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  • br Fig Bou inhibits the growth

    2020-08-18


    Fig. 5. Bou inhibits the growth of rectal cancer in a xenograft mouse model. HCT-116 BMS-936558 (1 × 107 cells per mouse) were injected subcutaneously into the dorsal flank of nude mice. Once the tumors reached approximately 75 mm3, mice received a daily i.p. injection of either vehicle control (1% DMSO in normal saline) or Bou (50 mg/kg). After the treatment for 16 days, the mice were sacrificed, and relevant organs were collected for examination. (A) Tumor volumes during the period of treatment and end-point tumor weight. (B) Body weight; (C) H&E examination of tumors and liver, immunohistochemistry analysis of Ki-67 and COX IV (a marker of mitochondria). Original magnification, 200 × . (D) Mitochondria copies. (E) Oxygen consumption of mitochondria isolated from tumors. (F) Mitochondrial complex I activity. (G) SIRT1 activity. (H) Protein level of SIRT1-PGC-1α-UCP2 axis determined by Western blot. *P < 0.05, **P < 0.01, ***P < 0.001, vs control group mice. Data were expressed as Mean ± S.E.M. from 10 mice in each group.
    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 cells in vitro.
    4. Discussion
    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.