolic activity (p < 0.001) and Sirt3 further enhanced the inducing effect of E2 ( Figure 3A), while ICI effectively abolished E2-induced metabolic activity to control levels in both cell lines.…”

Cytotoxicity Evaluation of Three Biocompatible and Bioactive Retrofilling Materials Using Periodontal Ligament Stem Cells: An In-Vitro Study

“…The fourth Cell passage were used in this study. During passaging, fibroblasts were transported from flask to another flask with trypsenization process using Trypsin EDTA solution aiming to de-adhering cells from the flask (11,12) .…”

“…Once the fibroblast cultures were found at 80-90% confluence, they were separated from the flask with trypsin-EDTA. Then 5×10 4 cells were overlaid in 100 µL of culture medium onto insert culture plates and incubated for 5h period at 99F, 6% CO 2 , and 97% humidity (12) . Initially, the complete media was added without material extract, to allow adaptation of the cells to the plate for 24h.…”

“…Then, the plates were incubated inside the biological incubator for 2h at 99F at 97% humidity and 5% CO 2 . During this period, the metabolically active cells bio-reduced MTT salt to formazan (12) . After this period MTT solution was removed from the cells and 100ul of dimethyl sulfoxide solution was added to each group to dissolve the insoluble formazan crystal formed inside the cells.…”