The use of pharmacologic ascorbate in the treatment of hepatocellular carcinoma: mechanism of cytotoxicity and synergism with the multikinase inhibitor sorafenib
Pharmacologic ascorbate’s use as an adjuvant chemotherapeutic in advanced cancer has a controversial history. In the past decade, interest in this treatment was renewed based on a re-assessment of the pharmacokinetics of ascorbate, which led to the discovery that ascorbate’s selective cytotoxicity only occurs at plasma levels achievable through intravenous administration, but not through oral administration. Hepatocellular carcinoma (HCC) is the 2nd leading cause of cancer deaths worldwide. It is often diagnosed at a late stage when few treatment options remain. The current standard of care for unresectable HCC is sorafenib, a multikinase inhibitor targeting Raf-1 and VEGFR-2 and -3, as well as other kinases. New combination therapies are urgently needed. Here we investigated the mechanism of selective ascorbate-induced cytotoxicity in tumor cells, including Hep G2 cells, compared to primary hepatocytes. Additionally, we examined the combination of ascorbate and sorafenib in Hep G2 cells and performed a preliminary assessment of this combination in Huh-7 and SNU-449 cells. H2O2 formation was required for ascorbate cytotoxicity, as extracellular catalase treatment protected tumor cells. H2O 2 generated by glucose oxidase treatment also caused cell killing, but treatment with a pharmacological dose (5-20 mM) of ascorbate was significantly more cytotoxic at comparable rates of H2O2 production, suggesting that ascorbate enhanced H2O2 cytotoxicity. Ascorbate treatment of Hep G2 cells 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. This observation prompted us to performed preliminary experiments using extracellular flux analysis (Seahorse Bioscience) to investigate sorafenib’s effects on the mitochondrial electron transport chain and mitochondrial energy conservation. Here we provide evidence that sorafenib may be a reversible general inhibitor of the ETC. Preliminary results of the combination of ascorbate and sorafenib in Huh-7 and SNU-449 cells varied in effectiveness compared to the synergistic response seen in Hep G2 cells, suggesting the net effects of sorafenib result from multiple actions of the drug on different cellular functions, the outcome of which may vary depending on the specific properties of these cells. 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.
Rouleau, Lauren L, "The use of pharmacologic ascorbate in the treatment of hepatocellular carcinoma: mechanism of cytotoxicity and synergism with the multikinase inhibitor sorafenib" (2016). ETD Collection for Thomas Jefferson University. AAI10190418.