Diverse antitumor effects of ascorbic acid on cancer cells and the tumor microenvironment.

Introduction

Ascorbic acid, also known as vitamin C, is a low-molecular-weight compound with the chemical formula C 6 H 8 O 6 and a molecular weight of 176.12 g/mol. It is an essential water-soluble vitamin that cannot be synthesized in the human body ( 1 ). Instead, this vitamin must be acquired by consuming food. Inadequate provision of dietary vitamin C can lead to deficiencies such as scurvy ( 2 – 4 ). Ascorbic acid acts in vivo as an antioxidant and cofactor in various enzymatic reactions but has also attracted substantial attention for its potential antitumor effects ( 5 , 6 ). However, the clinical efficacy of ascorbic acid as an anticancer treatment, and the mechanism behind its effects, have not yet been confirmed.

Cancer maintains its characteristic growth and progression by interacting with surrounding cells, forming a cancer microenvironment composed of various cells. Among these cells, cancer-associated fibroblasts (CAFs) play a significant role in cancer cell proliferation, invasion, and metastasis by providing growth factors and nutrients to cancer cells and reorganizing the extracellular matrix of the peri-cancer stroma ( 7 – 11 ). However, the effect of ascorbic acid on the cancer microenvironment is unclear. Moreover, the heterogeneity phenotype of fibroblasts in the peritumoral stroma of some carcinomas promotes tumor growth ( 12 , 13 ). Therefore, elucidation of the heterogeneity of fibroblasts is urgently required for the effective destruction of cancer cells.

In this review, we discuss the differences between the antioxidant and oxidant-promoting effects of ascorbic acid, including the role of ascorbic acid concentration. Our current understanding of the concentration-dependent actions and processes of ascorbic acid is also explained. We then provide future perspectives on the antitumor effects of ascorbic acid on cancer cells and its effects on CAFs, which form a key cancer microenvironment.

Administration route and vascular concentration of ascorbic acid

Orally ingested ascorbic acid is absorbed by transporters of sodium-dependent vitamin C transporters (SVCTs) and glucose transporters (GLUTs) in the small intestine and excreted via the kidneys ( 14 ). In vivo , ascorbic acid exists as reduced ascorbic acid or oxidized ascorbic acid (dehydroascorbic acid (DHA)), which are respectively taken into cells through SVCTs and GLUTs ( 15 – 17 ). Rat experiments revealed variations between the oral and intravenous administration of ascorbic acid, whereby oral administration of 5 mg/g of body weight did not raise blood ascorbic acid concentrations, but intravenous administration of 5 mg/g boosted ascorbic acid concentrations to approximately 10 mM ( 18 ). However, since mice and rats can synthesize ascorbic acid in their bodies (<100 μM), it is necessary to be careful in applying the results of experiments with mice and rats to humans, whose systems cannot synthesize ascorbic acid. In human studies, oral administration of 400 mg or more of ascorbic acid maintained steady-state blood concentrations of 50–80 µM ( 19 ), with oral administration of 3 g of ascorbic acid every 4 h increasing the maximum blood concentration to approximately 220 µM. Conversely, intravenous administration of 50 g of ascorbic acid was predicted to increase the maximum blood concentration to approximately 13.4 mM ( 20 ). The half-life of ascorbic acid in the blood is 2.0 ± 0.6 h ( 21 ). Furthermore, in a report on patients with cancer, ascorbic acid concentrations in the blood reached 20.3–49.0 mM with intravenous administration of 60–70 g/m 2 or 1.5 g/kg of ascorbic acid ( 21 – 23 ). In other words, blood concentrations of ascorbic acid vary widely depending on the route of administration. Thus, the pharmacological effects of ascorbic acid resulting from the low concentrations achieved by oral administration (several hundred μM) may differ from those resulting from the high pharmacological concentrations achieved by intravenous administration (>1 mM). As such, the intended administration route of ascorbic acid must be considered. Adverse effects of ascorbic acid include effects on renal function and hemolysis caused by a deficiency of glucose-6-phosphate dehydrogenase (G6PD). Oral doses of more than 1000 mg per day increase renal excretion of urate and oxalate compared to lower doses, so caution should be exercised when administering high doses ( 19 ). G6PD is required for the proper function of glutathione peroxidase, especially in erythrocytes ( 24 ). However, many clinical trials in which high concentrations of intravenous ascorbic acid were administered as monotherapy or in combination with anticancer agents have shown no serious adverse effects ( 21 , 25 – 27 ). Therefore, ascorbic acid is considered a drug with very low toxicity to the human body.

In vivo effects of ascorbic acid

Recent studies have demonstrated that ascorbic acid absorbed in vivo has both antioxidant and oxidant-promoting effects ( 28 , 29 ). Ascorbic acid also exhibits various physiological effects by catalyzing Fe(II)- and 2-oxoglutarate-dependent dioxygenase reactions ( 14 ).

Ascorbic acid and reactive oxygen species

Ascorbic acid degrades reactive oxygen species (ROS) at average blood concentrations of 40–80 μM, reducing low-density lipoprotein oxidation associated with atherosclerosis and lipid oxidation of cell membranes ( 30 – 32 ). However, high pharmacological concentrations of ascorbic acid achieved via intravenous administration produce H 2 O 2 in vivo ( 18 , 33 , 34 ) and then hydroxyl radicals via the Fenton reaction ( 35 ). Intravascularly, ROS produced by high concentrations of ascorbic acid are degraded by catalase in serum, whereas extravascularly, ROS accumulate without degradation by ascorbic acid and act as a pro-oxidant. Thus, ascorbic acid is notable for its paradoxical activity, serving as an antioxidant at low doses and a pro-oxidant at high doses ( 28 , 29 ). In addition, oral administration of ascorbic acid does not reach the same pharmacological concentrations as intravenous treatment ( 19 , 20 ); therefore, intravenous administration of ascorbic acid is required for pro-oxidant activity to occur. In a rat study, intravenous administration of 0.5 mg/g of ascorbic acid increased the H 2 O 2 concentration in the extracellular fluid from undetectable to 20 μM, and intraperitoneal injection of the same dose increased H 2 O 2 concentration to approximately 5 μM. In contrast, no increase in H 2 O 2 concentration in the extracellular fluid was detected after oral administration of ascorbic acid ( 18 ). In addition, in a mouse subcutaneous transplantation model, intraperitoneal administration of 4 mg/g of ascorbic acid increased the H 2 O 2 concentration in the extracellular fluid around the tumor to approximately 150 μM ( 34 , 35 ).

Ascorbic acid as a cofactor for dioxygenase

Members of the Fe(II) and 2-oxoglutarate-dependent dioxygenase families catalyze many oxidation reactions throughout biology. Ascorbic acid acts as a coenzyme that catalyzes the reactions that produce hydroxylation products using 2-oxoglutarate and oxygen as substrates ( 36 ). Particularly well-known are the reactions in collagen ( 37 ) and HIFα, which is a master regulator of the cellular hypoxia response pathway ( 38 ). The reaction in collagen is mediated by one of the proline hydroxylases, collagen prolyl-4-hydroxylase (C-P4H), which hydroxylates the procollagen proline ( 37 ). C-P4H has a high binding capacity to oxygen and is not affected by the oxygen concentration. Conversely, in the reactions in HIFα, ascorbic acid catalyzes two types of reactions: PHD1-3 in proline hydroxylase ( 38 – 40 ) and factor inhibiting HIF-1 (FIH-1) in asparagine hydroxylase ( 41 – 43 ). In the PHD reaction, ascorbic acid degrades HIFα via ubiquitination by pVHL proteins ( 44 – 46 ). In the reaction of FIH-1, it suppresses the interaction with CBP/p300, which is a transcriptional cofactor, and suppresses the transcriptional activity of HIFs ( 42 ). These reactions are dependent on the oxygen concentration; thus, ascorbic acid acts as an oxygen sensor in the cell because the reaction is reduced in a hypoxic environment and HIF is not degraded ( 36 ). The concentrations of ascorbic acid necessary to sustain enzymatic activity of PHDs and FIH-1 are 140-180 uM and 260 uM, respectively ( 37 ) and are well above the steady-state blood concentrations of 40–80 μM, suggesting that these reactions require sufficient blood and tissue concentrations of ascorbic acid ( 14 , 47 ). Ascorbic acid also acts as a cofactor for ten-eleven translocations (TETs) of DNA hydroxylases; TETs are proteins that convert 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC) ( 48 , 49 ). Ascorbic acid promotes DNA demethylation by accelerating the reaction of TETs ( 50 ).

Antitumor effect of ascorbic acid

Ascorbic acid exhibits antitumor effects in various carcinomas ( 5 , 6 , 51 ); however, clinical studies have not yet produced any significant evidence of these effects ( 52 ). Ascorbic acid exhibits antitumor effects through ROS-mediated mechanisms and as a cofactor. The mechanisms of ascorbic acid as a cofactor include effects on HIFα via PHDs and FIH-1 and epigenetic effects via DNA demethylases ( 6 , 49 ). ( Table 1 ) Ascorbic acid can also modulate metabolism and epigenetic gene expression in immune cells as well as cancer cells ( 64 – 67 ). Ascorbic acid is also known to inhibit EMT of tumor cells ( 58 , 59 ). Here, we discuss the known antitumor effects of ascorbic acid.

