The Association Between Ascorbate and the Hypoxia-Inducible Factors in Human Renal Cell Carcinoma Requires a Functional Von Hippel-Lindau Protein

Abstract

Hypoxia-inducible transcription factors (HIFs) drive angiogenesis and cancer cell growth, contributing to an aggressive tumor phenotype. HIF-α protein levels and activity are controlled at the post-translational level by HIF hydroxylases. Hydroxylated HIF-α is recognized by the von Hippel Lindau (VHL) tumor suppressor and targeted for degradation. The HIF hydroxylases are members of the iron and 2-oxoglutarate-dependent dioxygenases, which require ascorbate as cofactor for activity. Clear cell renal cell carcinomas (ccRCC) harbor mutations in the VHL gene, whereas papillary RCC (pRCC) have a functional VHL. These natural occurring VHL variants in RCC enable the testing, in clinical samples, of the hypothesis that ascorbate modulates HIF-α levels through its role as a cofactor for the HIF hydroxylases. We measured ascorbate, HIF-1α, and HIF-2α protein and HIF downstream targets BNIP3, CA9, cyclin D1, GLUT1, and VEGF (combined to generate the HIF pathway score) in VHL-defective ccRCC (n = 73) and VHL-proficient pRCC human tumor tissue (n = 41). HIF and ascorbate levels were increased in ccRCC and pRCC tumors compared to matched renal cortex. HIF-1 and total HIF pathway activation scores were decreased with higher ascorbate in pRCC tumors (Spearman r = −0.38, p < 0.05 and r = −0.35, p < 0.05). This was not evident for ccRCC tumors. In mechanistic studies in vitro, ascorbate influenced HIF-1 activity in VHL-proficient, but not VHL-defective ccRCC cells. Our results indicate that ccRCC, which lacks a functional VHL, does not respond to ascorbate-mediated modulation of the HIF response. This contrasts with the demonstrated association between ascorbate content and the HIF pathway observed in pRCC and other tumors with a functional VHL. The results support a role for ascorbate as a modulator of HIF activity and tumor aggression in cancer types with a functional hypoxic response.

Vitamin C administration by intravenous infusion increases tumour ascorbate content in patients with colon cancer: a clinical intervention study

“…The tissue protein expression levels showed a similar inverse relationship between ascorbate and markers of hypoxia as we have previously observed in retrospective analyses of human endometrial, breast, renal and colorectal tumour samples (22,35,36,47). A similar nding was described in thyroid cancer (48).…”

“…For GLUT1, the same positive control (20 µg protein of 1% O 2 treated T24 cell lysate) was run on each gel to normalize signals between the blots. For CA-IX, a clear cell renal cell carcinoma tissue sample (University of Otago Human Ethics committee H14/020, (36), adjusted to 1.5 µg DNA per well, was similarly run on each gel. For γH2AX, a positive control (WiDr cells from ATCC treated for 4 h with 20 mM ascorbate, 20 µg protein) was run on each gel.…”

“…Protein bands were visualized using the ECL Prime Western Blotting Detection Reagent (GE Healthcare, Chicago, USA), and quanti ed with the Alliance 4.7 imaging system and the ImageJ software. (35,36) VEGF ELISA VEGF protein levels were measured in tissue lysates using the human VEGF DuoSET ELISA Development Kit (R&D Systems, DY293B) according to the manufacturer´s instructions.…”

“…Expression of HIF-dependent proteins and markers of oxidative stress following infusion. We monitored the levels of VEGF, GLUT1 and CA-IX as markers of HIF activation in the surgical resection samples, as previously (22,36) and have compared tumour and adjacent normal mucosal tissue in control and infusion patient samples. Hypoxic marker protein levels were measured by western blotting (GLUT1, CA-IX) ( Supplementary Fig.…”

Intravenous vitamin C for reduction of cytokines storm in acute respiratory distress syndrome

“…In some cases, there have been no benefits. However, new knowledge regarding the pharmacokinetic properties of Vit-C, and recent preclinical studies, have revived interest in the utilization of high-dose Vit-C for cancer treatment [132][133][134][135][136][137][138][139][140][141][142][143][144][145]. Similar is the case of using IV Vit-C as antiviral, especially for the recent Covid19 [146][147][148][149][150].…”