Source: Wang Xin, Rao Benqiang. High-Dose Vitamin C Precision Therapy for Tumors [J/CD]. Journal of Tumor Metabolism and Nutrition, 2023, 10(3): 307-312.
Article link:
http://182.92.200.144/CN/abstract/abstract1016.shtml
Expert Introduction
Rao Benqiang, Deputy Director of the Second Department of Gastrointestinal Surgery at Beijing Shijitan Hospital, Capital Medical University, Deputy Director of the National Market Supervision Key Laboratory (Tumor Special Medical Food), Professor / Chief Physician, PhD Supervisor. He specializes in tumor nutritional metabolism therapy and holds several academic positions, including Executive Member of the Professional Committee of Integrated Chinese and Western Medicine of the Chinese Anti-Cancer Association, Chairman of the Tumor and Microecology Professional Committee, and Honorary Chairman of the Professional Committee of Inheritance and Innovation of Traditional Chinese Medicine of the Chinese Society of Geriatrics. He has presided over 16 national and provincial projects, published 115 papers, authored 6 monographs, and received 10 provincial scientific and technological awards, as well as 2 United Nations Peace Medals, and is recognized as a “hundred, thousand and ten thousand” talent in Jiangxi Province.
Abstract: High-dose vitamin C (HVC) therapy is a relatively safe and inexpensive treatment for tumors, capable of exerting anti-tumor effects through pathways such as promoting oxidative stress, epigenetic modification, inhibiting hypoxia-inducible factors (HIF-α), and immune regulation. However, HVC alone has a weak inhibitory effect on tumors, and the therapeutic effect is closely related to the tumor metabolic phenotype. HVC is effective in tumors with high expression of KRS/BRAF, HIF, TETs/IDH/WT1 mutations, and OMM Cyb5R3/VDAC1 complexes, while it has no significant inhibitory effect on tumors with low expression of KRS/BRAF, indicating that HVC requires precision therapy targeting tumor metabolic phenotypes. Combinations of high-dose vitamins, nano-formulated vitamin C, oncolytic viruses, immune checkpoint inhibitors such as PD-1/PD-L1 monoclonal antibodies, radiotherapy, cytotoxic drug therapy, and traditional Chinese medicine can significantly enhance the anti-tumor efficacy of HVC, suggesting that precision-enhanced therapy is an important research direction for HVC treatment, but more clinical trial evidence is needed to support this.
Since Cameron E and Pauling L reported in 1976 that high-dose vitamin C (HVC) therapy extended the survival of cancer patients, debates about tumor HVC treatment have continued. Although a publication by Moertel CG et al. from the Mayo Clinic in 1985 denied the therapeutic efficacy of tumor HVC, most preclinical trials still show that HVC can exert anti-tumor effects through various mechanisms such as promoting oxidative stress and epigenetic regulation. Because malignant tumor patients often have concurrent malnutrition, HVC therapy is a relatively safe and effective treatment method. With the advancement of research on the pharmacokinetics and anti-tumor mechanisms of vitamin C, HVC clinical protocols have become increasingly standardized. However, HVC alone has minimal efficacy in treating tumors, and for some tumors with specific metabolic phenotypes, or in combination with other methods, better efficacy can be achieved; thus, HVC requires precision-enhanced tumor therapy.
1 Clinical Advantageous Protocols for High-Dose Vitamin C Treatment
The effect of HVC on tumors depends on the intracellular concentration and retention time of vitamin C. The physiological plasma concentration of vitamin C is 26 ~ 84 μmol/L, exerting antioxidant effects; 100 μmol/L~0.5 mmol/L vitamin C regulates epigenetic dysregulation; while concentrations above 0.5 mmol/L mainly inhibit tumors through pro-oxidative reactions. After oral administration of 200 mg, the plasma concentration can reach 80 μmol/L; when the oral dose exceeds 200 mg, the absorption rate decreases and urinary excretion increases, and the plasma concentration of vitamin C still cannot reach millimolar levels, only by intravenous injection can pro-oxidative blood drug concentrations be achieved. At a dose of 75 g, it exhibits first-order pharmacokinetics, and at 100 g, the maximum plasma concentration tends to stabilize. Due to the half-life of vitamin C being only 2 hours, a scientifically reasonable intravenous administration protocol is 75 g, 2 times/day.
