VC plays a significant role in regulating gene expression, contributing to the prevention of malignant transformation and inhibiting the progression of tumors through various pathways (Figure 5A): (1) Downregulation of oncogenes. VC can downregulate the expression of the cellular myelocytomatosis oncogene (c-myc), which is associated with tumor growth and proliferation (74, 75). Dey et al. (76) found that VC inhibits the activation of epidermal growth factor receptor (EGFR) and its downstream target, c-myc, thereby reducing lung cell proliferation; (2) Activation of tumor suppressor genes. VC enhances the activities of glutathione peroxidase (GPX) and aldehyde dehydrogenase (ALDH) by transcriptionally activating the tumor suppressor gene p53. This activation is facilitated by the ubiquitination of murine double minute 2 (MDM2), which leads to the stabilization and activation of p53, promoting programmed cell death in cancer cells (77); (3) Cell cycle arrest. VC can activate the cyclin-dependent kinase inhibitor p21 and upregulates p53, leading to cell cycle arrest at the G0/G1 phase and thus limiting their proliferation (78); (4) Modulation of apoptotic factors. High doses of VC can downregulate the expression of the anti-apoptotic gene Bcl-2 and upregulate pro-apoptotic factors such as Bax and cleaved caspase-3 (79). This shift in the balance between pro- and anti-apoptotic factors ultimately leads to increased apoptosis in tumor cells.

Cancer cells often display significant DNA and RNA methylation due to elevated levels of DNA methyltransferases (DNMTs) and reduced activity of the TET proteins responsible for demethylation. VC can enhance the residual demethylation activity of TET proteins in cancer cells, leading to the re-expression of tumor suppressor genes, thereby interfering with tumor survival and enhancing sensitivity to other cancer treatments (80). Preclinical studies have indicated that high-dose VC can block the abnormal proliferation of hematopoietic stem cells by activating cellular responses to changes in energy metabolism, oxygen, and iron levels (81, 82). This occurs through the restoration activity of TET and the enhancement of Jumonji C domain (JmjC)-containing histone demethylase (JHDM) (Figure 5A).

Cancer cells often exhibit mitochondrial dysfunction, an unstable intracellular iron pool, low catalase activity, and inadequate ROS scavenging capabilities. High concentrations of VC can induce cancer cell death through prooxidant effects for several reasons (Figure 5B): (1) Impact on mitochondrial function. High concentrations of VC can influence cytochrome C and the nicotinamide adenine dinucleotide (NADH) ubiquinone reductase complex I within the mitochondrial electron transport chain (83–85). This effect is mediated by altering Fe²⁺-dependent dioxygenase activity in cancer cells, which upregulates the endoplasmic reticulum unfolded protein response and proteins related to translation inhibition, such as eukaryotic initiation factor 2α (eIF2α) and protein kinase R (PKR)/PKR pTr-446. This leads to increased mitochondrial and endoplasmic reticulum (ER) stress responses; (2) Iron metabolism alterations. VC can promote the synthesis of ferritin and inhibit its degradation by inhibit nuclear factor kappa-B (NF-κB) in cancer cell, resulting in an increase in Fe³⁺ within the cells and its subsequent reduction to Fe²⁺. The elevated unstable iron pool exacerbates the Fenton reaction, generating more ROS and heightening intracellular oxidative stress (8). The ROS produced can inhibit the phosphorylation of inhibitor of nuclear factor-κB kinase (IKK) α/β and inhibitory subunit of NF kappa B alpha (IκBα), creating a positive feedback loop that blocks the activation of the IKK/IκB/NF-κB pathway (86). Additionally, DHA, as the oxidized form of VC, can be reduced back to VC by consuming GSH within the cell, further amplifying oxidative stress levels. Excessive ROS in cancer cells can inhibit and kill these cells through several mechanisms (87, 88) (Figure 5B): (1) Cellular damage. High levels of ROS can damage DNA, proteins, and lipids, leading to oxidative stress in the endoplasmic reticulum and mitochondria of cancer cells; (2) Inhibition of kinase activity. ROS can inhibit the activity of key intracellular kinases, including mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinases (ERK), phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT), and BRAF, which are crucial for cell survival and proliferation; (3) Cysteine modification. ROS can oxidize and modify cysteine-containing proteins during translation and the cell cycle, resulting in the arrest of cancer cells in the S phase. This is especially relevant in tumors that rely on antioxidants to maintain their intracellular redox balance. Ma et al. (89) demonstrated that high doses of VC administered via tail vein injection in glioma model mice led to increased activity of checkpoint kinase 2 (CHK2) and histone 2AX (H2AX), indicating that high-dose VC caused DNA damage by elevating intracellular ROS levels. Moreover, VC can catalyze the reaction between superoxide anions and Fe²⁺ to produce hydrogen peroxide outside the cell, contributing to cancer cell death. Notably, high-dose VC appears to spare normal cells in vivo from damage while exerting its anticancer effects. This difference is primarily due to the lower unstable iron pool and stronger ROS scavenging capabilities found in normal cells compared to cancer cells. The above selective toxicity underscores the potential of VC as a therapeutic agent in cancer treatment.

