DOES HIGH-DOSE VITAMIN C ENHANCE IMMUNOTHERAPY?

Cancer is a multifaceted disease that involves several systems. Cancer makes its own ecosystem known as a tumor microenvironment (TME). This micro-ecosystem is highly heterogeneous and consists of blood vessels, fibroblasts, nearby immune cells, and the extracellular matrix (ECM). The embedded cancer cells get their nutritional support and signals for stimulating growth through TME (1). Hence, TME is now deliberated as a therapeutic target to put a stop to the growth of tumors, their metastasis, and the resistance they show against drugs. Currently, the main emphasis of cancer treatment has shifted progressively to a TME-centric model instead of a cancer-centric one. It means therapies are not targeting components of TME instead of focusing on whole cancer.

Among these, impressive success has been observed for targeted therapies and immunotherapies. The paramount TME hallmark is the suppression of the immune system. Tregs or Regulatory T-cells; a part of white blood cells, myeloid-derived suppressor cells (MDSCs); immature myeloid cells that suppress the immune system, and tumor-associated macrophages (TAMs); cells that create TME by forming chemokines, cytokines, and growth factors, all these penetrate TME and cause the release of immunosuppressive mediators in large amounts. This process results in the depletion of tumor effector cells and the conversion of phenotype M1 to M2. Moreover, a decrease in the formation of new blood vessels results in low pH, low oxygen levels within the tumor, and high blood pressure (2).

VITAMIN C AS AN ANTI-CANCER AGENT

Investigation into vitamin C and its anti-cancer properties has been carried out for a long time both as a monotherapy and in combination (3). Although, the effectiveness of vitamin C and its anti-cancer properties have been a controversial topic. Vitamin C is a six-carbon molecule that is formed from glucose and found abundantly in vegetables, fruits, and in the liver and kidney of the majority of animals (4). However, humans, fruit bats, and guinea pigs are not able to manufacture vitamin C inside their bodies. This is because a gene that encodes L-gulonolactone oxide, an enzyme required as a catalyst for the last step of vitamin C formation is mutated (5,6).

The oxidative stress prompted by reactive oxygen species (ROS) is understood to be the mechanism through which vitamin C exerts anti-cancer action (7–12) as well as through the modifying influence of epigenetic programs (13,14). Epigenetics means how a cell controls the activity of a gene without causing any change in the sequence of DNA. Cancer cells are preferentially affected by these mechanisms and the doses at which vitamin C produces these effects are not toxic for normal cells. Without a doubt, earlier research has reported a beneficial and critical role of vitamin C in immune cells because high levels of vitamin C are present intracellularly in lymphocytes (15,16). In the case, the body is deficient in vitamin C which impairs immunity (17). These functions of vitamin C that modulate the immune system seem to be controlled at the epigenetic level (18).

THE EFFECTS OF VITAMIN C ON IMMUNE CELLS

Through the epigenetic and oxidative mechanism, vitamin C performs its function on tumor cells. Research data indicated that similar mechanisms are also used by vitamin C to exert its action on immune cells. The activation and signaling of cells are regulated crucially at low doses by free radicals and other (ROS). Certainly, signaling pathways of the T-cell receptor (TCR) are positively regulated by small amounts of ROS, therefore promoting multiplying and activation of T-cells. This is supported by the fact that events associated with TCR signaling require ROS (19,20). For example, when ROS are generated in moderate amounts after the TCR-signaling, the phosphorylation (addition of phosphate molecule or ion) of the extracellular signal-regulated kinases (Erk) 1/2 is modulated by it (21,22). Moreover, the IκB kinase complex (IKK) can be activated by ROS such as hydrogen peroxide (20).

VITAMIN C TARGETS TME TO EMPLOY ANTICANCER ACTIVITY

Earlier studies concentrated on the part where vitamin C targeted the tumor cells in sequestration. With the appearance of the influence of vitamin C on the neighboring healthy cells and their secretions, further studies were conducted to understand the mechanism behind the dedicated TME more deeply. The tumor immune microenvironment (TIME) is further reprogrammed by the signaling pathways and epigenetic programs regulated by vitamin C. Several studies explored the prospects of vitamin C in regulating the immunosuppressive and immunoreactive cells along with their secretions. Therefore, vitamin C looks very promising when it comes to changing cold cancer i.e., immunosuppressed into a hot one (immunoreactive). Immunosuppressed means an ineffective immune system while immunoreactive means an immune system that reacts to particular substances.

The immunosuppressive TME may be reversed by vitamin C at the molecular as well as the cellular level. The penetration of T-cells into the TME is increased by vitamin C and hence, promotes the formation of immune memory. Further research elaborates that the ratio of CD8+/CD4+ increases in the penetrated T-lymphocytes and this reflects a noteworthy development in the antitumor effect (23,24). During the early differentiation of NK cells, the frequency of gene expression of KIR is increased by vitamin C by increasing the KIR promoter demethylation. KIR is a marker of mature NK cells (25). Activated NK cells secrete granzyme B and perforin in larger quantities under the influence of a high dose of vitamin C (26). Perforin is a glycoprotein that forms pores in the cell membranes of the targeted cells. Granzyme B is an enzyme that collaborates with perforin to cause the death of the targeted cell.