The Fenton reaction is a pro-oxidative stress response pattern of vitamin C. Vitamin C participates in the Fenton reaction by providing electrons, oxidizing extracellular Fe3+ to Fe2+, while generating H2O2, O2, and ascorbic acid free radicals (AFR); intracellularly, 1 mol of H2O2 reacts with 1 mol of Fe in the Fenton reaction to generate 1 mol of Fe3+, along with 1 mol of OH- and 1 mol of hydroxyl radicals, inducing lipid peroxidation leading to cell death. The intensity of the Fenton reaction also depends on the intracellular Fe2+ pool content; low levels of serum ferritin or free iron can affect HVC efficacy, and iron supplements can be administered appropriately 2-3 days prior to use.
After intravenous HVC treatment, continuing to take 10 g/day of vitamin C can prevent acute vitamin C deficiency after sudden withdrawal, allowing the patient’s vitamin C plasma concentration to reach 100 μmol/L ~ 0.5 mmol/L to exert epigenetic regulatory effects and thus enhance the anti-tumor effects of vitamin C. However, even at an oral dose of 1.25 g/kg, the plasma concentration of vitamin C can only reach 200 μmol/L, and further studies are needed to determine whether the designed dosage can achieve therapeutic goals.
2 Precision Treatment of Tumors with High-Dose Vitamin C
The effectiveness of HVC in treating tumors is related to specific tumor phenotypes. Mapping the metabolic and molecular profiles associated with HVC treatment sensitivity is conducive to achieving precise HVC treatment for tumors. Preliminary evidence shows that the metabolic molecular phenotypes sensitive to HVC treatment include vitamin C transporters, OMM Cyb5R3/VDAC1 complexes, KRS and BRAF mutations, TET2/IDH1/WT1 mutations, and HIF mutations. 2.1 Vitamin C Transporters Vitamin C exists in two forms: reduced ascorbic acid and oxidized dehydroascorbic acid (DHA). Ascorbic acid is actively transported through sodium-dependent vitamin C transporter (SVCT) 1 and enters cells via SVCT2; DHA is primarily transported into cells via glucose transporters (GLUT) such as GLUT 1 and GLUT 3. Cells with high expression of SVCT2 also exhibit high expression of stem cell-related genes Sox-2, Oct-4, Lin28, or tumor stem cell (CSC) markers CD133, making tumors more sensitive to HVC. There are significant differences in vitamin C transporters among different tissue types, and single nucleotide polymorphisms in the SVCT gene can affect plasma vitamin C concentrations and intracellular content; analyzing tumor cell transporters can help predict HVC efficacy.
2.2 OMM Cyb5R3/VDAC1 Complex The OMM Cyb5R3/VDAC1 redox complex is expressed on the outer mitochondrial membrane, utilizing cytosolic NADH as an electron donor to catalyze the rapid conversion of AFR to ascorbate, restoring the ascorbate “pool”, maintaining the intracellular NAD+/NADH ratio and ATP production, thereby protecting mitochondria from oxidative damage. Normal cells maintain low levels of reactive oxygen species (ROS) and normal reductive levels, while tumor cells exhibit excessive ROS production, hypoxic environments, and elevated cytosolic NADH; compensatory overexpression of OMM Cyb5R3/VDAC1 protects tumor cells, which is the “protective mode” of OMM Cyb5R3/VDAC1. During HVC treatment, AFR permanently accumulates in tumor cells, and OMM Cyb5R3/VDAC1 is feedback inhibited, accompanied by decreased AFR in the mitochondrial intermembrane space and cytosolic NAD+/NADH, causing respiratory chain electron flow and proton flow disturbances, imbalance in coenzyme Q (CoQ) “pool”, and initiating reverse electron transfer leading to cell death, which is the “destructive mode” of OMM Cyb5R3/VDAC1. OMM Cyb5R3 can serve as an efficacy marker for HVC treatment of tumors.