In solid tumors, the mismatch between blood vessel formation and tumor growth often leads to hypoxia, as tumor cells obstruct surrounding blood vessels and inhibit new capillary formation (90) (Figure 5C). The hypoxic microenvironment inhibits the hydroxylation of proline residues on HIF-1α by proline hydroxylase (PHD), which prevents HIF-1α from binding to the tumor suppressor protein VHL, thereby avoiding its degradation by E3 ubiquitinase. Simultaneously, hypoxia also impairs the hydroxylation of asparagine residues on HIF-1α by factor-inhibiting HIF (FIH), facilitating its binding to p300. These processes collectively enhance the transcriptional activity of HIF-1α, activating various genes that promote tumor survival. Importantly, HIF hydroxylase, an α-KG-dependent dioxygenase, requires VC as a cofactor for iron cycling (91). Therefore, VC supplement can enhance HIF hydroxylase function, thereby reducing HIF-1α expression and inhibiting tumor progression. For example, previous study showed a significant negative correlation between intratumoral HIF-1α levels and VC in endometrial, renal, and colon cancers, with higher VC levels associated with better postoperative outcomes (92). The potential mechanism could be ascribed to high-dose VC decreasing HIF-1α and its downstream targets (GLUT-1 and VEGF). Zhao et al. (93) and Zeng et al. (94) fund that high-dose VC can decrease vimentin levels and increase E-cadherin expression, effectively reversing the epithelial–mesenchymal transition (EMT) in tumor cells. This reversal is achieved by VC inactivating the HIF-1α, which inhibits cell migration and invasion, thereby potentially limiting tumor progression and metastasis. Tumor neovascularization is marked by high permeability and discontinuity, which can facilitate distant metastasis while also obstructing immune cell infiltration and drug delivery. VC has been shown to downregulate the expression of VEGF-A and VEGF-D, thereby inhibiting abnormal angiogenesis (95, 96). Additionally, VC promotes vessel normalization and reduces vascular permeability by inducing endothelial cell contraction and preventing endothelial cell apoptosis. These multifaceted actions of VC may enhance the efficacy of therapies targeting tumors by improving drug delivery and immune response.