ROS-mediated antitumor effects of ascorbic acid

The ROS-mediated mechanism is the most well-known mechanism of the antitumor effect of ascorbic acid in various carcinoma. Intravenous administration of high ascorbic acid concentrations acts as a pro-oxidant in vivo , producing ROS through the Fenton reaction ( 18 ). H 2 O 2 , a ROS formed outside of the cell, diffuses rapidly inside the cell ( 68 ) where it consumes antioxidants such as reduced glutathione and NADPH. In addition, in colorectal cancer with KRAS or BRAF mutations, lung cancer with KRAS mutations, and pancreatic cancer, GLUT1 expression is increased because of an accelerated glycolytic pathway, resulting in higher DHA absorption ( 69 – 72 ). ROS accumulation increases oxidative stress, such as DNA damage, and DNA damage increases PARP activity, thereby decreasing NAD + levels and limiting glycolytic system processes ( 73 , 74 ). In addition, GAPDH, an enzyme of the glycolytic system, is inhibited in its enzymatic function by the reversible binding of oxidized glutathione to cystein152, which is reactive to oxidative stress ( 75 ). As a result, the glycolytic pathway produces less adenosine triphosphate (ATP), and cells suffer apoptosis. Indeed, in a report of metabolic changes induced by ascorbate in a colon cancer cell line with KRAS or BRAF mutations, metabolomic analysis using LC/MS/MS showed that upstream metabolites in the glycolytic reaction catalyzed by NAD + and GAPDH were accumulated, whereas downstream metabolites were reduced ( 74 ). Because of redox imbalance, cancer cells are susceptible to ROS and the effects of ascorbic acid ( 76 ). In conclusion, the pro-oxidant effect of high doses of ascorbic acid induces cell death by generating ROS in cancer cells and limiting ATP generation through the glycolytic pathway.

Conversely, the balance of oxidative stress and antioxidant activity plays a crucial role in tumor development and progression. In melanoma, ROS are overproduced by mitochondria or NADPH oxidase, which promotes tumor development and progression through DNA damage-induced mutation of oncogenes and signal transduction via NF-κB ( 77 , 78 ). In addition, melanoma acquires metastatic potential due to enhanced production of antioxidant enzymes such as catalase and tolerance to oxidative stress ( 78 , 79 ). Ascorbic acid has a dual impact on melanoma, with high concentrations triggering cell death and low amounts promoting tumor growth ( 80 ).

Despite the above reported antitumor effects of ascorbic acid at high concentrations, the ROS-mediated antitumor effects of ascorbic acid remain insufficient for the following reasons. First, the Fenton reaction-mediated ROS-generating effect of ascorbic acid, which is recognized in vitro , may be inhibited at in vivo concentrations of Fe2 + and Fe3 + ( 81 ). Second, in vivo , iron ions are always chelated, so the Fenton reaction may not occur ( 30 ). Finally, the inhibitory effect of ascorbic acid on ATP synthesis, even in the presence of PARP inhibitors in vitro , may be exerted by ascorbic acid regardless of the reduction of NAD + levels by PARP ( 82 ). In conclusion, it is possible that in vitro results of the ROS-mediated antitumor effects of ascorbic acid are not compatible with its in vivo mode of action, suggesting that alternative anticancer mechanisms may be involved.

HIFα-mediated antitumor effects of ascorbic acid as a coenzyme

HIFα, which is expressed in many tumors such as melanoma, leukemia, and carcinomas, including colon, pancreatic, and lung cancer ( 83 – 87 ), is involved in angiogenesis and regulation of the glycolytic system, which are crucial processes for cancer growth and progression, suggesting that HIFα may be a novel cancer therapeutic strategy ( 88 , 89 ). Ascorbic acid is a cofactor for Fe(II)- and 2-oxoglutarate-dependent dioxygenases and has various physiological effects, catalyzing the interaction of PHDs and FIH-1 and degrading the HIFα activity ( 44 – 46 ). Ascorbic acid concentrations in human tumor samples were negatively connected with HIF1α expression in colon cancer, with higher ascorbic acid concentrations associated with prolonged recurrence-free survival ( 83 ). In human endometrial tumors, patients with higher ascorbic acid levels in tumors had lower protein expression of HIF1α, VEGF, and GLUT1 and lower malignancy ( 90 ). In human pancreatic cancer cell lines, in vitro , low ascorbic acid concentrations (25 μM) reduced HIF1α expression and suppressed tumor growth under hypoxic conditions ( 57 ). In a model of subcutaneous lung tumor transplantation in rats, intraperitoneal injection of ascorbic acid (1 g/kg) suppressed HIF1α expression in tumors and decreased tumor growth and vascular density ( 91 ). In a mouse model of human B cell lymphoma implanted subcutaneously, oral treatment of ascorbic acid (5 g/L) reduced HIF1α expression and prevented tumor development ( 92 ).

Thus, the activity of ascorbic acid as a coenzyme may suppress HIFα expression and activity in tumor cells and may inhibit tumor cell proliferation by inhibiting angiogenesis.

Ascorbic acid regulates epigenomic modifications

Ascorbic acid catalyzes the reaction of DNA hydroxylase TETs and Jumonji C domain-containing histone demethylases (JHMDs), thereby having epigenetic antitumor effects ( 6 , 49 , 93 ). TET is a member of the same family of iron- and 2-oxoglutarate-dependent dioxygenases as PHDs, which convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine, promoting histone demethylation and contributing to oncogene suppression and the re-expression of tumor suppressor genes ( 94 ). In hematologic tumors such as acute myeloid leukemia and myelodysplastic syndromes, loss-of-function mutations in TET2 are known to occur frequently, resulting in decreased and hypermethylated 5hmC. In these hematologic tumors, administration of several hundred μM ascorbic acid has a gene reprogramming effect that restores TET function and increases 5hmC levels, suppressing cell proliferation and promoting myeloid progenitor cell differentiation ( 60 , 94 ). In malignant melanoma, 5hmC is known to decrease as the disease progresses, and administration of 100 μM ascorbic acid restores 5hmC via TET, induces apoptosis in tumor cells, and shows antitumor effects ( 61 , 62 ). For colon cancer, administration of 1 mM ascorbic acid has also been reported to increase 5hmC via TET in vitro , showing antitumor effects when combined with an inhibitor of isocitrate dehydrogenase (IDH) mutations ( 63 ). JHDMs are histone demethylases that use Fe2 + and α-ketoglutarate as cofactors to demethylate histones and regulate gene expression ( 95 ). Isocitrate dehydrogenases (IDH) mutations reduce α-ketoglutarate, a substrate for TETs and JHDMs, and promote DNA methylation in cells with IDH mutations, regulating gene expression that leads to carcinogenesis such as glioma ( 96 ). Ascorbic acid is necessary for the proper activity of JHDMs and may correct gene expression that promotes oncogenesis by promoting histone demethylation ( 93 , 97 ). Essentially, ascorbic acid has antitumor effects by improving the hypermethylation state observed in tumors via TETs and JHDMs, and by reprogramming gene expression.

Ascorbic acid downregulates EMT

Ascorbic acid regulates the epithelial-mesenchymal transition (EMT), which is important for metastatic tumor potential. In vitro , ascorbic acid suppressed EMT in human pancreatic cancer cells by decreasing Snail and increasing E-cadherin at concentrations of 1-1.5 mM ( 98 ). Ascorbic acid, in conjunction with 5-azacytidine (5-AZA), a potent DNA methyltransferase inhibitor, regulated EMT inhibition and cell cycle progression in human HCC cells in vitro by suppressing Snail expression via TET ( 58 ). Interestingly, ascorbic acid produced two distinct reactions in human breast cancer in vitro . A low dose (100 μM) of ascorbic acid decreased E-cadherin and increased the mesenchymal marker vimentin, while a high dose (2 mM) of ascorbic acid conversely increased E-cadherin and decreased vimentin, reversing TGF-β 1-induced EMT and, as a result, suppressing the formation of lung metastases in vivo ( 59 ). Ascorbic acid at concentrations of 1 mM or higher is thought to suppress EMT in tumors, possibly by inhibiting the effect of TGF-β1 or by regulating Snail expression by TETs.

Effects of ascorbic acid on fibroblasts

Ascorbic acid is known to enhance collagen synthesis ( 99 , 100 ) and wound healing ( 101 ), reduce UV-induced damage ( 102 , 103 ), and exhibit anti-inflammatory effects ( 104 , 105 ); however, these effects are primarily skin-confined. Recently, the CAF cancer microenvironment has attracted considerable attention ( 10 , 11 ), although few studies have described the effects of ascorbic acid on CAFs. Here, we describe the effects of ascorbic acid on fibroblasts.

Ascorbic acid and dermal fibroblasts

Ascorbic acid acts as a cofactor for C-P4Hs when taken up by human dermal fibroblasts and promotes collagen production by stabilizing the three-dimensional structure of procollagen through hydroxylation of its proline ( 4 , 106 , 107 ). In vitro , a low concentration of ascorbic acid (100 μM) in human skin fibroblasts increases the expression of type1,3,4 collagen and SVCT2 at the mRNA level ( 53 – 55 ) as well as increasing proliferation ( 56 ), suggesting a direct effect on fibroblasts. In human clinical data, oral ascorbic acid intake with exercise stimulation doubled the amino-terminal propeptide of collagen I in the blood, indicating enhanced collagen production ( 108 ). In addition, ascorbic acid concentrations as low as 0.17 mM in human skin fibroblasts increase the contractile phenotype of myofibroblasts in the presence of TGF-β1 through enhancement of the expression of TGFb1-responsive genes, but do not increase such a phenotype in the absence of TGF-β1 ( 109 ). Ascorbic acid promotes collagen production and proliferation of skin fibroblasts as a coenzyme. Moreover, in these studies, ascorbic acid increases in collagen synthesis and secretion occurred at concentrations as low as several hundred μM.

Ascorbic acid and other fibroblast reports

According to a study on tumor stroma, intraperitoneal administration of ascorbic acid at a high dose of 4 g/kg in an orthotopically transplanted mouse model of human pancreatic cancer resulted in tumor reduction, reduced metastasis, and enhanced tumor stroma due to increased collagen secretion ( 98 ). In the 4T1 breast cancer orthotopic model utilizing ascorbic acid-deficient (gulonolactone oxidase knockout mouse) mice, oral administration of ascorbic acid increased type 1 collagen to form a capsule around the tumor, and tumor boundaries were more clearly defined than in the control group ( 110 ). Thus, ascorbic acid may increase collagen production in the tumor stroma at both high and low doses. However, it is unknown whether this effect is on tumor cells or CAFs, and further research is needed to determine whether ascorbic acid activates CAFs in the tumor microenvironment and increases collagen production. In contrast, hepatic stellate cells, which are responsible for liver fibrosis, were inhibited in vitro by low doses of ascorbic acid (50-200 M), which decreased intracellular TGF-1 in rat cell lines ( 111 ). In a report examining the development of pulmonary fibrosis by paraquat treatment, intraperitoneal administration of 150 mg/kg of ascorbic acid inhibited pulmonary fibrosis in a mouse model by inhibiting inflammatory cell infiltration into the bronchoalveolar lavage fluid, suppressing apoptosis by increasing antioxidant activity in the lung, and inhibiting TGF-β in the lung ( 112 ). As a result, ascorbic acid may inhibit fibrosis by inhibiting inflammatory cell infiltration and reduction of TGF-β in tissues. In our study, we also found that in vitro , human pancreatic-derived fibroblasts, whose proliferation is promoted when co-cultured with cancer cells, receive high doses (>1 mM) of ascorbic acid for growth inhibition. ( Figure 1 ) In conclusion, the effects of low and high doses of ascorbic acid on CAFs, such as enhanced collagen production and inhibition of fibrosis development, differ from organ to organ or disease model to disease model and remain unclear.