2.3 KRS/BRAF Mutations KRS/BRAF mutations activate downstream MAPK pathways, leading to upregulation of GLUT1 expression, enhancing the ability and rate of cells to uptake DHA. KRS/BRAF mutant tumor cells severely rely on glycolysis for survival and growth; inhibiting glycolysis may deplete ATP, trigger an energy crisis, and lead to cell death, making KRS/BRAF mutant tumors particularly sensitive to HVC. A clinical trial involving 442 colorectal cancer patients indicated that compared to the control group, there was no difference in progression-free survival (PFS), effective rate, and overall survival between HVC treatment and standard chemotherapy; however, patients with KRS mutations receiving chemotherapy combined with HVC treatment had a longer PFS than those receiving chemotherapy alone (9.2 months vs. 7.8 months).
2.4 TETs/IDH/WT1 Mutations Vitamin C acts as a cofactor for demethylases (TETs), directly interacting with the C-terminal catalytic domain of TETs to reduce Fe3+ to Fe2+ and activate TETs to participate in DNA demethylation, thereby inhibiting tumors. TETs include TET1, TET2, and TET3, with significant individual differences in expression in tumors, especially high mutation rates of TET2 in myeloid and lymphoid tumors. However, TET2 mutations are mutually exclusive with mutations in isocitrate dehydrogenases 1/2 (IDH1/2) or WT1 genes. IDH1/2 catalyze the oxidative decarboxylation of isocitrate to alpha-ketoglutarate, which is essential for maintaining the activity of TETs and other dioxygenases; mutations lead to excessive production of the metabolite 2-HG, which competitively inhibits TET2 function. WT1 is a transcription factor that regulates signaling pathways such as WNT and MAPK, participates in cell differentiation and tumor suppression, and interacts with TET2 to promote tumor demethylation. WT1 mutations hinder the binding of WT1 to TET2 and activate the transcription of WT1 target genes, leading to low TET2 activity and low levels of 5-hydroxymethylcytosine (5 hmC), which is considered an independent marker of poor prognosis and an abnormal marker of TET regulation. HVC can reduce DNA methylation and restore 5 hmC DNA levels by enhancing TET2 activity, thereby inhibiting epigenetic dysregulation associated with tumor progression. In IDH1 mutant tumor cells, daily administration of vitamin C (100 μg/ml) can overcome the effects of IDH1 mutations, stimulate TET2 to promote DNA demethylation and epigenetic remodeling of transcription factor binding sites, thus inducing tumor cell differentiation, with a synergistic effect on TET activity with IDH1 inhibitors.
2.5 Expression of Hypoxia-Inducible Factors Vitamin C acts as a cofactor for hypoxia-inducible factor (HIF) hydroxylase, inducing the von Hippel-Lindau (VHL) tumor suppressor protein to recognize HIF-1α, which is then ubiquitinated and degraded by the proteasome. Under hypoxic conditions, HIF-1α undergoes hydroxylation at specific proline and asparagine residues, inhibiting proteasomal degradation and translocating to the nucleus, where it dimerizes with HIF-1β and activates target genes regulating various cellular functions such as proliferation, apoptosis, angiogenesis, and glucose transport. High HIF-1α activity can inhibit TET2 expression; HVC treatment of tumors with high HIF-1α expression will increase HIF hydroxylase activity, thereby degrading HIF-1α and inhibiting the tumor-promoting effects of this transcription factor. However, in clear cell renal cancer patients with VHL gene mutations, the hydroxylation and degradation of HIF-α leads to the accumulation of transcription factors, and the activity of HIF-α is not associated with ascorbic acid content, making HVC ineffective against tumors with VHL gene defects. Conversely, in tumors with normal VHL genes, where hypoxic conditions increase HIF-α activity, HVC can achieve better efficacy by stimulating HIF hydroxylase activity to enhance HIF-α degradation.
