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.
Highlights
The search of an effective radio-protector for protection of normal tissue toxicity during radio-therapy and nuclear accidents; and a newer, more potent radio-sensitizer to achieve enhanced therapeutic gain in radio resistant tumors, are the primary goals of radiation oncologists and radiation biologist
While studying the radiation induced growth inhibition in various cell lines using MTT assay and counting cell number, we found that results obtained from metabolic viability based assays do not correlate with actual cell number at different time points after radiation exposure
The amount of formazan formed (i.e MTT index) at 48 hrs after irradiation in all three cell lines (A549, MDA-MB-231 and HeLa) showed 20 to 35% reduction as compared to un-irradiated cells, while the decrease in cell numbers was in the range of 70 to 90% (Supplementary Fig. 1Aii to Cii), clearly indicating a lack of correlation between metabolic viability (MTT index) and growth inhibition at all the radiation doses; the two radiation response parameters analyzed
Summary
The search of an effective radio-protector for protection of normal tissue toxicity during radio-therapy and nuclear accidents; and a newer, more potent radio-sensitizer to achieve enhanced therapeutic gain in radio resistant tumors, are the primary goals of radiation oncologists and radiation biologist. Similar observations were reported in the literature earlier while studying the growth inhibitory effects of polyphenols[1,18,19] It appears, metabolic viability based assays do not give the real picture of cell viability or proliferation when compared with the actual cell numbers, in case of polyphenols[1,18,19] and radiation (this study). Metabolic viability based assays do not give the real picture of cell viability or proliferation when compared with the actual cell numbers, in case of polyphenols[1,18,19] and radiation (this study) These observations driven us to understand the limitation of MTT assay in the precise analysis of radiation induced growth inhibition. Our study highlights the limitation of MTT assay with mechanistic evidences of mitochondrial biogenesis
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