Clinical trials on ascorbic acid

In the 1970s, clinical trials involving ascorbic acid revealed that a small sample of patients treated with intravenous and oral ascorbic acid lived longer than a control group ( 113 , 114 ). At that time, the mechanism of the antitumor effect of ascorbic acid efficacy remained unclear, and subsequent randomized, double-blind, placebo-controlled trials with oral ascorbic acid failed to demonstrate a survival benefit ( 115 , 116 ). Therefore, the antitumor effect of ascorbic acid was viewed unfavorably. Multiple mechanisms of ascorbic acid’s antitumor effect were subsequently proven in vitro , along with differences in ascorbic acid blood levels between oral and intravenous administration methods. Furthermore, the fact that blood levels of ascorbic acid were decreased in cancer patients ( 117 , 118 ) and that the adverse effects associated with ascorbic acid administration were extremely low, led to the expectation that ascorbic acid could be used for therapeutic applications. ( Table 2 ) There were a few scattered case reports showing tumor shrinkage with ascorbic acid treatment ( 131 , 153 – 158 ), and there were also reports of antitumor effects in a small number of studies ( 25 , 124 , 159 , 160 ). Ascorbic acid in combination with chemotherapeutic agents has also been researched, and some reports of reduced side effects and improved quality of life have been observed ( 21 , 161 , 162 ). In contrast, there have been no large-scale clinical trials that have demonstrated an additional antitumor effect by ascorbic acid ( 6 , 76 , 131 , 163 ), and several ongoing clinical trials of ascorbic acid alone or in combination with chemotherapeutic agents for advanced colon cancer, pancreatic cancer, lung cancer, and other malignancies are expected to provide results in the near future ( 140 , 148 , 149 , 151 ) ( Table 2 ).

Discussion

Ascorbic acid is a medicine that has been widely investigated and used for a long time; however, its beneficial effects against cancer have not yet been proven by clinical trials. The contrasting in vivo effects of ascorbic acid may explain this. That is, the oxidative-promoting impact at high concentrations is detrimental to cancer cells, whereas the antioxidant effect at low concentrations may promote cancer ( 164 ). Because of this paradoxical effect, the administration route of ascorbic acid should be carefully considered. In addition, future research should explain the different activities of multiple dioxylases as cofactors, such as HIFα degradation, immune cell modulation, and epigenetic regulation of gene expression, in relation to the cancer microenvironment ( Figure 2 ).

Additionally, research on the effects of ascorbic acid on CAFs implies the existence of novel therapeutic possibilities. Since the diversity of gene expression in fibroblasts in vivo differs among organs and pathological conditions ( 165 ), the effects of ascorbic acid on CAFs are also expected to vary among organs and pathological conditions. One of the potential effects of ascorbic acid may be the inhibition of tumor-promoting CAFs. Tumor-promoting CAFs support cancer growth by supplying cancer cells with nutrients and growth factors ( 7 – 11 ). Moreover, tumor-promoting CAFs control ECM secretion and protease secretion, remodel the ECM, and generate invasive routes necessary for solid tumor invasion ( 166 , 167 ). Furthermore, in tumors with a high stromal component, such as pancreatic and breast cancer, the stromal fluid pressure in the tumor area increases, reducing drug delivery and indicating resistance to treatment ( 168 , 169 ). Tumor-promoting CAFs promote cancer through cross-talk functions with cancer cells, ECM remodeling functions, and physical drug barrier functions. Ascorbic acid has an inhibitory effect on fibroblasts through a reduction in TGF at low doses and an inhibitory effect on cell proliferation via a prooxidant effect at higher doses, suggesting that it may have an inhibitory effect on tumor-promoting CAFs.

Conversely, collagen is known to form a barrier that physically obstructs cell migration without protease degradation ( 170 , 171 ). In a mouse model lacking -SMA-positive fibroblasts, the tumor suppressive effects of CAFs have been demonstrated to induce an undifferentiated tumor phenotype and dramatically reduce survival ( 172 ). The increase in cancer stroma, tumor shrinkage, and metastasis inhibition effects of ascorbic acid may be attributed to the activation of tumor suppressive fibroblasts and the formation of collagen barriers that inhibit tumor progression.

Ascorbic acid may also affect CAFs via suppression of HIF1α. Tumor-induced ROS-mediated “pseudo-hypoxia” in CAFs leads to the accumulation of HIF1α and enhanced aerobic glycolysis ( 173 , 174 ). Furthermore, high expression of HIF1α in CAFs induces protein expression in myofibroblasts in CAFs, and inhibition or knockout of HIF1α improves their phenotype ( 175 ). Stimulation by TGF-β or PDGF also suppresses IDH3 expression and decreases 2-oxoglutarate in fibroblasts, resulting in HIF1α accumulation and regulating fibroblast differentiation into CAFs ( 176 ). Ascorbic acid may inhibit the accumulation of HIF1α by promoting the reaction of 2-oxoglutarate-dependent dioxygenases such as PHDs and FIH-1, thereby suppressing fibroblast differentiation into CAFs. The JAK1/STAT3 pathway is also an important pathway that maintains actomyosin contractility and the CAF phenotype ( 177 ), and methylation of the promoter of protein tyrosine phosphatase non-receptor type 6 (SHP-1), which negatively regulates the JAK/STAT pathway, allowing for sustained signaling ( 167 ). For this epigenetic reorganization, DNA demethylase-mediated effects such as TETs of ascorbic acid may be exerted. However, CAFs have an enhanced glycolytic system due to chronic hypoxia in the tumor microenvironment and subsequent epigenetic reorganization by demethylation of HIF1α and promoters of enzymes of the glycolytic system ( 178 ), and there may be unexpected epigenetic effects of ascorbic acid that should be clarified in the future. Ascorbic acid may have a tumor suppressive effect by affecting CAFs and reprogramming them into normal fibroblasts. ( Figure 3 ) It is possible that the antitumor effect of ascorbic acid can be improved by examining the method of administration and adapting it to the expression status of HIF in tumors and CAFs. Furthermore, elucidating the effects of ascorbic acid targeting not only tumor cells but also tumor microenvironments such as CAFs may help to reveal further antitumor effects of ascorbic acid.

Author contributions

TMa and TMi drafted the manuscript, which was subsequently critically revised by MT and SU. All authors contributed to the article and approved the submitted version.

Funding

This work was supported by JSPS KAKENHI (grant Nos. 22K08769).

Acknowledgments

The authors would like to thank Ikuko Arikawa, Yumiko Ito, and Sachiko Sawada for technical assistance with the experiments. Furthermore, we would like to thank Editage ( www.editage.jp ) for English language editing.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Publisher’s note

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References

1 Nishikimi M Koshizaka T Ozawa T Yagi K . Occurrence in humans and guinea pigs of the gene related to their missing enzyme l-gulono-gamma-lactone oxidase . Arch Biochem Biophys ( 1988 ) 267 : 842–6 . doi: 10.1016/0003-9861(88)90093-8 3214183

2 Valdés F . [Vitamin c] . Actas Dermo Sifiliogr ( 2006 ) 97 : 557–68 . doi: 10.1111/odi.12446 17173758

3 Padayatty SJ Katz A Wang Y Eck P Kwon O Lee JH . Vitamin c as an antioxidant: evaluation of its role in disease prevention . J Am Coll Nutr ( 2003 ) 22 : 18 – 35 . doi: 10.1080/07315724.2003.10719272 12569111

4 Peterkofsky B . Ascorbate requirement for hydroxylation and secretion of procollagen: relationship to inhibition of collagen synthesis in scurvy . Am J Clin Nutr ( 1991 ) 54 : 1135S–40S . doi: 10.1093/ajcn/54.6.1135s 1720597

5 Villagran M Ferreira J Martorell M Mardones L . The role of vitamin c in cancer prevention and therapy: A literature review . Antioxid (Basel) ( 2021 ) 10 : 1894 . doi: 10.3390/antiox10121894 PMC8750500 34942996

6 Böttger F Vallés-Martí A Cahn L Jimenez CR . High-dose intravenous vitamin c, a promising multi-targeting agent in the treatment of cancer . J Exp Clin Cancer Res ( 2021 ) 40 : 343 . doi: 10.1186/s13046-021-02134-y 34717701 PMC8557029

7 Sousa CM Biancur DE Wang X Halbrook CJ Sherman MH Zhang L . Pancreatic stellate cells support tumour metabolism through autophagic alanine secretion . Nature ( 2016 ) 536 : 479–83 . doi: 10.1038/nature19084 PMC5228623 27509858

8 Parker SJ Amendola CR Hollinshead KER Yu Q Yamamoto K Encarnación-Rosado J . Selective alanine transporter utilization creates a targetable metabolic niche in pancreatic cancer . Cancer Discov ( 2020 ) 10 : 1018–37 . doi: 10.1158/2159-8290.CD-19-0959 PMC7334074 32341021

9 Ikuta D Miyake T Shimizu T Sonoda H Mukaisho KI Tokuda A . Fibrosis in metastatic lymph nodes is clinically correlated to poor prognosis in colorectal cancer . Oncotarget ( 2018 ) 9 : 29574–86 . doi: 10.18632/oncotarget.25636 PMC6049853 30038705

