Rouleau Lauren, Antony Anil Noronha, Bisetto Sara, Newberg Andrew, Doria Cataldo, Levine Mark, Monti Daniel A, Hoek Jan B
MitoCare Center, Department of Pathology, Anatomy and Cell Biology, Thomas Jefferson University, Philadelphia, PA 19107, USA.
Jefferson-Myrna Brind Center of Integrative Medicine, Thomas Jefferson University Hospital, Philadelphia, PA 19107, USA.
Free Radic Biol Med. 2016 Jun;95:308-322. doi: 10.1016/j.freeradbiomed.2016.03.031. Epub 2016 Mar 30.
We investigated the mechanism of selective ascorbate-induced cytotoxicity in tumor cells, including Hep G2 cells, compared to primary hepatocytes. H2O2 formation was required for ascorbate cytotoxicity, as extracellular catalase treatment protected tumor cells. H2O2 generated by glucose oxidase treatment also caused cell killing, but treatment with a pharmacologic dose (5-20mM) of ascorbate was significantly more cytotoxic at comparable rates of H2O2 production, suggesting that ascorbate enhanced H2O2 cytotoxicity. This was further supported by the finding that ascorbate at a non-cytotoxic dose (1mM) enhanced cell killing caused by glucose oxidase. Consistent with this conclusion, ascorbate treatment caused deregulation of cellular calcium homeostasis, resulting in massive mitochondrial calcium accumulation. Ascorbate acted synergistically with the chemotherapeutic sorafenib in killing Hep G2 cells, but not primary hepatocytes, suggesting adjuvant ascorbate treatment can broaden sorafenib's therapeutic range. Sorafenib caused mitochondrial depolarization and prevented mitochondrial calcium sequestration. Subsequent ascorbate addition further deregulated cellular calcium homeostasis promoting cell death. Additionally, we present the case of a patient with hepatocellular carcinoma (HCC) who had prolonged regression of a rib metastasis upon combination treatment with ascorbate and sorafenib, indicating that these studies have direct clinical relevance.
我们研究了与原代肝细胞相比，选择性抗坏血酸诱导肿瘤细胞（包括Hep G2细胞）产生细胞毒性的机制。抗坏血酸的细胞毒性需要过氧化氢（H2O2）的形成，因为细胞外过氧化氢酶处理可保护肿瘤细胞。葡萄糖氧化酶处理产生的H2O2也会导致细胞死亡，但在产生H2O2速率相当的情况下，用药理剂量（5 - 20mM）的抗坏血酸处理的细胞毒性显著更高，这表明抗坏血酸增强了H2O2的细胞毒性。抗坏血酸在非细胞毒性剂量（1mM）下增强葡萄糖氧化酶引起的细胞杀伤这一发现进一步支持了这一点。与此结论一致，抗坏血酸处理导致细胞钙稳态失调，导致大量线粒体钙积累。抗坏血酸与化疗药物索拉非尼协同作用杀死Hep G2细胞，但不杀死原代肝细胞，这表明抗坏血酸辅助治疗可拓宽索拉非尼的治疗范围。索拉非尼导致线粒体去极化并阻止线粒体钙螯合。随后添加抗坏血酸进一步破坏细胞钙稳态，促进细胞死亡。此外，我们报告了一例肝细胞癌（HCC）患者，该患者在抗坏血酸和索拉非尼联合治疗后，肋骨转移灶出现了长期缓解，这表明这些研究具有直接的临床相关性。
