Cornell 大学：维生素C对直结肠癌细胞有选择性的毒性作用 Vitamin C is selective cytotoxic to colorectal cancer cells

康奈尔大学：维生素C对直结肠癌细胞有选择性的毒性作用

Vitamin C inhibits the survival and growth of colorectal cancer cells with KRAS or BRAF mutations

尽管超过一半的结直肠癌患者伴有KRAS或BRAF突变，但对这一亚组癌症患者的靶向治疗是缺乏的。

我们之前的研究表明，同种的人类结肠直肠癌细胞(CRC)含有KRAS或BRAF突变，上调葡萄糖转运体1(GLUT1)的表达，增加葡萄糖的摄取和糖酵解。

在本研究中，我们继续展示KRAS或BRAF突变细胞对维生素C的摄取有显著的增加，主要是在其氧化的形式，脱氢抗坏血酸(DHA)，相对于它们的野生型对应的GLUT1。值得注意的是，CRCs没有摄取还原性的维生素C，这可能是由于它的已知转运体，钠维生素C转运蛋白(SVCTs)在CRC中普遍不存在。在突变细胞中，GLUT1的缺失，野生型细胞中GLUT1的过表达，以及葡萄糖和DHA之间的竞争，证实了GLUT1对CRC中维生素C的摄取是必要的，也是足够的。突变体和野生型细胞在低葡萄糖(1 – 2mM)中都能以相似的速度生长。

在同样的情况下，我们发现维生素C对KRAS和BRAF突变细胞有选择性的毒性，导致它们发生细胞凋亡。与此相反，在高糖水平(> 10 mM)中，类似浓度的维生素C对突变细胞的存活和生长没有影响。此外，维生素C可以显著抑制体内异种移植模型中KRAS或BRAF突变的癌细胞生长。

为了找到维生素C在KRAS或BRAF突变细胞中诱导细胞死亡的机制，我们使用LC / MS进行了靶向代谢组学。无标记的总代谢物分析和C13 -葡萄糖同位素的通量分析强烈建议，维生素C将葡萄糖的使用从有氧糖酵解和非氧化性戊糖磷酸途径(PPP)重新路由到氧化性PPP，以弥补NADPH的较低水平，而NADPH被迅速消耗，还原GSSH为GSH。仅摄取DHA，氧化态维生素C，突变细胞立即使用GSH还原维生素C，导致GSSH的积累，伴随GSH(谷胱甘肽，细胞内主要的抗氧化剂）的迅速耗竭。将葡萄糖分流到氧化PPP中会降低有氧糖酵解，降低ATP水平，降低葡萄糖合成代谢，不支持核酸、脂类和氨基酸的合成，这些都是突变细胞存活和生长所必需的。

令人惊讶的是，在维生素C治疗后，反应性氧化应激(ROS)的水平基本没有改变，因为在突变细胞中，维生素C的含量增加了抗氧化剂的水平。目前有超过三个临床试验阶段I / II检查高剂量维生素C对淋巴瘤或包括CRC在内的固体肿瘤患者静脉注射的效果。

我们的研究结果将证明CRC中的KRAS或BRAF突变可以作为维生素C治疗的预测生物标志物。

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参考文献：

Vitamin C inhibits the survival and growth of colorectal cancer cells with KRAS or BRAF mutations

来源：http://cancerres.aacrjournals.org/content/73/19_Supplement/C60

Abstract C60: Vitamin C inhibits the survival and growth of colorectal cancer cells with KRAS or BRAF mutations

AUTHOR INFORMATION

Jihye Yun1, Adam Kavalier1, Jatin Roper2, Steve Gross1, Carlo Rago3, Nickolas Papadopoulos3, Bert Vogelstein3, and Lewis Cantley1

1Weill Cornell Medical College, New York, NY,

2Division of Gastroenterology, Department of Medicine, Tufts Medical Center, Boston. MA,

3The Ludwig Center and The Howard Hughes Medical Institute at Johns Hopkins Kimmel Cancer Center, Baltimore, MD.

Although more than half of colorectal cancer patients have either KRAS or BRAF mutations, targeted therapies for these subgroups of cancer patients are lacking. We previously showed that isogenic human colorectal cancer cell (CRC) lines harboring a KRAS or BRAF mutation up-regulate the expression of glucose transporter 1 (GLUT1), and increase glucose uptake and glycolysis. In this study, we go on to show that KRAS or BRAF mutant cells exhibit a significant increase in the uptake of vitamin C, mainly in its oxidized form, dehydroascorbic acid (DHA), relative to their wild-type counterparts through GLUT1. Notably, CRCs did not uptake vitamin C in its reduced form, possibly due to the fact that its known transporters, sodium vitamin C cotransporters (SVCTs) were generally not present in CRCs. Deletion of GLUT1 in mutant cells, overexpression of GLUT1 in wild-type cells and competition assays between glucose and DHA confirmed that GLUT1 is both necessary and sufficient for the uptake of vitamin C in CRCs. Both mutant and wild-type cells were able to grow in low glucose (1-2 mM) at a similar rate. In the same condition, we found vitamin C to be selectively toxic to KRAS and BRAF mutant cells, causing them to undergo apoptosis. In contrast, in high glucose levels (>10 mM), a similar concentration of vitamin C had no effect on the survival and growth of mutant cells. In addition, vitamin C significantly inhibited the growth of cancer cells with KRAS or BRAF mutations in vivo xenografts models. To find the mechanism by which vitamin C induces cell death in KRAS or BRAF mutant cells, we performed targeted metabolomics using LC/MS. Both unlabeled total metabolite analysis and flux analysis with C13-Glucose isotope strongly suggested that vitamin C re-routes glucose usage from aerobic glycolysis and non-oxidative pentose phosphate pathway (PPP) to the oxidative PPP to compensate for the lower levels of NADPH which was rapidly consumed to reduce GSSH to GSH. Having taken up exclusively DHA, the oxidized state of vitamin C, the mutant cells immediately use GSH to reduce Vitamin C, resulting in the accumulation of GSSH. Shunting glucose to the oxidative PPP reduces aerobic glycolysis, lowering ATP levels, and decreases glucose anabolism, disfavoring the synthesis of nucleic acids, lipid and amino acids, all needed for the survival and growth of mutant cells. Surprisingly the level of reactive oxidative stress (ROS) was largely unchanged following vitamin C treatment possibly because increased levels of vitamin C served as an anti-oxidant in mutant cells. There are currently more than three clinical trials phase I/II examining the effect of high dose vitamin C via intravenous administration in patients against lymphoma or solid cancers including CRCs. Our findings will provide proof of concept that KRAS or BRAF mutations in CRC can be predictive biomarkers for vitamin C therapy.

Citation Format: Jihye Yun, Adam Kavalier, Jatin Roper, Steve Gross, Carlo Rago, Nickolas Papadopoulos, Bert Vogelstein, Lewis Cantley. Vitamin C inhibits the survival and growth of colorectal cancer cells with KRAS or BRAF mutations. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C60.