10 Sahai E Astsaturov I Cukierman E DeNardo DG Egeblad M Evans RM . A framework for advancing our understanding of cancer-associated fibroblasts . Nat Rev Cancer ( 2020 ) 20 : 174–86 . doi: 10.1038/s41568-019-0238-1 PMC7046529 31980749

11 Kalluri R Zeisberg M . Fibroblasts in cancer . Nat Rev Cancer ( 2006 ) 6 : 392 – 401 . doi: 10.1038/nrc1877 16572188

12 Maehira H Miyake T Iida H Tokuda A Mori H Yasukawa D . Vimentin expression in tumor microenvironment predicts survival in pancreatic ductal adenocarcinoma: heterogeneity in the fibroblast population . Ann Surg Oncol ( 2019 ) 26 : 4791–804 . doi: 10.1245/s10434-019-07891-x 31583548

13 Mizutani Y Kobayashi H Iida T Asai N Masamune A Hara A . Meflin-positive cancer-associated fibroblasts inhibit pancreatic carcinogenesis . Cancer Res ( 2019 ) 79 : 5367–81 . doi: 10.1158/0008-5472.CAN-19-0454 31439548

14 Padayatty SJ Levine M . Vitamin c physiology: the known and the unknown and goldilocks . Oral Dis ( 2016 ) 22 : 463–93 . doi: 10.1111/odi.12446 PMC4959991 26808119

15 Tsukaguchi H Tokui T Mackenzie B Berger UV Chen XZ Wang Y . A family of mammalian na+-dependent l-ascorbic acid transporters . Nature ( 1999 ) 399 : 70–5 . doi: 10.1038/19986 10331392

16 Wang Y Mackenzie B Tsukaguchi H Weremowicz S Morton CC Hediger MA . Human vitamin c (L-ascorbic acid) transporter SVCT1 . Biochem Biophys Res Commun ( 2000 ) 267 : 488–94 . doi: 10.1006/bbrc.1999.1929 10631088

17 Muñoz-Montesino C Peña E Roa FJ Sotomayor K Escobar E Rivas CI . Transport of vitamin c in cancer . Antioxid Redox Signal ( 2021 ) 35 : 61 – 74 . doi: 10.1089/ars.2020.8166 33607936

18 Chen Q Espey MG Sun AY Lee JH Krishna MC Shacter E . Ascorbate in pharmacologic concentrations selectively generates ascorbate radical and hydrogen peroxide in extracellular fluid in vivo . Proc Natl Acad Sci USA ( 2007 ) 104 : 8749–54 . doi: 10.1073/pnas.0702854104 PMC1885574 17502596

19 Levine M Conry-Cantilena C Wang Y Welch RW Washko PW Dhariwal KR . Vitamin c pharmacokinetics in healthy volunteers: evidence for a recommended dietary allowance . Proc Natl Acad Sci USA ( 1996 ) 93 : 3704–9 . doi: 10.1073/pnas.93.8.3704 PMC39676 8623000

20 Padayatty SJ Sun H Wang Y Riordan HD Hewitt SM Katz A . Vitamin c pharmacokinetics: implications for oral and intravenous use . Ann Intern Med ( 2004 ) 140 : 533–7 . doi: 10.7326/0003-4819-140-7-200404060-00010 15068981

21 Stephenson CM Levin RD Spector T Lis CG . Phase I clinical trial to evaluate the safety, tolerability, and pharmacokinetics of high-dose intravenous ascorbic acid in patients with advanced cancer . Cancer Chemother Pharmacol ( 2013 ) 72 : 139–46 . doi: 10.1007/s00280-013-2179-9 PMC3691494 23670640

22 Nielsen TK Højgaard M Andersen JT Poulsen HE Lykkesfeldt J Mikines KJ . Elimination of ascorbic acid after high-dose infusion in prostate cancer patients: A pharmacokinetic evaluation . Basic Clin Pharmacol Toxicol ( 2015 ) 116 : 343–8 . doi: 10.1111/bcpt.12323 25220574

23 Hoffer LJ Levine M Assouline S Melnychuk D Padayatty SJ Rosadiuk K . Phase I clinical trial of i.v. ascorbic acid in advanced malignancy . Ann Oncol ( 2008 ) 19 : 1969–74 . doi: 10.1093/annonc/mdn377 18544557

24 Quinn J Gerber B Fouche R Kenyon K Blom Z Muthukanagaraj P . Effect of high-dose vitamin c infusion in a glucose-6-Phosphate dehydrogenase-deficient patient . Case Rep Med ( 2017 ) 2017 : 5202606 . doi: 10.1155/2017/5202606 29317868 PMC5727655

25 Monti DA Mitchell E Bazzan AJ Littman S Zabrecky G Yeo CJ . Phase I evaluation of intravenous ascorbic acid in combination with gemcitabine and erlotinib in patients with metastatic pancreatic cancer . PLoS One ( 2012 ) 7 : e29794 . doi: 10.1371/journal.pone.0029794 22272248 PMC3260161

26 Hoffer LJ Robitaille L Zakarian R Melnychuk D Kavan P Agulnik J . High-dose intravenous vitamin c combined with cytotoxic chemotherapy in patients with advanced cancer: a phase I-II clinical trial . PLoS One ( 2015 ) 10 : e0120228 . doi: 10.1371/journal.pone.0120228 25848948 PMC4388666

27 Nauman G Gray JC Parkinson R Levine M Paller CJ . Systematic review of intravenous ascorbate in cancer clinical trials . Antioxid (Basel) ( 2018 ) 7 : 89 . doi: 10.3390/antiox7070089 PMC6071214 30002308

28 Du J Cullen JJ Buettner GR . Ascorbic acid: chemistry, biology and the treatment of cancer . Biochim Biophys Acta ( 2012 ) 1826 : 443–57 . doi: 10.1016/j.bbcan.2012.06.003 PMC3608474 22728050

29 Kaźmierczak-Barańska J Boguszewska K Adamus-Grabicka A Karwowski BT . Two faces of vitamin c-antioxidative and pro-oxidative agent . Nutrients ( 2020 ) 12 : 1501 . doi: 10.3390/nu12051501 PMC7285147 32455696

30 Ďuračková Z . Some current insights into oxidative stress . Physiol Res ( 2010 ) 59 : 459–69 . doi: 10.33549/physiolres.931844 19929132

31 Santos KLB Bragança VAN Pacheco LV Ota SSB Aguiar CPO Borges RS . Essential features for antioxidant capacity of ascorbic acid (vitamin c) . J Mol Model ( 2021 ) 28 : 1 . doi: 10.1007/s00894-021-04994-9 34862566

32 Gale CR Ashurst HE Powers HJ Martyn CN . Antioxidant vitamin status and carotid atherosclerosis in the elderly . Am J Clin Nutr ( 2001 ) 74 : 402–8 . doi: 10.1093/ajcn/74.3.402 11522566

33 Chen Q Espey MG Krishna MC Mitchell JB Corpe CP Buettner GR . Pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues . Proc Natl Acad Sci USA ( 2005 ) 102 : 13604–9 . doi: 10.1073/pnas.0506390102 PMC1224653 16157892

34 Chen Q Espey MG Sun AY Pooput C Kirk KL Krishna MC . Pharmacologic doses of ascorbate act as a pro-oxidant and decrease growth of aggressive tumor xenografts in mice . Proc Natl Acad Sci USA ( 2008 ) 105 : 11105–9 . doi: 10.1073/pnas.0804226105 PMC2516281 18678913

35 Pawlowska E Szczepanska J Blasiak J . Pro- and antioxidant effects of vitamin c in cancer in correspondence to its dietary and pharmacological concentrations . Oxid Med Cell Longev ( 2019 ) 2019 : 7286737 . doi: 10.1155/2019/7286737 31934267 PMC6942884

36 Ozer A Bruick RK . Non-heme dioxygenases: cellular sensors and regulators jelly rolled into one ? Nat Chem Biol ( 2007 ) 3 : 144–53 . doi: 10.1038/nchembio863 17301803

37 Myllyharju J Kivirikko KI . Characterization of the iron- and 2-oxoglutarate-binding sites of human prolyl 4-hydroxylase . EMBO J ( 1997 ) 16 : 1173–80 . doi: 10.1093/emboj/16.6.1173 PMC1169716 9135134

38 Bruick RK McKnight SL . A conserved family of prolyl-4-hydroxylases that modify HIF . Science ( 2001 ) 294 : 1337–40 . doi: 10.1126/science.1066373 11598268

39 Epstein ACR Gleadle JM McNeill LA Hewitson KS O’Rourke J Mole DR . C. elegans EGL-9 and mammalian homologs define a family of dioxygenases that regulate HIF by prolyl hydroxylation . Cell ( 2001 ) 107 : 43 – 54 . doi: 10.1016/S0092-8674(01)00507-4 11595184

40 Ivan M Haberberger T Gervasi DC Michelson KS Günzler V Kondo K . Biochemical purification and pharmacological inhibition of a mammalian prolyl hydroxylase acting on hypoxia-inducible factor . Proc Natl Acad Sci USA ( 2002 ) 99 : 13459–64 . doi: 10.1073/pnas.192342099 PMC129695 12351678

41 Hewitson KS McNeill LA Riordan MV Tian YM Bullock AN Welford RW . Hypoxia-inducible factor (HIF) asparagine hydroxylase is identical to factor inhibiting HIF (FIH) and is related to the cupin structural family . J Biol Chem ( 2002 ) 277 : 26351–5 . doi: 10.1074/jbc.C200273200 12042299

42 Lando D Peet DJ Gorman JJ Whelan DA Whitelaw ML Bruick RK . FIH-1 is an asparaginyl hydroxylase enzyme that regulates the transcriptional activity of hypoxia-inducible factor . Genes Dev ( 2002 ) 16 : 1466–71 . doi: 10.1101/gad.991402 PMC186346 12080085

43 Rani S Roy S Singh M Kaithwas G . Regulation of transactivation at c-TAD domain of HIF-1 alpha by factor-inhibiting HIF-1 alpha (FIH-1): A potential target for therapeutic intervention in cancer . Oxid Med Cell Longev ( 2022 ) 2022 : 2407223 . doi: 10.1155/2022/2407223 35592530 PMC9113874

