Tetrazolium-based colorimetric assays underestimat the direct antitumor effects of anti-VEGF agent bevacizumab

The direct antitumor effect of bevacizumab (BEV) has long been debated. Assessment of the direct cytotoxic activities of drugs is usually conducted via in vitro experiments, of which tetrazolium-based colorimetric assays are widely employed to measure the direct antitumor activity of BEV. This study aimed to investigate whether tetrazolium-based colorimetric assays are applicable when evaluating the cytotoxicity of BEV against tumor cells. Our results showed that BEV significantly augmented tumor-cell mitochondrial metabolism. Enhanced mitochondrial metabolism caused changes in cellular oxidation-and-reduction environment and upregulated succinate dehydrogenase, which in turn promoted the reduction of tetrazolium to produce formazan. Increased formazan formation resulted in underestimation of the in vitro direct antitumor effect of BEV. Furthermore, inhibition of mitochondrial hypermetabolism partially corrected the underestimation of colorimetric assays in evaluating the direct antitumor activity of BEV. Our findings suggest that tetrazolium-based colorimetric assays are unsuitable for accurately assessing the in vitro cytotoxicity of anti-VEGF drugs and may be the methodological reason for the controversial direct antitumor effect of BEV.