Carboxyamidotriazole-orotate inhibits proliferation and mitochondrial metabolism in human pancreatic cancer gemcitabine-resistant cell strain

Objective To investigate the effects of carboxyamidotriazole-orotate (CTO) on proliferation, apoptosis and metabolism of human pancreatic cancer gemcitabine-resistant cell line (ASPC-1-GEM, AG cells). Methods Cell viability was detected by sulforhodamine B(SRB); apoptosis was detected by annexin V/PI staining and flow cytometry; cell division rate was examined by CFSE microscopy and flow cytometry; The oxygen consumption rate (OCR) was detected by Seahorse bio-energy assay; intracellular NAD+ and NADH contents were detected with MTT and NAD+/NADH ratio was calculated. Autophagy-related protein expression was detected by Western blot. Results Comparing with the control group, the living AG cells were significantly reduced and the proportion of apoptotic cells was increased in CTO-treated group. The drug effect was time-dose dependent. The fluorescence intensity of CFSE staining of AG cells was significantly increased in the 20 μmol/L CTO group (P＜0.05); Compared with the control group, intracellular nicotinamide adenine dinucleotidehydrogen(NADH) content was increased after incubation with different concentrations of CTO treatment(P＜0.05 or P＜0.01) and no significant change in the content of nicotinamide adenine dinucleotide (NAD+) NDA+/NADH ratio was decreased(P＜0.01). The OCR in AG cells was significantly reduced and the expression of autophagy-related proteins was significantly inhibited in the CTO-treated group (P＜0.05). Conclusions CTO inhibits the proliferation of gemcitabine-resistant pancreatic cancer cells by inducing apoptosis, impairing mitochondrial respiration, and down-regulating the expression of autophagy-related proteins in AG cells.