br Consistent with the data in cellular Bou treatment
Consistent with the data in cellular, Bou treatment increased SIRT1 activity leading to a decrease in total acetylation level and acetylated PGC-1α in tumors, resulting in PGC-1α-UCP2 axis activation (Fig. 5G and H). These in vivo eﬀects on metabolic reprogramming and cancer growth were similar to those observed in cultured HCT-116 Adrucil in vitro.
The present study examined the therapeutic eﬀects of Bou on rectal cancer cells in relation to metabolic reprogramming of the Warburg eﬀect. Our results show that Bou is eﬃcacious in promoting the oxi-dation of glucose and in decreasing glycolysis. In cellular, Bou treat-ment eﬃciently suppresses HCT-116 cell proliferation, clone sphere expansion and cycling without apoptosis. At the same time, HCT-116 cells treated with Bou increased glucose uptake and oxidative utilization of glucose. Metabolic studies revealed that Bou treatment decreased EACR and increased oxygen consumption in HCT-116 cells, indicating that Bou induces a metabolic reprogramming toward to aerobic metabolism. Further results show that Bou treatment upregu-lated UCP2 through PGC-1α enrichment in its promoter with SIRT1 as an upstream target. These metabolic reprogramming and anti-cancer eﬀects of Bou were reproduced in UCP2-overexpressing cells. Finally, a mouse model of rectal cancer showed that Bou substantially reduced the growth of rectal cancer and confirmed the metabolic reprogram-ming eﬀect and upregulation of UCP2 in the tumor tissue of mice. These data indicated that Bou is capable of suppressing the proliferation of rectal cancer by inducing metabolic reprogramming of the Warburg eﬀect via the upregulation of UCP2 (Fig. 6).
The growth of cancer cells is metabolically dependent on the Warburg eﬀect, which shifts the fuel metabolism away from mi-tochondrial oxidation and into the glycolytic pathway. The Warburg eﬀect produces substrates for the proliferation and growth of cancer cells, provides an acidic environment preferred by cancer cells (Koppenol et al., 2011; Grippo and Maker, 2017). In light of this, re-versing the Warburg eﬀect back to mitochondrial oxidation can sup-press the survival and progression of cancer cells. Bou is a derivative we have recently developed from rutaecarpine. This molecule can stimu-late the oxidation of cellular fuels by promoting mitochondrial oxida-tion via uncoupling the oxidation of fuel metabolites from the genera-tion of ATP (Rao et al., 2017). These metabolic eﬀects suggest that Bou may exert therapeutic eﬃcacy on cancer cells by abolishing the
Fig. 6. Proposed mechanism for the therapeutic eﬀect of Bou for rectal cancer.
Warburg eﬀect via redirecting glycolysis to the oxidation of glucose. As lactate is a major product of glycolysis and its increase is a hallmark of the Warburg eﬀect, which preferentially forms an acidic micro-environment and also favored by cancer cells for survival and invasion (Doherty and Cleveland, 2013; Nenu et al., 2017). Therapies that di-rectly or indirectly target the acidity of the tumor microenvironment are becoming widespread and eﬃciently inhibit tumor growth (Imai et al., 2017). Indeed, we found that Bou treatment decreased ECAR in HCT-116 cells, instead, the OCR was increased, indicating that the Warburg eﬀect in Bou-treated cells was inhibited.
Therefore, we examined the eﬀects of Bou on aerobic (or oxidative) metabolism in cultured HCT-116 cells. Interestingly, cells treated with Bou increased mitochondria content, mitochondrial complexes (I and
II) activity as well as oxidation capacity. In contrast to the improved oxidation capacity, Bou treatment decreased the levels of incomplete oxidation products, including MDA and reactive oxygen species in HCT-116 cells. More importantly, we found that Bou treatment decreased the ATP and NADH levels. ATP is mainly produced by oxidation phosphorylation using the energy of the electron transfer chain, which generated from the oxidation of energy-rich substrates (such as NADH) originating from the metabolism of fuels such as glucose and fatty acids. Combined with the results of decreased ATP and increased mitochon-drial oxidation, we concluded that Bou induced a metabolic repro-gramming of cancer cells to surpass the Warburg eﬀect as result of in-creased the oxidative path possibly in cells by uncoupling oxidation from phosphorylation in mitochondria.