3 High-Dose Vitamin C Enhanced Therapy for Tumors
3.1 High-Dose Vitamin C Combined with Radiotherapy and Chemotherapy HVC and radiotherapy (RT) can selectively promote oxidative stress overload in tumor cells by utilizing the differences in redox status between normal cells and tumor cells, producing large amounts of ROS and causing mitochondrial dysfunction. The synergistic anti-tumor mechanisms of both HVC and radiotherapy can increase the level of unstable iron pools in tumor cells, and both can inhibit the expression of RelB in tumor cells. Radiotherapy can promote the generation of H2O2, thereby enhancing ROS-mediated cytotoxicity. Currently, HVC combined with radiotherapy as a standard treatment protocol in clinical trials has resulted in nearly a 50% reduction in overall symptom intensity scores during radiotherapy. HVC combined with radiotherapy and gemcitabine has extended both progression-free survival (PFS) and overall survival (OS) in pancreatic cancer (13.7 months vs. 4.6 months, 21.7 months vs. 11.1 months).
In 23 completed clinical trials of HVC combined with chemotherapy drugs, the chemotherapy drugs primarily included arsenic trioxide, gemcitabine, carboplatin, paclitaxel, temozolomide, and standard chemotherapy regimen FOLFOX. Welsh JL et al. studied 9 advanced pancreatic cancer subjects who received intravenous vitamin C twice a week, achieving plasma levels ≥20 mmol/L after infusion, with subjects completing at least 2 cycles (8 weeks) of gemcitabine treatment showing a PFS of 6.5 months and an average survival of 13 months, compared to historical controls of 3.7 months and 6.7 months. Furqan M et al. completed a clinical trial of HVC combined with carboplatin-paclitaxel for advanced non-small cell lung cancer (NSCLC), with 75 g of vitamin C administered intravenously twice a week, and carboplatin and paclitaxel every 3 weeks for a total of 4 cycles, showing an objective response rate of 34.2%, disease control rate of 84.2%, and PFS and OS of 5.7 months and 12.8 months, respectively.
3.2 High-Dose Vitamin C Combined with Targeted Therapy Targeted drugs combined with HVC include anti-angiogenic drugs, kinase inhibitors (such as gefitinib), mitochondrial inhibitors (such as metformin), PARP inhibitors, glycolysis inhibitors, and clusterin (CLU) inhibitors. Mustafi S et al. achieved better results using 100 μmol/L vitamin C combined with CLU inhibitors for melanoma, with the synergistic mechanism being epigenetic regulation rather than pro-oxidative stress. Cetuximab-induced tumor cell transition from glycolysis to oxidative phosphorylation is easily influenced by HVC-induced oxidative stress; relatively high-dose vitamin C (0.5 g/kg) can eliminate cetuximab resistance, and SVCT2 can serve as a potential marker for combined treatment of KRAS mutant colorectal cancer (CRC) patients. Polyadenosine diphosphate-ribose polymerase (PARP) inhibitors can inhibit DNA repair in BRCA mutant cells, and combined with vitamin C can induce single-strand breaks in DNA, activating base excision repair (BER) pathways through the mediation of TET proteins, resulting in dual DNA damage effects. Targeting mitochondrial metabolic drugs such as berberine combined with relatively low concentrations of vitamin C (250 μmol/L) can inhibit over 90% of tumor stem cell growth.