44 Jaakkola P Mole DR Tian YM Wilson MI Gielbert J Gaskell SJ . Targeting of HIF-alpha to the von hippel-lindau ubiquitylation complex by O2-regulated prolyl hydroxylation . Science ( 2001 ) 292 : 468–72 . doi: 10.1126/science.1059796 11292861

45 Cockman ME Masson N Mole DR Jaakkola P Chang GW Clifford SC . Hypoxia inducible factor-alpha binding and ubiquitylation by the von hippel-lindau tumor suppressor protein . J Biol Chem ( 2000 ) 275 : 25733–41 . doi: 10.1074/jbc.M002740200 10823831

46 Kuiper C Dachs GU Currie MJ Vissers MC . Intracellular ascorbate enhances hypoxia-inducible factor (HIF)-hydroxylase activity and preferentially suppresses the HIF-1 transcriptional response . Free Radic Biol Med ( 2014 ) 69 : 308–17 . doi: 10.1016/j.freeradbiomed.2014.01.033 24495550

47 Lykkesfeldt J Tveden-Nyborg P . The pharmacokinetics of vitamin c . Nutrients ( 2019 ) 11 : 2412 . doi: 10.3390/nu11102412 PMC6835439 31601028

48 Tahiliani M Koh KP Shen Y Pastor WA Bandukwala H Brudno Y . Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1 . Science ( 2009 ) 324 : 930–5 . doi: 10.1126/science.1170116 PMC2715015 19372391

49 Camarena V Wang G . The epigenetic role of vitamin c in health and disease . Cell Mol Life Sci ( 2016 ) 73 : 1645–58 . doi: 10.1007/s00018-016-2145-x PMC4805483 26846695

50 Blaschke K Ebata KT Karimi MM Zepeda-Martínez JA Goyal P Mahapatra S . Vitamin c induces tet-dependent DNA demethylation and a blastocyst-like state in ES cells . Nature ( 2013 ) 500 : 222–6 . doi: 10.1038/nature12362 PMC3893718 23812591

51 Hamaguchi R Ito T Narui R Morikawa H Uemoto S Wada H . Effects of alkalization therapy on chemotherapy outcomes in advanced pancreatic cancer: A retrospective case-control study . In Vivo ( 2020 ) 34 : 2623–9 . doi: 10.21873/invivo.12080 PMC7652496 32871792

52 van Gorkom GNY Lookermans EL Van Elssen CHMJ Bos GMJ . The effect of vitamin c (ascorbic acid) in the treatment of patients with cancer: A systematic review . Nutrients ( 2019 ) 11 : 977 . doi: 10.3390/nu11050977 PMC6566697 31035414

53 Kishimoto Y Saito N Kurita K Shimokado K Maruyama N Ishigami A . Ascorbic acid enhances the expression of type 1 and type 4 collagen and SVCT2 in cultured human skin fibroblasts . Biochem Biophys Res Commun ( 2013 ) 430 : 579–84 . doi: 10.1016/j.bbrc.2012.11.110 23228664

54 Tajima S Pinnell SR . Ascorbic acid preferentially enhances type I and III collagen gene transcription in human skin fibroblasts . J Dermatol Sci ( 1996 ) 11 : 250–3 . doi: 10.1016/0923-1811(95)00640-0 8785178

55 Maione-Silva L de Castro EG Nascimento TL Cintra ER Moreira LC Cintra BAS . Ascorbic acid encapsulated into negatively charged liposomes exhibits increased skin permeation, retention and enhances collagen synthesis by fibroblasts . Sci Rep ( 2019 ) 9 : 522 . doi: 10.1038/s41598-018-36682-9 30679479 PMC6345870

56 Phillips CL Combs SB Pinnell SR . Effects of ascorbic acid on proliferation and collagen synthesis in relation to the donor age of human dermal fibroblasts . J Invest Dermatol ( 1994 ) 103 : 228–32 . doi: 10.1111/1523-1747.ep12393187 7518857

57 Chen C Tang P Yue J Ren P Liu X Zhao X . Effect of siRNA targeting HIF-1alpha combined l-ascorbate on biological behavior of hypoxic MiaPaCa2 cells . Technol Cancer Res Treat ( 2009 ) 8 : 235–40 . doi: 10.1177/153303460900800309 19445542

58 Sajadian SO Tripura C Samani FS Ruoss M Dooley S Baharvand H . Vitamin c enhances epigenetic modifications induced by 5-azacytidine and cell cycle arrest in the hepatocellular carcinoma cell lines HLE and Huh7 . Clin Epigenet ( 2016 ) 8 : 46 . doi: 10.1186/s13148-016-0213-6 PMC4851801 27134688

59 Zeng LH Wang QM Feng LY Ke YD Xu QZ Wei AY . High-dose vitamin c suppresses the invasion and metastasis of breast cancer cells via inhibiting epithelial-mesenchymal transition . Onco Targets Ther ( 2019 ) 12 : 7405–13 . doi: 10.2147/OTT.S222702 PMC6753468 31571901

60 Mingay M Chaturvedi A Bilenky M Cao Q Jackson L Hui T . Vitamin c-induced epigenomic remodelling in IDH1 mutant acute myeloid leukaemia . Leukemia ( 2018 ) 32 : 11 – 20 . doi: 10.1038/leu.2017.171 28663574 PMC5770587

61 Lian CG Xu Y Ceol C Wu F Larson A Dresser K . Loss of 5-hydroxymethylcytosine is an epigenetic hallmark of melanoma . Cell ( 2012 ) 150 : 1135–46 . doi: 10.1016/j.cell.2012.07.033 PMC3770275 22980977

62 Mustafi S Sant DW Liu ZJ Wang G . Ascorbate induces apoptosis in melanoma cells by suppressing clusterin expression . Sci Rep ( 2017 ) 7 : 3671 . doi: 10.1038/s41598-017-03893-5 28623268 PMC5473908

63 Gerecke C Schumacher F Berndzen A Homann T Kleuser B . Vitamin c in combination with inhibition of mutant IDH1 synergistically activates TET enzymes and epigenetically modulates gene silencing in colon cancer cells . Epigenetics ( 2020 ) 15 : 307–22 . doi: 10.1080/15592294.2019.1666652 PMC7028341 31505989

64 van Gorkom GNY Klein Wolterink RGJ Van Elssen CHMJ Wieten L Germeraad WTV Bos GMJ . Influence of vitamin c on lymphocytes: an overview . Antioxid (Basel) ( 2018 ) 7 : 41 . doi: 10.3390/antiox7030041 PMC5874527 29534432

65 Ang A Pullar JM Currie MJ Vissers MCM . Vitamin c and immune cell function in inflammation and cancer . Biochem Soc Trans ( 2018 ) 46 : 1147–59 . doi: 10.1042/BST20180169 PMC6195639 30301842

66 McPherson RC Konkel JE Prendergast CT Thomson JP Ottaviano R Leech MD . Epigenetic modification of the PD-1 (Pdcd1) promoter in effector CD4+ T cells tolerated by peptide immunotherapy . Elife ( 2014 ) 3 : e03416 . doi: 10.7554/eLife.03416 PMC4297948 25546306

67 Minor EA Court BL Young JI Wang G . Ascorbate induces ten-eleven translocation (Tet) methylcytosine dioxygenase-mediated generation of 5-hydroxymethylcytosine . J Biol Chem ( 2013 ) 288 : 13669–74 . doi: 10.1074/jbc.C113.464800 PMC3650403 23548903

68 Antunes F Cadenas E . Estimation of H2O2 gradients across biomembranes . FEBS Lett ( 2000 ) 475 : 121–6 . doi: 10.1016/S0014-5793(00)01638-0 10858501

69 Kawada K Nakamoto Y Kawada M Hida K Matsumoto T Murakami T . Relationship between 18F-fluorodeoxyglucose accumulation and KRAS/BRAF mutations in colorectal cancer . Clin Cancer Res ( 2012 ) 18 : 1696–703 . doi: 10.1158/1078-0432.CCR-11-1909 22282467

70 Yun J Rago C Cheong I Pagliarini R Angenendt P Rajagopalan H . Glucose deprivation contributes to the development of KRAS pathway mutations in tumor cells . Science ( 2009 ) 325 : 1555–9 . doi: 10.1126/science.1174229 PMC2820374 19661383

71 Sasaki H Shitara M Yokota K Hikosaka Y Moriyama S Yano M . Overexpression of GLUT1 correlates with kras mutations in lung carcinomas . Mol Med Rep ( 2012 ) 5 : 599 – 602 . doi: 10.3892/mmr.2011.736 22200795

72 Bryant KL Mancias JD Kimmelman AC Der CJ . KRAS: feeding pancreatic cancer proliferation . Trends Biochem Sci ( 2014 ) 39 : 91 – 100 . doi: 10.1016/j.tibs.2013.12.004 24388967 PMC3955735

73 Alano CC Garnier P Ying W Higashi Y Kauppinen TM Swanson RA . NAD + depletion is necessary and sufficient for poly(ADP-ribose) polymerase-1-mediated neuronal death . J Neurosci ( 2010 ) 30 : 2967–78 . doi: 10.1523/JNEUROSCI.5552-09.2010 PMC2864043 20181594

74 Yun J Mullarky E Lu C Bosch KN Kavalier A Rivera K . Vitamin c selectively kills KRAS and BRAF mutant colorectal cancer cells by targeting GAPDH . Science ( 2015 ) 350 : 1391–6 . doi: 10.1126/science.aaa5004 PMC4778961 26541605

75 Hwang NR Yim SH Kim YM Jeong J Song EJ Lee Y . Oxidative modifications of glyceraldehyde-3-phosphate dehydrogenase play a key role in its multiple cellular functions . Biochem J ( 2009 ) 423 : 253–64 . doi: 10.1042/BJ20090854 19650766

76 Ngo B Van Riper JM Cantley LC Yun J . Targeting cancer vulnerabilities with high-dose vitamin c . Nat Rev Cancer ( 2019 ) 19 : 271–82 . doi: 10.1038/s41568-019-0135-7 PMC6526932 30967651