Cancer cells frequently exhibit a dependency on aerobic glycolysis, also known as the Warburg effect, and glutamine addiction to procure glucose and glutamine from their microenvironment for energy feeding (97) (Figure 5D). VC has been shown to impede these energy acquisition pathways through multiple mechanisms. It induces elevated levels of ROS within cancer cells, which in turn suppresses glycolysis by inhibiting GAPDH activity. VC promotes a metabolic shift in glutamine utilization toward the synthesis of GSH, thereby replenishing intracellular GSH levels and competitively inhibiting glutamine-dependent energy production. VC facilitates the degradation of glutamine synthetase, which diminishes the endogenous supply of glutamine for metabolic energy production. These alterations compel cancer cells to rely on the more energetically demanding oxidative phosphorylation pathway for energy acquisition. As described in section “2.1 Biological properties and functions,” it was established that cancer cells expressing GLUT1 transport DHA at a faster rate than glucose, and that the ROS released by cancer cells in response to DHA can further enhance its uptake. Consequently, for tumors with KRAS or BRAF mutations (such as colon and pancreatic cancers) that exhibit high GLUT1 expression and low SVCTs expression, extracellular DHA treatment competitively inhibits glucose uptake by cancer cells. This impairment of glycolysis consequently reduces the cells’ energy production. Simultaneously, the treatment creates a significant concentration gradient of DHA across the cancer cell membrane. The accelerated influx of DHA into cancer cells leads to the generation of copious amounts of ROS. The production of ROS depletes intracellular NADPH and disrupts the GAPDH-mediated glycolytic flux between glyceraldehyde 3-phosphate (GAP) and 1,3-bisphosphoglyceric acid (1,3-BPG). Furthermore, the elevated ROS levels increase pentose phosphate pathway (PPP) enzyme activity while diminishing the tricarboxylic acid (TCA) cycle and downstream glycolytic products (98). These combined effects result in decreased intracellular ATP production, culminating in an energy crisis and the demise of the cancer cells.

Most immune cells rely on high intracellular concentrations of VC to exert antioxidant effects, maintain redox balance, and provide electrons necessary for the activity of iron and copper-dependent dioxygenases, which are crucial for normal immune function (Figure 5E). However, the uptake of VC from TME by cancer cells can lead to a deficiency in immune cells, resulting in immune dysfunction. Therefore, supplementing with VC can help restore the antitumor immune activity of these cells. This includes promoting the differentiation and polarization of myeloid and T cells, enhancing T cell maturation and activation, increasing T cell infiltration, and improving the CD8+/CD4+ T cell ratio (99, 100). VC supplementation can also support the development and chemotaxis of B cells, boosts cytokine production, induces apoptosis in M2-type macrophages, and enhances natural killer (NK) cell-mediated tumor killing (101–103).

Cancer-associated fibroblasts (CAFs) play a crucial role in extracellular matrix (ECM) remodeling by producing type I, III, and V collagen, as well as fibronectin, and secreting matrix metalloproteinases (MMPs) (104) (Figure 5F). This remodeling contributes to tumor tissue stiffness and provides a supportive scaffold for tumor cell survival, proliferation, and metastasis. During the early stages of metastasis, cancer cells can detect changes in the mechanical properties of the ECM, transmitting mechanical signals that trigger cytoskeletal remodeling and EMT. VC is pivotal in the above processes. In terms of ECM formation, VC significantly downregulates the expression of genes associated with collagen synthesis, cell adhesion, and the ECM in CAFs (105, 106). As a cofactor for hydroxylase enzymes, VC enhances proline and lysine hydroxylation, increasing collagen content in normal tissue and promoting the formation of a stable three-dimensional collagen structure, which helps prevent cancer cell invasion (107). VC can inhibit ECM remodeling through several mechanisms: (1) Reducing the mRNA expression of various MMPs in CAFs, thereby blocking ECM remodeling and the secretion of tumor-promoting growth factors (105); (2) Inhibiting tumor cell EMT by modulating integrin and YAP/TAZ mechanical signaling (108); (3) Enhancing proline hydroxylation and further promoting collagen-integrin adhesion and ERK1/2 phosphorylation in cell stretching pathways (109).

