Vitamin C Administration by Intravenous Infusion Increases Tumor Ascorbate Content in Patients With Colon Cancer: A Clinical Intervention Study

Abstract

The use of high dose ascorbate infusions in cancer patients is widespread, but without evidence of efficacy. Several mechanisms whereby ascorbate could affect tumor progression have been proposed, including: (i) the localized generation of cytotoxic quantities of H₂O₂; (ii) ascorbate-dependent activation of the 2-oxoglutarate-dependent dioxygenases that control the hypoxia-inducible factors (HIFs) and that are responsible for the demethylation of DNA and histones; (iii) increased oxidative stress induced by dehydroascorbic acid. We hypothesize that the dysfunctional vasculature of solid tumors results in compromised delivery of ascorbate to poorly perfused regions of the tumor and that this ascorbate deficit acts as an additional driver of the hypoxic response via upregulation of HIFs. Using a randomized "therapeutic window of opportunity" clinical study design we aimed to determine whether ascorbate infusions affected tumor ascorbate content and tumor biology. Patients with colon cancer were randomized to receive infusions of up to 1 g/kg ascorbate for 4 days before surgical resection (n = 9) or to not receive infusions (n = 6). Ascorbate was measured in plasma, erythrocytes, tumor and histologically normal mucosa at diagnostic colonoscopy and at surgery. Protein markers of tumor hypoxia or DNA damage were monitored in resected tissue. Plasma ascorbate reached millimolar levels following infusion and returned to micromolar levels over 24 h. Pre-infusion plasma ascorbate increased from 38 ± 10 µM to 241 ± 33 µM (p < 0.0001) over 4 days and erythrocyte ascorbate from 18 ± 20 µM to 2509 ± 1016 µM (p < 0.005). Tumor ascorbate increased from 15 ± 6 to 28 ± 6 mg/100 g tissue (p < 0.0001) and normal tissue from 14 ± 6 to 21 ± 4 mg/100 g (p < 0.001). A gradient of lower ascorbate was evident towards the tumor centre in both control and infusion samples. Lower expression of hypoxia-associated proteins was seen in post-infusion tumors compared with controls. There were no significant adverse events and quality of life was unaffected by ascorbate infusion. This is the first clinical study to demonstrate that tumor ascorbate levels increase following infusion, even in regions of poor diffusion, and that this could modify tumor biology.

Clinical trial registration: ANZCTR Trial ID ACTRN12615001277538 (https://www.anzctr.org.au/).

Keywords: colorectal cancer; hypoxia-inducible factor; normal mucosa; plasma; tumor; tumor hypoxia; vitamin C.

Figure 1

Plasma and red blood cell ascorbate levels in patients at baseline, immediately post-infusion and over the course of high dose ascorbate infusion. IVC was administered daily for 4 days and blood taken pre- and post-IVC infusion as indicated. The first infusion comprised 25 g ascorbate and subsequent infusions were at 1 g/kg body weight, with a maximum of 75 g. (A) Baseline plasma ascorbate from patients in the control (○) and infusion (⬤) cohorts at colonoscopy (1) and at surgery (2) (control patients) or immediately prior to first infusion (Infusion patients). Horizontal bars show the mean value. (B) Plasma ascorbate in samples taken prior to first infusion and immediately after infusion with high dose ascorbate. Horizontal bars show the mean value. (○) sample following 25 g infusion. ****p < 0.0001 (unpaired t-test). (C) Plasma ascorbate immediately prior to consecutive high dose ascorbate infusions on days 1–4. Horizontal bars show the mean value. Significant differences were observed between day 1 plasma ascorbate and the subsequent days ***p < 0.005, ****p < 0.0001 (unpaired t-tests). There was a significant difference between concentrations at days 1 and 4, *p < 0.05 and a one-way ANOVA indicated a significant difference between days 1 and 4, p < 0.0001. (D) Red cell ascorbate concentrations pre- and post-infusion; means ± SD. Unpaired Student’s t test showed no significant differences between red cell ascorbate concentrations on day 2 post-infusion and pre-infusion day 3, and post-infusion day 3 and pre-infusion day 4. (E) Plasma (▪) and red cell (⬤) ascorbate concentrations measured prior to infusion each day; means ± SD. A mixed-effects model indicated a significant difference between days, with day 1 different to day 2, 3, and 4; p < 0.005 for red cells and p < 0.05 for plasma.

Figure 2

Ascorbate levels in patients with colorectal cancer in normal mucosa and in tumor tissue. Tissue levels in control (A, B) and infusion patients (C, D), with samples taken at diagnostic colonoscopy and after resection. Ascorbate levels were unchanged in normal mucosa (A) and in tumor tissue (B) from control patients, but were significantly increased in both normal mucosa (C) and tumor tissue (D) following infusions. Tumor lesions were sampled at multiple locations as follows: edge/periphery (Peri), mid-tumor (Mid) and central core (Cent). Not every part of the tumor was sampled in every patient and hence sample numbers may vary for the tumor regions (B, D). Individual data for control (n ≤ 6) and infusion cohorts (n ≤ 9) with means are shown. Results were compared using paired t-tests for changes following infusion, as shown. *p < 0.05, **p < 0.01, n.s., not significant.

Figure 3

Associations between tumor and normal tissue ascorbate, and between tissue and plasma ascorbate levels in patients with colon cancer. (A) At biopsy, tumor and normal mucosa tissue ascorbate is closely correlated (Pearson r = 0.82, p = 0.0002). (B) Association between plasma ascorbate levels and normal mucosa (○) and tumor (▪) tissue in patients at biopsy (least squares fit, R² for best fit 0.64 and 0.62 for normal (^…….) and tumor (—), respectively). (C) At resection, a correlation between tumor periphery and normal tissue is seen (Pearson r = 0.87, p < 0.0001). (D) Association between plasma levels (pre-infusion on day prior to resection) and normal mucosa (○) and tumor periphery (▪), with non-linear saturation curve fit indicated (R² for best fit 0.85 and 0.69 for normal and tumor, respectively). Individual data for control (n = 6) and infusion cohorts (n = 9). (E) Comparison of normal mucosa (N) and tumor (T) tissue ascorbate levels with increasing plasma levels. Box plots show medians and the interquartile range with whiskers representing the 10–90 percentiles. There is a significant difference between mucosal and tumor tissue when plasma levels exceed the normal physiological maximum of 100 µM (unpaired t-test, **p = 0.008).

Figure 4

Protein expression associated with the hypoxic response and oxidative stress response in bowel mucosal tissue and in tumor tissue in control and infusion cohorts. Relative levels of (A) VEGF, (B) GLUT1, (C) CA-IX, were monitored in normal mucosal and tumor tissue from each individual. Expression of each protein was elevated in tumor tissue relative to control. Levels were lower in tumor tissue (all tumor regions) from the infusion cohort when compared with the levels in control tumor tissue (unpaired t-test results shown). (D) HIF Pathway score was derived from the combined measurement of the three proteins for each tumor sample. (E) Relative levels of γH2AX in tumor and normal mucosal tissues. Comparative statistics for combined tumor samples from peripheral, mid and central regions of the tumors are shown (unpaired t-tests). In addition, one way ANOVA with fitting for a mixed effects model (Brown-Forsythe and Welch test) indicated a significant increase in the tumor regions from periphery to central for the combined HIF Pathway score (p = 0.02).