Cenigaonandia-Campillo Aiora, Serna-Blasco Roberto, Gómez-Ocabo Laura, Solanes-Casado Sonia, Baños-Herraiz Natalia, Puerto-Nevado Laura Del, Cañas Jose Antonio, Aceñero María Jesús, García-Foncillas Jesús, Aguilera Óscar
Translational Oncology Division, Oncohealth Institute, IIS-Fundacion Jimenez Diaz-UAM (Madrid), Spain.
Hospital Puerta De Hierro Majadahonda, Majadahonda (Madrid), Spain.
Theranostics. 2021 Jan 25;11(8):3595-3606. doi: 10.7150/thno.51265. eCollection 2021.
In hypoxic tumors, positive feedback between oncogenic KRAS and HIF-1α involves impressive metabolic changes correlating with drug resistance and poor prognosis in colorectal cancer. Up to date, designed KRAS-targeting molecules do not show clear benefits in patient overall survival (POS) so pharmacological modulation of aberrant tricarboxylic acid (TCA) cycle in hypoxic cancer has been proposed as a metabolic vulnerability of KRAS-driven tumors. Annexin V-FITC and cell viability assays were carried out in order to verify vitamin C citotoxicity in KRAS mutant SW480 and DLD1 as well as in Immortalized Human Colonic Epithelial Cells (HCEC). HIF1a expression and activity were determined by western blot and functional analysis assays. HIF1a direct targets GLUT1 and PDK1 expression was checked using western blot and qRT-PCR. Inmunohistochemical assays were perfomed in tumors derived from murine xenografts in order to validate previous observations . Vitamin C dependent PDH expression and activity modulation were detected by western blot and colorimetric activity assays. Acetyl-Coa levels and citrate synthase activity were assessed using colorimetric/fluorometric activity assays. Mitochondrial membrane potential (Δ) and cell ATP levels were assayed using fluorometric and luminescent test. PDK-1 in mutant CRC cells and murine xenografts was downregulated using pharmacological doses of vitamin C through the proline hydroxylation (Pro402) of the Hypoxia inducible factor-1(HIF-1)α, correlating with decreased expression of the glucose transporter 1 (GLUT-1) in both models. Vitamin C induced remarkable ATP depletion, rapid mitochondrial Δ dissipation and diminished pyruvate dehydrogenase E1-α phosphorylation at Serine 293, then boosting PDH and citrate synthase activity. We report a striking and previously non reported role of vitamin C in the regulation of the pyruvate dehydrogenase (PDH) activity, then modulating the TCA cycle and mitochondrial metabolism in mutant colon cancer. Potential impact of vitamin C in the clinical management of anti-EGFR chemoresistant colorectal neoplasias should be further considered.
在缺氧肿瘤中，致癌性KRAS与HIF-1α之间的正反馈涉及显著的代谢变化，这与结直肠癌的耐药性和不良预后相关。迄今为止，设计的靶向KRAS的分子在患者总生存期（POS）方面未显示出明显益处，因此，有人提出对缺氧癌症中异常的三羧酸（TCA）循环进行药理学调节是KRAS驱动肿瘤的一种代谢脆弱性。进行了膜联蛋白V-异硫氰酸荧光素（Annexin V-FITC）和细胞活力测定，以验证维生素C对KRAS突变型SW480和DLD1以及永生化人结肠上皮细胞（HCEC）的细胞毒性。通过蛋白质免疫印迹法和功能分析测定法确定HIF1α的表达和活性。使用蛋白质免疫印迹法和定量逆转录聚合酶链反应（qRT-PCR）检测HIF1α的直接靶标葡萄糖转运蛋白1（GLUT1）和丙酮酸脱氢酶激酶1（PDK1）的表达。为了验证先前的观察结果，对源自小鼠异种移植瘤的肿瘤进行了免疫组织化学测定。通过蛋白质免疫印迹法和比色活性测定法检测维生素C依赖性丙酮酸脱氢酶（PDH）的表达和活性调节。使用比色/荧光活性测定法评估乙酰辅酶A水平和柠檬酸合酶活性。使用荧光和发光测试测定线粒体膜电位（Δ）和细胞三磷酸腺苷（ATP）水平。在突变型结直肠癌细胞和小鼠异种移植瘤中，通过缺氧诱导因子-1（HIF-1）α的脯氨酸羟化（Pro⁴⁰²），使用药理学剂量的维生素C下调PDK-1，这与两种模型中葡萄糖转运蛋白1（GLUT-1）表达的降低相关。维生素C诱导显著的ATP消耗、线粒体Δ的快速消散以及丝氨酸²⁹³处丙酮酸脱氢酶E1-α磷酸化的减少，进而增强PDH和柠檬酸合酶的活性。我们报道了维生素C在调节丙酮酸脱氢酶（PDH）活性方面一个显著且此前未被报道的作用，进而调节突变型结肠癌中的TCA循环和线粒体代谢。应进一步考虑维生素C在抗表皮生长因子受体（EGFR）化疗耐药性结直肠肿瘤临床管理中的潜在影响。
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