Long-term high rates of aerobic glycolysis often leads to the accumulation of acidic substances (such as lactic acid and H⁺) in TME, resulting in an acidic milieu with a pH of 6.7 to 7.1 (110) (Figure 5G). This acidic TME adversely affects various aspects of malignant tumors, including immunosuppression, interstitial degradation, metastatic spread, and drug resistance. VC, known as a glycolysis inhibitor, can directly inhibit HIF-α-mediated glycolysis and the expression of downstream genes associated with acidic metabolites in both cancer cells and stromal cells within the microenvironment. This action helps reduce the levels of endogenous acidic metabolites. Additionally, VC downregulates the gene expression of hypoxia-induced carbonic anhydrase 9 (CAIX) and monocarboxylate transporter (MCT), thereby decreasing H⁺ generation and inhibiting the efflux of H⁺ and lactic acid (111, 112).

In addition to neurons, glial cells, such as Schwann cells and oligodendrocytes, play a key role in neuro-tumor communication. Zhang et al. (113) found that Schwann cells promote the neural invasion of cancer cells through autophagy based on a study of pancreatic cancer. Englard and Seifter (114) reported that VC is abundant in neurons and can be used as a neuromodulator to promote the biosynthesis of norepinephrine in nerve cells by enhancing the activity of dopamine β hydroxylase (Figure 5H). Huff et al. (115) demonstrated that VC can boost proline hydroxylase activity in Schwann cells, promoting collagen synthesis and inducing demyelination of Schwann cell myelin genes to enhance myelin formation. Some studies have also shown that VC can promote the storage and release of neurotransmitters (dopamine, acetylcholine, glutamic acid, etc.), promote neural growth and development, and mediate the metabolic coupling between glial cells and neurons through the ascorbic acid cycle (116). Conversely, Ferrada et al. (117) found that a physiological dose of VC (200 μM) could induce necrotic apoptosis in neurons in vitro. While VC plays a role in maintaining neurobiological function within the tumor microenvironment, strong evidence supporting its regulatory effects on tumor-associated nerves is still lacking, and further exploration of the related functions and mechanisms is needed.

The immune-tumor-microorganism axis has emerged as a key focus in tumor diagnosis and treatment research. The microbial TME includes intestinal and intratumoral microorganisms and their metabolites, which significantly influence tumor development (118). VC can modulate tumor progression by regulating microbial balance both in the intestine and within tumors. Some studies indicated that oral VC administration increases the abundance of Collinsella and Lachnospiraceae bacteria in feces, leading to heightened secretion of short-chain fatty acids (SCFAs) that enhance the immunogenicity of various cancers (119, 120) (Figure 5I). This suggests a novel role for VC in improving cancer immunotherapy. Notably, VC can also strengthen the tumor’s physical barrier, inhibit microbial colonization, and promote the transcription of retroviral genes in tumor cells, thereby enhancing the effectiveness of anti-PD-1 immune checkpoint therapy and epigenetic treatments (103). Moreover, the research by Ma et al. (121) demonstrated that ROS generated by high-dose VC can induce immunogenic cell death mediated by oncolytic viruses, transforming the tumor into a “hot” environment conducive to immune response.