77 Brar SS Kennedy TP Whorton AR Sturrock AB Huecksteadt TP Ghio AJ . Reactive oxygen species from NAD(P)H:quinone oxidoreductase constitutively activate NF-kappaB in malignant melanoma cells . Am J Physiol Cell Physiol ( 2001 ) 280 : C659–76 . doi: 10.1152/ajpcell.2001.280.3.C659 11171586

78 Sagwal SK Bekeschus S . ROS pleiotropy in melanoma and local therapy with physical modalities . Oxid Med Cell Longev ( 2021 ) 2021 : 6816214 . doi: 10.1155/2021/6816214 34777692 PMC8580636

79 Baldi A Lombardi D Russo P Palescandolo E De Luca A Santini D . Ferritin contributes to melanoma progression by modulating cell growth and sensitivity to oxidative stress . Clin Cancer Res ( 2005 ) 11 : 3175–83 . doi: 10.1158/1078-0432.CCR-04-0631 15867210

80 Yang G Yan Y Ma Y Yang Y . Vitamin c at high concentrations induces cytotoxicity in malignant melanoma but promotes tumor growth at low concentrations . Mol Carcinog ( 2017 ) 56 : 1965–76 . doi: 10.1002/mc.22654 28370562

81 Murayama Y Kashiyagura R Ohmomi E Ishida Y Shinada T Satoh T . Fe 2+ as a physiological and selective inhibitor of vitamin c-induced cancer cell death . React Oxygen Species ( 2020 ) 10 : 180–96 . doi: 10.20455/ros.2020.833

82 Du J Martin SM Levine M Wagner BA Buettner GR Wang SH . Mechanisms of ascorbate-induced cytotoxicity in pancreatic cancer . Clin Cancer Res ( 2010 ) 16 : 509–20 . doi: 10.1158/1078-0432.CCR-09-1713 PMC2807999 20068072

83 Kuiper C Dachs GU Munn D Currie MJ Robinson BA Pearson JF . Increased tumor ascorbate is associated with extended disease-free survival and decreased hypoxia-inducible factor-1 activation in human colorectal cancer . Front Oncol ( 2014 ) 4 : 10 . doi: 10.3389/fonc.2014.00010 24551593 PMC3912592

84 Dang DT Chen F Gardner LB Cummins JM Rago C Bunz F . Hypoxia-inducible factor-1A promotes nonhypoxia-mediated proliferation in colon cancer cells and xenografts . Cancer Res ( 2006 ) 66 : 1684–693 . doi: 10.1158/0008-5472.CAN-05-2887 16452228

85 Shukla SK Purohit V Mehla K Gunda V Chaika NV Vernucci E . MUC1 and HIF-1alpha signaling crosstalk induces anabolic glucose metabolism to impart gemcitabine resistance to pancreatic cancer . Cancer Cell ( 2017 ) 32 : 71 – 87.e7 . doi: 10.1016/j.ccell.2017.06.004 28697344 PMC5533091

86 Giatromanolaki A Koukourakis MI Sivridis E Turley H Talks K Pezzella F . Relation of hypoxia inducible factor 1 alpha and 2 alpha in operable non-small cell lung cancer to angiogenic/molecular profile of tumours and survival . Br J Cancer ( 2001 ) 85 : 881–90 . doi: 10.1054/bjoc.2001.2018 PMC2375073 11556841

87 Semenza GL . Defining the role of hypoxia-inducible factor 1 in cancer biology and therapeutics . Oncogene ( 2010 ) 29 : 625–34 . doi: 10.1038/onc.2009.441 PMC2969168 19946328

88 Jin X Dai L Ma Y Wang J Liu Z . Implications of HIF-1α in the tumorigenesis and progression of pancreatic cancer . Cancer Cell Int ( 2020 ) 20 : 273 . doi: 10.1186/s12935-020-01370-0 32587480 PMC7313137

89 Semenza GL . HIF-1 mediates metabolic responses to intratumoral hypoxia and oncogenic mutations . J Clin Invest ( 2013 ) 123 : 3664–71 . doi: 10.1172/JCI67230 PMC3754249 23999440

90 Kuiper C Molenaar IG Dachs GU Currie MJ Sykes PH Vissers MC . Low ascorbate levels are associated with increased hypoxia-inducible factor-1 activity and an aggressive tumor phenotype in endometrial cancer . Cancer Res ( 2010 ) 70 : 5749–58 . doi: 10.1158/0008-5472.CAN-10-0263 20570889

91 Campbell EJ Vissers MCM Wohlrab C Hicks KO Strother RM Bozonet SM . Pharmacokinetic and anticancer properties of high dose ascorbate in solid tumours of ascorbate-dependent mice . Free Radic Biol Med ( 2016 ) 99 : 451–62 . doi: 10.1016/j.freeradbiomed.2016.08.027 27567539

92 Gao P Zhang H Dinavahi R Li F Xiang Y Raman V . HIF-dependent antitumorigenic effect of antioxidants in vivo . Cancer Cell ( 2007 ) 12 : 230–8 . doi: 10.1016/j.ccr.2007.08.004 PMC2084208 17785204

93 Lee Chong T Ahearn EL Cimmino L . Reprogramming the epigenome with vitamin c . Front Cell Dev Biol ( 2019 ) 7 : 128 . doi: 10.3389/fcell.2019.00128 31380368 PMC6646595

94 Kroeze LI van der Reijden BA Jansen JH . 5-hydroxymethylcytosine: An epigenetic mark frequently deregulated in cancer . Biochim Biophys Acta ( 2015 ) 1855 : 144–54 . doi: 10.1016/j.bbcan.2015.01.001 25579174

95 Franci G Ciotta A Altucci L . The jumonji family: past, present and future of histone demethylases in cancer . Biomol Concepts ( 2014 ) 5 : 209–24 . doi: 10.1515/bmc-2014-0010 25372754

96 Turcan S Rohle D Goenka A Walsh LA Fang F Yilmaz E . IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype . Nature ( 2012 ) 483 : 479–83 . doi: 10.1038/nature10866 PMC3351699 22343889

97 Tsukada Y Fang J Erdjument-Bromage H Warren ME Borchers CH Tempst P . Histone demethylation by a family of JmjC domain-containing proteins . Nature ( 2006 ) 439 : 811–6 . doi: 10.1038/nature04433 16362057

98 Polireddy K Dong R Reed G Yu J Chen P Williamson S . High dose parenteral ascorbate inhibited pancreatic cancer growth and metastasis: mechanisms and a phase I/IIa study . Sci Rep ( 2017 ) 7 : 17188 . doi: 10.1038/s41598-017-17568-8 29215048 PMC5719364

99 Wang K Jiang H Li W Qiang M Dong T Li H . Role of vitamin c in skin diseases . Front Physiol ( 2018 ) 9 : 819 . doi: 10.3389/fphys.2018.00819 30022952 PMC6040229

100 Boyera N Galey I Bernard BA . Effect of vitamin c and its derivatives on collagen synthesis and cross-linking by normal human fibroblasts . Int J Cosmet Sci ( 1998 ) 20 : 151–8 . doi: 10.1046/j.1467-2494.1998.171747.x 18505499

101 Vivcharenko V Wojcik M Palka K Przekora A . Highly porous and superabsorbent biomaterial made of marine-derived polysaccharides and ascorbic acid as an optimal dressing for exuding wound management . Mater (Basel) ( 2021 ) 14 : 1211 . doi: 10.3390/ma14051211 PMC7961991 33806657

102 Stewart MS Cameron GS Pence BC . Antioxidant nutrients protect against UVB-induced oxidative damage to DNA of mouse keratinocytes in culture . J Invest Dermatol ( 1996 ) 106 : 1086–9 . doi: 10.1111/1523-1747.ep12339344 8618044

103 Hakozaki T Date A Yoshii T Toyokuni S Yasui H Sakurai H . Visualization and characterization of UVB-induced reactive oxygen species in a human skin equivalent model . Arch Dermatol Res ( 2008 ) 300 : S51–6 . doi: 10.1007/s00403-007-0804-3 17968570

104 Cárcamo JM Pedraza A Bórquez-Ojeda O Golde DW . Vitamin c suppresses TNF alpha-induced NF-kappaB activation by inhibiting I kappa b alpha . Biochemistry ( 2002 ) 41 : 12995–3002 . doi: 10.1021/bi0263210 12390026

105 Farris PK . Topical vitamin c: a useful agent for treating photoaging and other dermatologic conditions . Dermatol Surg ( 2005 ) 31 : 814–8 . doi: 10.1111/j.1524-4725.2005.31725 16029672

106 Peterkofsky B Udenfriend S . Enzymatic hydroxylation of proline in microsomal polypeptide leading to formation of collagen . Proc Natl Acad Sci USA ( 1965 ) 53 : 335–42 . doi: 10.1073/pnas.53.2.335 PMC219517 14294066

107 Pinnell SR . Regulation of collagen biosynthesis by ascorbic acid: a review . Yale J Biol Med ( 1985 ) 58 : 553–9 . PMC2589959 3008449

108 Shaw G Lee-Barthel A Ross ML Wang B Baar K . Vitamin c-enriched gelatin supplementation before intermittent activity augments collagen synthesis . Am J Clin Nutr ( 2017 ) 105 : 136–43 . doi: 10.3945/ajcn.116.138594 PMC5183725 27852613

109 Piersma B Wouters OY de Rond S Boersema M Gjaltema RAF Bank RA . Ascorbic acid promotes a TGFβ1-induced myofibroblast phenotype switch . Physiol Rep ( 2017 ) 5 : e13324 . doi: 10.14814/phy2.13324 28904079 PMC5599854

110 Cha J Roomi MW Ivanov V Kalinovsky T Niedzwiecki A Rath M . Ascorbate supplementation inhibits growth and metastasis of B1 6FO melanoma and 4T1 . Int J Oncol ( 2013 ) 42 : 55 – 64 . doi: 10.3892/ijo.2012.1712 23175106 PMC3583641

