In previous work, we showed that cancer cells do not depend on glycolysis for ATP production, but they do on fatty acid oxidation. However, we found some cancer cells induced cell death after glucose deprivation along with a decrease of ATP production. We investigated the different response of glucose deprivation with two types of cancer cells including glucose insensitive cancer cells (GIC) which do not change ATP levels, and glucose sensitive cancer cells (GSC) which decrease ATP production in 24 h. Glucose deprivation-induced cell death in GSC by more than twofold after 12 h and by up to tenfold after 24 h accompanied by decreased ATP production to compare to the control (cultured in glucose). Glucose deprivation decreased the levels of metabolic intermediates of the pentose phosphate pathway (PPP) and the reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) in both GSC and GIC. However, glucose deprivation increased reactive oxygen species (ROS) only in GSC, suggesting that GIC have a higher tolerance for decreased NADPH than GSC. The twofold higher ratio of reduced/oxidized glutathione (GSH/GSSG) in GIS than in GSC correlates closely with the twofold lower ROS levels under glucose starvation conditions. Treatment with N-acetylcysteine (NAC) as a precursor to the biologic antioxidant glutathione restored ATP production by 70% and reversed cell death caused by glucose deprivation in GSC. The present findings suggest that glucose deprivation-induced cancer cell death is not caused by decreased ATP levels, but rather triggered by a failure of ROS regulation by the antioxidant system. Conclusion is clear that glucose deprivation-induced cell death is independent from ATP depletion-induced cell death.

In pancreatic ductal adenocarcinoma (PDAC), irinotecan (1μmol/L) chemotherapy triggers a dual-phase autophagy process that drives drug resistance. Although mTOR and autophagy exert suppressive effects on each other, coactivation of mTOR and autophagy has been observed when PDAC cells begin to regrow after treatment. Therefore, we hypothesized that the distinct temporal phases of autophagy are governed by independent upstream pathways. Initially, DNA damage activated AMPK, inducing early autophagy within 24 hours that fueled fatty acid oxidation (FAO), boosting ATP production. After 48 hours, elevated ATP levels inactivated AMPK and activated mTOR, which typically suppresses autophagy. However, autophagy and FAO activity persisted beyond 72 hours of irinotecan treatment via the JNK1–Beclin-1 pathway. This created a paradoxical state in which mTOR and autophagy were coactivated, promoting cell survival under irinotecan treatment. Irinotecan combined with FAO inhibition using KN510713 (a combination of KN510 targeting the carnitine–acylcarnitine transporter and KN713 targeting acetyl-CoA acyltransferase1/2) or FAO gene knockdown blocked autophagy flux and cell growth. FAO inhibition–induced fatty acid accumulation impaired autophagy flux and induced cytotoxicity, leading to cancer cell death. In xenograft models, combining irinotecan with KN510713 significantly prevented tumor regrowth compared with irinotecan alone. These findings suggest that targeting FAO induced by autophagy activation may overcome acquired drug resistance in PDAC while minimizing the toxic side effects associated with systemic inhibition of autophagy in healthy cells. Significance: Combined treatment with irinotecan and fatty acid oxidation inhibition reverses acquired drug resistance driven by autophagy in pancreatic cancer.

CEC50 values of KN510 and KN713 in PDAC cell lines

IC50 values of KN510 and KN713 for ATP reduction in PDAC cell lines.

Colby analysis for the synergistic effect of KN510 and KN713 in PDAC cell lines

Irinotecan treatment increased autophagy simultaneously with an increase in FAO