High-dose VC has been extensively studied as a potential monotherapy for various cancers, including hematological malignancies like leukemia, as well as solid tumors such as colorectal, pancreatic, and lung cancers. Mussa et al. (128) reported that VC induced dose-dependent tumor spheroid cell death, primarily due to elevated H₂O₂ production and oxidative stress imbalance caused by GSH depletion, with varying susceptibilities between MCF-7 and MDA-MB-231 spheroids. VC also showed efficacy against acute myeloid leukemia with IDH mutations, a common genetic alteration associated with poor prognosis (129). Furthermore, studies on triple-negative breast cancer stem cell lines (MDA-MB-231 and MDA-MB-468) showed differential but excellent therapeutic responses to VC, suggesting its potential as a targeted treatment strategy (130). Additionally, research by Cimmino et al. (131) demonstrated that daily high-dose VC injections in mice with low TET2 activity slowed leukemia progression by inhibiting hematopoietic stem cell proliferation and reducing leukocyte counts. Beyond hematologic cancers, VC has also shown promise in solid tumors. Vaishampayan and Lee (132) demonstrated that pharmacological VC induced non-apoptotic cancer cell death in osteosarcoma models via an intracellular ROS-iron-calcium crosstalk mechanism, leading to mitochondrial dysfunction and reduced ATP levels, with potential clinical utility in osteosarcoma treatment. A study published in Science in 2015 revealed that high-dose VC could trigger apoptosis of BRAF or KRAS mutant colon cancer cells by decreasing intracellular glucose and energy production due to intracellular ROS accumulation and GAPDH inhibition, which was not observed in wild type colon cancer cells (133). In genetically engineered mice with KRAS-driven colon tumors, VC treatment led to fewer and smaller tumors. Cenigaonandia-Campillo et al. (134) found that higher doses of VC (5–10 mM) inhibited KRAS mutant colon cancer growth in both in vivo and in vitro studies by reducing mitochondrial membrane potential and levels of ATP and GLUT1 in cancer cells. Previous research for brain and lung cancers have confirmed the safety of high-dose VC, showing alterations in cellular iron metabolism and increased reactive oxygen species, which contribute to cancer cell death (135). Clinical phase II studies are currently evaluating the effectiveness of high-dose VC monotherapy in metastatic colorectal, pancreatic, and lung cancers, particularly those with KRAS or BRAF mutations (136) (Table 3). Other clinical studies of VC monotherapy for cancer that have explored the antitumor effect of low-to-moderate doses or oral VC monotherapy (137–139). However, as mentioned earlier, most basic research has shown that low and medium doses of VC or oral VC have no anticancer effect. Only HDIVC can exert the independent anticancer effect of VC. Nevertheless, VC’s impact may not be as robust as first-line treatments like chemotherapy or radiotherapy, indicating its potential role as an adjuvant therapy to enhance the efficacy of existing cancer treatments. As RAF family protein kinases are a key node in the RAS/RAF pathways in the signaling cascade, KRAS mutations typically cause broad disruption to RAS pathway signaling (140). This includes impairment of the antioxidant response and a heightened reliance on glycolysis. In contrast, BRAF mutations often allow compensatory mechanisms within the pathway to remain functional, such as mitochondrial metabolism. Consequently, compared to BRAF-mutant tumors, KRAS-mutant tumors may demonstrate elevated vulnerability to oxidative stress and epigenetic modulation triggered by high-dose VC. However, the differential sensitivity of KRAS- versus BRAF-mutated tumors to high-dose VC remains unexplored empirically and thus warrants further investigation.

Although VC or DHA monotherapy has certain anticancer effects, it should be noted that their in vivo stability is poor, their half-life is very short, and their therapeutic effects are limited. Therefore, high-dose VC anticancer treatment often requires the combination of other anticancer treatment methods, including immunotherapy, chemotherapy, radiotherapy, and photothermal therapy, to achieve the best effect.

VC serves as a nutritional supplement that mitigates the adverse effects of cancer treatments rather than inducing toxicity. A retrospective study by Ou et al. (150) compared outcomes in advanced triple-negative breast cancer patients treated with gemcitabine and carboplatin alone versus in combination with VC. The study found that combining VC significantly reduced side effects such as bone marrow suppression, hair loss, nausea, vomiting, and constipation. Moreover, this combination enhanced the efficacy of chemotherapy, leading to an increase in progression-free survival (PFS) from 4.5 months to 7 months and overall survival (OS) from 18 months to 27 months. The percentage of patients surviving beyond 2 and 3 years rose to 54.3% and 14.3%, respectively, compared to 14.3% and 0% in the chemotherapy-only group. Additionally, Mikirova et al. (151) fund that HDIVC reduced inflammatory markers, including interleukin-1α, interleukin-2, interleukin-8, tumor necrosis factor-α, and C-reactive protein, and correlated with decreased tumor markers. A multicenter observational study further indicated that HDIVC improved cancer patients’ quality of life, providing significant relief from fatigue, pain, insomnia, and constipation after four weeks of treatment (152).