3.3 Vitamin C Nanotherapy IVC therapeutic efficacy on tumors depends on the compartmentalized blood drug concentration and duration. Vitamin C is unstable, with a half-life of only 2 hours, and is easily affected by factors such as the number of SVCT2 transport proteins in the plasma membrane, metabolic activity, administration route, cytoplasmic pH, and membrane potential. Nanotechnology can shield vitamin C from degradation by the external environment, increasing its stability, achieving controlled release, and prolonging its intratumoral retention time. Some studies have designed a nanocapsule formulation of “vitamin C – sodium tripolyphosphate – alcohol-soluble protein” with a sustained release time of over 14 days, with only 5% oxidation after 10 days. Amiri S et al. prepared vitamin C liposomes, which still achieved stable vitamin C release after 20 days. Preclinical studies have shown that the effective blood drug concentration and duration of compartmentalized vitamin C nanomedicine are approximately 10 times higher than that of free vitamin C, and low-concentration vitamin C nanocapsules can also achieve pro-oxidative effects.
3.4 High-Dose Vitamin Combinations High-dose vitamin combinations include vitamin C with vitamin K3 (M/A), vitamin C with vitamin A (A/A), and vitamin C with B vitamins (A/B). The quinone in vitamin K3 undergoes single-electron reduction or double-electron reduction reactions to generate superoxide free radicals and consume NADH, glutathione, and other reductive substances, and can selectively activate alkaline deoxyribonuclease in tumor cells, exacerbating DNA damage mediated by HVC. The toxicity of M/A to tumor cells is accompanied by dose-dependent increases in mitochondrial superoxide and decreases in ATP synthesis, ranging from 2-3 times (2/200 μmol/L) to 8-15 times (20/2000 μmol/L), with concentrations of 5/500 μmol/L being the critical threshold for M/A to shift from inhibitory to lethal effects. The toxicity of the M/A combination to tumor cells is also related to the UBIAD1 protein, which catalyzes the conversion of vitamin K3 to K2; UBIAD1 is highly expressed in normal cells but low in tumor cells, and low expression of UBIAD1 will inhibit the conversion of vitamin K3 to K2, while vitamin K2 has reduced cytotoxicity to tumor cells by two orders of magnitude. UBIAD1 expression is one of the metabolic phenotypes for M/A therapy. The M/A combination dose ratio is 1:100, and this combination treatment has been applied to 17 patients with rising prostate-specific antigen (PSA) levels after local standard treatment failure; after 12 weeks of treatment with 50 g of vitamin C and 50 mg of vitamin K3 daily, 13 patients showed decreased PSA levels, and among 15 patients who continued treatment after 12 weeks, only 1 died after 14 months of treatment. The M/A combination has no toxic effects on normal cells below 2 mmol/L vitamin C or its M/A combination, but the addition of alpha-tocopherol succinate (α-TOS) induced cell death, attributed to α-TOS targeting mitochondrial respiratory chains to generate superoxide anions, lysosomal disturbances, mitochondrial fission, and cytosolic release of cytochrome c. The A/A combination synergistically reduces methylcytosine levels, enhancing metabolic reprogramming of naive pluripotent stem cells. Both vitamin A and vitamin C can induce NKG2DLs expression and inhibit tumor cell methylation by downregulating DNA methyltransferase levels, promoting the binding of tumor cells with natural killer (NK) cells, enhancing the immune targeting of tumor cells, and showing good efficacy against TET2 or TET3 mutant tumors. The A/B combination’s metabolic basis is that the active form of B vitamins, thiamine pyrophosphate (TPP), is an important cofactor for the enzyme-catalyzed reactions of pyruvate dehydrogenase complexes, α-ketoglutarate dehydrogenase complexes, and the pentose phosphate pathway, with the main components of redox reactions in the body being NADH, NADPH, and glutathione, all produced during enzyme-catalyzed reactions involving TPP. B vitamins have a synergistic anti-tumor effect with vitamin C in regulating cellular glucose metabolism and maintaining oxidative metabolic balance.
