**4.** *In Vivo* **Studies**

Levine *et al.* reported that reaction products obtained from ascorbate *in vitro* are also found *in vivo* [34]. They showed that after i.v. injection, ascorbate baseline concentrations of 50–100 μM in blood and extracellular fluids peaked to >8 mM. After intraperitoneal injection, peaks approached 3 mM in both fluids. They hypothesized that *in vivo*, ascorbate was a prodrug for selective delivery of ascorbate radical and H2O2 to the extracellular space. Moreover, a regimen of daily pharmacologic ascorbate treatment significantly decreased growth rates of ovarian (*p* < 0.005), pancreatic (*p* < 0.05) and glioblastoma (*p* < 0.001) tumors established in mice [35]. In addition to inducing oxidative stress, high concentrations of ascorbic acid inhibited cell migration and the gap filling capacity of endothelial progenitor cells (EPCs) [36], and it has been reported that ascorbic acid inhibited corneal neovascularization in a rat model [37].

High concentrations of ascorbic acid also inhibited tumor growth in BALB/C mice implanted with sarcoma 180 cancer cells [38]. The survival rate increased by 20% in the group that received high doses of ascorbic acid, compared to controls. Gene expression studies from biopsy and wound healing analysis *in vivo* and *in vitro* suggested that the carcinostatic effect induced by high doses of ascorbic acid were related to inhibition of angiogenesis [39]. In addition, intraperitoneal administration of high doses of ascorbic acid quantitatively upregulated Raf kinase inhibitory protein (RKIP) and annexin A5 expression in a group of BALB/C mice implanted with S-180 sarcoma cancer cells. The increase in RKIP protein levels suggested that these proteins are involved in the ascorbic acid-mediated suppression of tumor formation [39]. Moreover, high doses of ascorbic acid (~1 mM) enhanced the apoptosis of cancer cells during co-treatment with paclitaxel, and the combinational treatment of paclitaxel and ascorbic acid ameliorated the side effects caused by paclitaxel in BALB/c mice implanted with or without sarcoma 180 cancer cells [40].