111 Su M Chao G Liang M Song J Wu K . Anticytoproliferative effect of vitamin c on rat hepatic stellate cells . Am J Transl Res ( 2016 ) 8 : 2820–5 . PMC4931176 27398165

112 Rodrigues da Silva M Schapochnik A Peres Leal M Esteves J Bichels Hebeda C Sandri S . Beneficial effects of ascorbic acid to treat lung fibrosis induced by paraquat . PLoS One ( 2018 ) 13 : e0205535 . doi: 10.1371/journal.pone.0205535 30395570 PMC6218022

113 Cameron E Pauling L . Supplemental ascorbate in the supportive treatment of cancer: Prolongation of survival times in terminal human cancer . Proc Natl Acad Sci USA ( 1976 ) 73 : 3685–9 . doi: 10.1073/pnas.73.10.3685 PMC431183 1068480

114 Cameron E Pauling L . Supplemental ascorbate in the supportive treatment of cancer: reevaluation of prolongation of survival times in terminal human cancer . Proc Natl Acad Sci USA ( 1978 ) 75 : 4538–42 . doi: 10.1073/pnas.75.9.4538 PMC336151 279931

115 Creagan ET Moertel CG O’Fallon JR Schutt AJ O’Connell MJ Rubin J . Failure of high-dose vitamin c (ascorbic acid) therapy to benefit patients with advanced cancer. a controlled trial . N Engl J Med ( 1979 ) 301 : 687–90 . doi: 10.1056/NEJM197909273011303 384241

116 Moertel CG Fleming TR Creagan ET Rubin J O’Connell MJ Ames MM . High-dose vitamin c versus placebo in the treatment of patients with advanced cancer who have had no prior chemotherapy. a randomized double-blind comparison . N Engl J Med ( 1985 ) 312 : 137–41 . doi: 10.1056/NEJM198501173120301 3880867

117 Carr AC Spencer E Das A Meijer N Lauren C MacPherson S . Patients undergoing myeloablative chemotherapy and hematopoietic stem cell transplantation exhibit depleted vitamin c status in association with febrile neutropenia . Nutrients ( 2020 ) 12 : 1879 . doi: 10.3390/nu12061879 PMC7353216 32599718

118 Mayland CR Bennett MI Allan K . Vitamin c deficiency in cancer patients . Palliat Med ( 2005 ) 19 : 17 – 20 . doi: 10.1191/0269216305pm970oa 15690864

119 ClinicalTrials.gov Identifier: NCT00954525 . Intravenous vitamin c in combination with standard chemotherapy for pancreatic cancer . Available at: https://www.clinicaltrials.gov/ct2/show/NCT00954525 [Accessed August 15, 2022 ].

120 ClinicalTrials.gov Identifier: NCT00006021 . Arsenic trioxide plus vitamin c in treating patients with recurrent or refractory multiple myeloma . ( 2003 ) Available at: https://clinicaltrials.gov/ct2/show/NCT00006021 [Accessed August 15, 2022 ]

121 Bahlis NJ McCafferty-Grad J Jordan-McMurry I Neil J Reis I Kharfan-Dabaja M . Feasibility and correlates of arsenic trioxide combined with ascorbic acid-mediated depletion of intracellular glutathione for the treatment of relapsed/refractory multiple myeloma . Clin Cancer Res ( 2002 ) 8 : 3658–68 . 12473574

122 ClinicalTrials.gov Identifier: NCT00317811 . Bortezomib, ascorbic acid, and melphalan in treating patients with newly diagnosed multiple myeloma . ( 2006 ). Available at: https://clinicaltrials.gov/ct2/show/NCT00317811 [Accessed August 15, 2022 ].

123 Berenson JR Yellin O Woytowitz D Flam MS Cartmell A Patel R . Bortezomib, ascorbic acid and melphalan (BAM) therapy for patients with newly diagnosed multiple myeloma: an effective and well-tolerated frontline regimen . Eur J Haematol ( 2009 ) 82 : 433–9 . doi: 10.1111/j.1600-0609.2009.01244.x 19226361

124 Welsh JL Wagner BA van’t Erve TJ Zehr PS Berg DJ Halfdanarson TR . Pharmacological ascorbate with gemcitabine for the control of metastatic and node-positive pancreatic cancer (PACMAN): results from a phase I clinical trial . Cancer Chemother Pharmacol ( 2013 ) 71 : 765–75 . doi: 10.1007/s00280-013-2070-8 PMC3587047 23381814

125 ClinicalTrials.gov Identifier: NCT01049880 . A research trial of high dose vitamin c and chemotherapy for metastatic pancreatic cancer . ( 2015 ). Available at: https://clinicaltrials.gov/ct2/show/NCT01049880 [Accessed August 15, 2022 ].

126 ClinicalTrials.gov Identifier: NCT01050621 . Trial of chemotherapy plus intravenous vitamin c in patients with advanced cancer for whom chemotherapy alone is only marginally effective . ( 2010 ). Available at: https://clinicaltrials.gov/ct2/show/NCT01050621 [Accessed August 15, 2022 ].

127 ClinicalTrials.gov Identifier: NCT01080352 . Vitamin c as an anti-cancer drug . ( 2010 ). Available at: https://clinicaltrials.gov/ct2/show/NCT01080352 [Accessed August 15, 2022 ].

128 Nielsen TK Højgaard M Andersen JT Jørgensen NR Zerahn B Kristensen B . Weekly ascorbic acid infusion in castration-resistant prostate cancer patients: a single-arm phase II trial . Transl Androl Urol ( 2017 ) 6 : 517–28 . doi: 10.21037/tau.2017.04.42 PMC5503969 28725594

129 ClinicalTrials.gov Identifier: NCT01364805 . New treatment option for pancreatic cancer . ( 2011 ). Available at: https://clinicaltrials.gov/ct2/show/NCT01364805 [Accessed August 15, 2022 ].

130 ClinicalTrials.gov Identifier: NCT00228319 . Treatment of newly diagnosed ovarian cancer with antioxidants . ( 2005 ). Available at: https://clinicaltrials.gov/ct2/show/NCT00228319 [Accessed August 15, 2022 ].

131 Ma Y Chapman J Levine M Polireddy K Drisko J Chen Q . High-dose parenteral ascorbate enhanced chemosensitivity of ovarian cancer and reduced toxicity of chemotherapy . Sci Transl Med ( 2014 ) 6 : 222ra18 . doi: 10.1126/scitranslmed.3007154 24500406

132 ClinicalTrials.gov Identifier: NCT02655913 . Safety and efficacy of vitamin c infusion in combination with local mEHT to treat non small cell lung cancer . ( 2016 ). Available at: https://clinicaltrials.gov/ct2/show/NCT02655913 [Accessed August 15, 2022 ].

133 Ou J Zhu X Chen P Du Y Lu Y Peng X . A randomized phase II trial of best supportive care with or without hyperthermia and vitamin c for heavily pretreated, advanced, refractory non-small-cell lung cancer . J Adv Res ( 2020 ) 24 : 175–82 . doi: 10.1016/j.jare.2020.03.004 PMC7190757 32368355

134 ClinicalTrials.gov Identifier: NCT01905150 . Ph 2 trial of vitamin c & G-FLIP (Low doses gemcitabine, 5FU, leucovorin, irinotecan, oxaliplatin) for pancreatic cancer . ( 2013 ). Available at: https://clinicaltrials.gov/ct2/show/NCT01905150 [Accessed August 15, 2022 ].

135 Bruckner H Hirschfeld A Gurell D Lee K . Broad safety impact of high-dose ascorbic acid and induction chemotherapy for high-risk pancreatic cancer . J Clin Oncol ( 2017 ) 35 ( 15_suppl ): e15711 . doi: 10.1200/JCO.2017.35.15_suppl.e15711

136 ClinicalTrials.gov Identifier: NCT01754987 . A study of vitamin c in the treatment of liver cancer to determine if it is safe and effective . ( 2012 ). Available at: https://clinicaltrials.gov/ct2/show/NCT01754987 [Accessed August 15, 2022 ].

137 ClinicalTrials.gov Identifier: NCT03410030 . Trial of ascorbic acid (AA) + nanoparticle paclitaxel protein bound + cisplatin + gemcitabine (AA NABPLAGEM) . ( 2018 ). Available at: https://clinicaltrials.gov/ct2/show/NCT03410030 [Accessed August 15, 2022 ].

138 ClinicalTrials.gov Identifier: NCT03964688 . Effect of vitamin c in autologous stem cell transplantations . ( 2019 ). Available at: https://clinicaltrials.gov/ct2/show/NCT03964688 [Accessed August 15, 2022 ].

139 ClinicalTrials.gov Identifier: NCT02905578 . A phase 2 trial of high-dose ascorbate for pancreatic cancer (PACMAN 2.1) . ( 2016 ). Available at: https://clinicaltrials.gov/ct2/show/NCT02905578 [Accessed August 15, 2022 ].

140 ClinicalTrials.gov Identifier: NCT03146962 . High dose vitamin c intravenous infusion in patients with resectable or metastatic solid tumor malignancies . ( 2017 ). Available at: https://clinicaltrials.gov/ct2/show/NCT03146962 [Accessed August 15, 2022 ].

141 ClinicalTrials.gov Identifier . Ascorbic acid and combination chemotherapy for the treatment of relapsed or refractory lymphoma or clonal cytopenia of undetermined significance . ( 2018 ). Available at: https://clinicaltrials.gov/ct2/show/NCT03418038 [Accessed August 15, 2022 ].

142 ClinicalTrials.gov Identifier: NCT03433781 . A phase Ib/IIa study evaluating the safety and tolerability of vitamin c in patients with intermediate or high risk myelodysplastic syndrome with TET2 mutations . ( 2018 ). Available at: https://clinicaltrials.gov/ct2/show/NCT03433781 [Accessed August 15, 2022 ].

143 ClinicalTrials.gov Identifier: NCT03508726 . High dose ascorbate with preoperative radiation in patients with locally advanced soft tissue sarcomas . ( 2018 ). Available at: https://clinicaltrials.gov/ct2/show/NCT03508726 [Accessed August 15, 2022 ].