3.5 High-Dose Vitamin C Combined with PD-1/PD-L1 Inhibitors HVC increases the expression of programmed cell death ligand 1 (PD-L1) in tumor cells, and combined treatment with PD-1/PD-L1 inhibitors can achieve better results, with this synergy depending on CD8+ T cell infiltration, related to HVC activation of the cGAS-STING pathway. cGAS is a DNA sensor of the innate immune system; HVC generates ROS that activate cGAS, promoting the secretion of its second messenger cGAMP and activating downstream interferon-stimulated genes (STING) molecules. Since STING is primarily expressed in tumor endothelial cells (not expressed in tumor cells), HVC activates the endothelial cell STING pathway (endothelial activation). Endothelial activation is an important marker of vascular normalization, promoting tumor vascular normalization and the expression of lymphocyte adhesion molecules, leading to CD8+ T cell infiltration and ultimately inducing an acquired anti-tumor immune response. HVC can serve as a STING agonist combined with PD-1/PD-L1 inhibitors for tumor treatment.
3.6 High-Dose Vitamin C Combined with Oncolytic Virus Therapy Oncolytic viruses (OVs) are a class of viruses that can selectively infect and lyse tumor cells. Due to their dual action of locally killing tumor cells and inducing systemic anti-tumor immune responses, they have become an attractive new class of tumor immunotherapy drugs. However, OVs are quickly cleared by antiviral antibodies and cytotoxic T cell-mediated immune responses, and physical barriers such as high interstitial pressure and extracellular matrix prevent OVs from infecting tumor cells, limiting the efficacy of OVs as a single therapy. ROS are important components regulating intracellular pathways of immunogenic cell death (ICD); HVC can induce tumor cells to produce large amounts of ROS, and combined with OVs, it can become a new strategy for HVC-enhanced tumor therapy. HVC-induced increases in ROS mediate the translocation of calreticulin from the cytoplasm to the cell membrane, upregulating the expression of two ICD markers, HSP90 and high mobility group box, promoting the maturation and activation of dendritic cells (DCs), extending and enhancing the ICD effects triggered by OVs. Moreover, HVC enhances the infection rate and lytic ability of OVs on tumor cells, while OVs enhance the oxidative stress effects induced by HVC, leading to selective replication of OVs in tumor cells, severely disrupting cellular antioxidant functions and causing sustained increases in ROS. Both HVC and OVs can activate DCs and CD8+ T cells, reducing the number of M2-TAM and Treg cells, transforming the immunosuppressive tumor microenvironment (TME) into an immune-permissive TME, and converting “cold tumors” into “hot tumors”. Animal experiments have shown that the combination of HVC and OVs can inhibit tumor growth and metastasis, resulting in complete tumor regression in 30% of CT26 tumor-bearing mice and 20% of 4T1 (a tumor cell line) tumor-bearing mice.
3.7 High-Dose Vitamin C Combined with Traditional Chinese Medicine The holistic view and syndrome differentiation in traditional Chinese medicine provide natural advantages for multi-targeted, multi-pathway, and spatiotemporal coordinated regulation of tumor metabolism. Traditional Chinese medicine can also synergistically enhance HVC therapy for tumors through multiple links and levels. Heat-clearing and detoxifying drugs enhance the pro-oxidative stress effects of HVC by regulating mitochondrial function in tumor cells, blood-activating and stasis-removing drugs intervene in tumor lipid metabolism to promote the epigenetic modification effects of HVC, while drugs that strengthen the body’s defenses mainly regulate amino acid metabolism to improve the immune inhibitory effects of HVC. The combination of HVC and traditional Chinese medicine for enhanced therapy shows promise.
4 Outlook Precision-enhanced therapy can significantly improve the anti-tumor effects of HVC and is a key research direction for the future. However, several critical issues remain to be addressed, such as how multiple enhancement protocols can optimize HVC synergistic treatment for tumors, and how various precision treatment markers can predict efficacy. Addressing these issues requires high-quality clinical big data research to provide direct evidence for achieving better efficacy of HVC in tumor treatment.