144 ClinicalTrials.gov Identifier: NCT03682029 . Epigenetics, vitamin c, and abnormal blood cell formation - vitamin c in patients with low-risk myeloid malignancies . ( 2018 ). Available at: https://clinicaltrials.gov/ct2/show/NCT03682029 [Accessed August 15, 2022 ].

145 ClinicalTrials.gov Identifier: NCT03799094 . Vitamin c and tyrosine kinase inhibitor in lung cancer patients with epidermal growth factor receptor mutations . ( 2019 ). Available at: https://clinicaltrials.gov/ct2/show/NCT03799094 [Accessed August 15, 2022 ].

146 ClinicalTrials.gov Identifier: NCT03999723 . Combining active and passive DNA hypomethylation . ( 2019 ). Available at: https://clinicaltrials.gov/ct2/show/NCT03999723 [Accessed August 15, 2022 ].

147 ClinicalTrials.gov Identifier: NCT04033107 . High dose vitamin c combined with metformin in the treatment of malignant tumors . ( 2019 ). Available at: https://clinicaltrials.gov/ct2/show/NCT04033107 [Accessed August 15, 2022 ].

148 ClinicalTrials.gov Identifier: NCT04046094 . Intravenous (IV) vitamin c with chemotherapy for cisplatin ineligible bladder cancer patients . ( 2019 ). Available at: https://clinicaltrials.gov/ct2/show/NCT04046094 [Accessed August 15, 2022 ].

149 ClinicalTrials.gov Identifier: NCT04516681 . IV ascorbic acid in peritoneal metastatic colorectal cancer . ( 2020 ). Available at: https://clinicaltrials.gov/ct2/show/NCT04516681 [Accessed August 15, 2022 ].

150 ClinicalTrials.gov Identifier: NCT04634227 . Gemcitabine plus ascorbate for sarcoma in adults (Pilot) . ( 2020 ). Available at: https://clinicaltrials.gov/ct2/show/NCT04634227 [Accessed August 15, 2022 ].

151 ClinicalTrials.gov Identifier: NCT04801511 . Preoperative IMRT with concurrent high-dose vitamin c and mFOLFOX6 in locally advanced rectal cancer . ( 2021 ). Available at: https://clinicaltrials.gov/ct2/show/NCT04801511 [Accessed August 15, 2022 ].

152 ClinicalTrials.gov Identifier: NCT02516670 . Docetaxel with or without ascorbic acid in treating patients with metastatic prostate cancer . ( 2022 ). Available at: https://clinicaltrials.gov/ct2/show/NCT02516670 [Accessed August 15, 2022 ].

153 Drisko JA Chapman J Hunter VJ . The use of antioxidants with first-line chemotherapy in two cases of ovarian cancer . J Am Coll Nutr ( 2003 ) 22 : 118–23 . doi: 10.1080/07315724.2003.10719284 12672707

154 González MJ Berdiel MJ Miranda-Massari JR López D Duconge J Rodriguez JL . High dose intravenous vitamin c and metastatic pancreatic cancer: two cases . Integr Cancer Sci Ther ( 2016 ) 3 : 1 – 2 . doi: 10.15761/ICST.1000219

155 Padayatty SJ Riordan HD Hewitt SM Katz A Hoffer LJ Levine M . Intravenously administered vitamin c as cancer therapy: three cases . CMAJ ( 2006 ) 174 : 937–42 . doi: 10.1503/cmaj.050346 PMC1405876 16567755

156 Riordan HD Riordan NH Jackson JA Casciari JJ Hunninghake R González MJ . Intravenous vitamin c as a chemotherapy agent: a report on clinical cases . P R Health Sci J ( 2004 ) 23 : 115–8 . 15377059

157 Seo MS Kim JK Shim JY . High-dose vitamin c promotes regression of multiple pulmonary metastases originating from hepatocellular carcinoma . Yonsei Med J ( 2015 ) 56 : 1449–52 . doi: 10.3349/ymj.2015.56.5.1449 PMC4541681 26256994

158 Drisko JA Serrano OK Spruce LR Chen Q Levine M . Treatment of pancreatic cancer with intravenous vitamin c: a case report . Anticancer Drugs ( 2018 ) 29 : 373–9 . doi: 10.1097/CAD.0000000000000603 PMC5882293 29438178

159 Schoenfeld JD Sibenaller ZA Mapuskar KA Wagner BA Cramer-Morales KL Furqan M . O2 ·- and H 2 O 2 -mediated disruption of fe metabolism causes the differential susceptibility of NSCLC and GBM cancer cells to pharmacological ascorbate . Cancer Cell ( 2017 ) 31 : 487 – 500 . doi: 10.1016/j.ccell.2017.02.018 28366679 PMC5497844

160 Zhao H Zhu H Huang J Zhu Y Hong M Zhu H . The synergy of vitamin c with decitabine activates TET2 in leukemic cells and significantly improves overall survival in elderly patients with acute myeloid leukemia . Leuk Res ( 2018 ) 66 : 1 – 7 . doi: 10.1016/j.leukres.2017.12.009 29331774

161 Vollbracht C Schneider B Leendert V Weiss G Auerbach L Beuth J . Intravenous vitamin c administration improves quality of life in breast cancer patients during chemo-/radiotherapy and aftercare: results of a retrospective, multicentre, epidemiological cohort study in Germany . In Vivo ( 2011 ) 25 : 983–90 . 22021693

162 Takahashi H Mizuno H Yanagisawa A . High-dose intravenous vitamin c improves quality of life in cancer patients . Pers Med Univ ( 2012 ) 1 : 49 – 53 . doi: 10.1016/j.pmu.2012.05.008

163 Jacobs C Hutton B Ng T Shorr R Clemons M . Is there a role for oral or intravenous ascorbate (vitamin c) in treating patients with cancer? a systematic review . Oncologist ( 2015 ) 20 : 210–23 . doi: 10.1634/theoncologist.2014-0381 PMC4319640 25601965

164 Cockfield JA Schafer ZT . Antioxidant defenses: A context-specific vulnerability of cancer cells . Cancers (Basel) ( 2019 ) 11 : 1208 . doi: 10.3390/cancers11081208 PMC6721511 31434226

165 Buechler MB Pradhan RN Krishnamurty AT Cox C Calviello AK Wang AW . Cross-tissue organization of the fibroblast lineage . Nature ( 2021 ) 593 : 575–9 . doi: 10.1038/s41586-021-03549-5 33981032

166 Gaggioli C Hooper S Hidalgo-Carcedo C Grosse R Marshall JF Harrington K . Fibroblast-led collective invasion of carcinoma cells with differing roles for RhoGTPases in leading and following cells . Nat Cell Biol ( 2007 ) 9 : 1392–400 . doi: 10.1038/ncb1658 18037882

167 Albrengues J Bertero T Grasset E Bonan S Maiel M Bourget I . Epigenetic switch drives the conversion of fibroblasts into proinvasive cancer-associated fibroblasts . Nat Commun ( 2015 ) 6 : 10204 . doi: 10.1038/ncomms10204 26667266 PMC4682161

168 Provenzano PP Cuevas C Chang AE Goel VK Von Hoff DD Hingorani SR . Enzymatic targeting of the stroma ablates physical barriers to treatment of pancreatic ductal adenocarcinoma . Cancer Cell ( 2012 ) 21 : 418–29 . doi: 10.1016/j.ccr.2012.01.007 PMC3371414 22439937

169 Nathanson SD Nelson L . Interstitial fluid pressure in breast cancer, benign breast conditions, and breast parenchyma . Ann Surg Oncol ( 1994 ) 1 : 333–8 . doi: 10.1007/BF03187139 7850532

170 Liotta LA . Tumor invasion and metastases–role of the extracellular matrix: Rhoads memorial award lecture . Cancer Res ( 1986 ) 46 : 1 – 7 . 2998604

171 Egeblad M Rasch MG Weaver VM . Dynamic interplay between the collagen scaffold and tumor evolution . Curr Opin Cell Biol ( 2010 ) 22 : 697 – 706 . doi: 10.1016/j.ceb.2010.08.015 20822891 PMC2948601

172 Özdemir BC Pentcheva-Hoang T Carstens JL Zheng X Wu CC Simpson TR . Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival . Cancer Cell ( 2014 ) 25 : 719–34 . doi: 10.1016/j.ccr.2014.04.005 PMC4180632 24856586

173 Yoshida GJ Azuma A Miura Y Orimo A . Activated fibroblast program orchestrates tumor initiation and progression; molecular mechanisms and the associated therapeutic strategies . Int J Mol Sci ( 2019 ) 20 : 2256 . doi: 10.3390/ijms20092256 PMC6539414 31067787

174 Yoshida GJ . Metabolic reprogramming: the emerging concept and associated therapeutic strategies . J Exp Clin Cancer Res ( 2015 ) 34 : 111 . doi: 10.1186/s13046-015-0221-y 26445347 PMC4595070

175 Zhang Y Bian Y Wang Y Wang Y Duan X Han Y . HIF-1α is necessary for activation and tumour-promotion effect of cancer-associated fibroblasts in lung cancer . J Cell Mol Med ( 2021 ) 25 : 5457–69 . doi: 10.1111/jcmm.16556 PMC8184678 33943003

176 Zhang D Wang Y Shi Z Liu J Sun P Hou X . Metabolic reprogramming of cancer-associated fibroblasts by IDH3α downregulation . Cell Rep ( 2015 ) 10 : 1335–48 . doi: 10.1016/j.celrep.2015.02.006 25732824

177 Sanz-Moreno V Gaggioli C Yeo M Albrengues J Wallberg F Viros A . ROCK and JAK1 signaling cooperate to control actomyosin contractility in tumor cells and stroma . Cancer Cell ( 2011 ) 20 : 229–45 . doi: 10.1016/j.ccr.2011.06.018 21840487

178 Becker LM O’Connell JT Vo AP Cain MP Tampe D Bizarro L . Epigenetic reprogramming of cancer-associated fibroblasts deregulates glucose metabolism and facilitates progression of breast cancer . Cell Rep ( 2020 ) 31 : 107701 . doi: 10.1016/j.celrep.2020.107701 32492417 PMC7339325