Cancer is promoted by overcoming the surveillance of the immune system when TAMS are changed into M2 phenotype as a reaction to immunosuppressive cytokines present in the TME. M2 phenotypes of TAM give rise to tumor progression and also provide resistance against chemotherapies (27). A 2020 study demonstrated that apoptosis (cell death) of M2-type TAM that penetrates the tumor nodules is induced by vitamin C (28). The intracellular availability of vitamin C influences the gene expression of proteins and monocytes (a type of white blood cell). Although the exact nature of the impact is not fully determined, scientists found that when vitamin C is included in the treatment, alterations take place in protein secretions and gene expression associated with M2 (29). Treg cells are the primary cells responsible to maintain immune tolerance. The methylation of conserved noncoding sequence 2 (CNS2) decreases due to vitamin C which in turn increases the promotion of a gene called Foxp3+. The methylation of CNS2 indicates a robust capacity for immunosuppression. The Foxp3+ gene makes the protein that controls the tolerance of the immune system (30).

DOES THE EFFICACY OF IMMUNOTHERAPY INCREASE WITH A HIGH DOSE OF VITAMIN C?

An animal study was carried out in Italy on mice demonstrating the potential of using high doses of vitamin C to encourage the immune system to get a better response to cancer drugs used as immune therapy called checkpoint inhibitors. These are Keytruda, Optivo, Yervoy, etc. The study observed the effect of vitamin C while considering a component of white blood cells called T-cells or lymphocytes in the adaptive immune system. The experimental study suggested that vitamin C can play a critical role in preparing the immune system as well as destroying cancer linked with lymphocytes. The number of tumor-penetrating lymphocytes (T-cells) increases due to vitamin C and the activation of CD8 and CD4 T-cells also improves. These both are important indicators of cancer-killing through the immune system. This may help to develop an understanding of why vitamin C on its own slows tumor growth in those individuals having a complete immune system (23).

The efficacy of combined checkpoint inhibitors such as anti–PD-1 blockade and anti–CTLA-4 increased in mice models with colorectal, pancreatic, and breast cancer due to the administration of high doses of vitamin C. In the majority of cases, the combination therapy not only delayed the growth of the tumor but in some mice, the tumor completely disappeared. Besides, the checkpoint inhibitors in colorectal cancer seem to have a greater role in the immune system, the combination therapy with vitamin C led to the doorway where a significant response was produced as a result of the long-lasting shrinkage of the tumor.  When vitamin C was used alone as monotherapy, these cancers also showed sensitivity to it as well. The animals did not exhibit any signs of toxicities or adverse events related to the immune system when vitamin C was used as treatment along with checkpoint inhibitors. This indicates that combination therapy is hypothesized to be tolerated by humans as well. The doses of vitamin C exhibiting these responses can only be achieved through intravenous administration (23).

THE BOTTOM LINE

Vitamin C is a small molecule but it has emerged in recent times as a potent immunomodulatory substance. It acts on the cells of the immune system through its well-known epigenetic programs and antioxidant action along with direct effects of signaling pathways. It is being suggested by the increasing scientific evidence that vitamin C can help to achieve great benefits therapeutically when used in combination as a part of immunotherapies. Anticancer properties are present in vitamin C when administered intravenously in pharmacologic concentrations, but for tumor clearance, there may requirement for combinatorial immunotherapeutic approaches.

REFERENCES

1. Whiteside TL. The tumor microenvironment and its role in promoting tumor growth. Oncogene. 2008 Oct 6;27(45):5904–12.
2. Jin MZ, Jin WL. The updated landscape of tumor microenvironment and drug repurposing. Signal Transduct Target Ther. 2020 Aug 25;5(1):166.
3. Nauman G, Gray JC, Parkinson R, Levine M, Paller CJ. Systematic Review of Intravenous Ascorbate in Cancer Clinical Trials. Antioxidants. 2018 Jul;7(7):89.
4. Linster CL, Van Schaftingen E. Vitamin C. FEBS J. 2007;274(1):1–22.
5. Nishikimi M, Fukuyama R, Minoshima S, Shimizu N, Yagi K. Cloning and chromosomal mapping of the human nonfunctional gene for L-gulono-gamma-lactone oxidase, the enzyme for L-ascorbic acid biosynthesis missing in man. J Biol Chem. 1994 May 6;269(18):13685–8.
6. Nishikimi M, Kawai T, Yagi K. Guinea pigs possess a highly mutated gene for L-gulono-gamma-lactone oxidase, the key enzyme for L-ascorbic acid biosynthesis missing in this species. J Biol Chem. 1992 Oct 25;267(30):21967–72.
7. Chen Q, Espey MG, Krishna MC, Mitchell JB, Corpe CP, Buettner GR, et al. Pharmacologic ascorbic acid concentrations selectively kill cancer cells: action as a pro-drug to deliver hydrogen peroxide to tissues. Proc Natl Acad Sci U S A. 2005 Sep 20;102(38):13604–9.
8. Chen Q, Espey MG, Sun AY, Pooput C, Kirk KL, Krishna MC, et al. Pharmacologic doses of ascorbate act as a prooxidant and decrease growth of aggressive tumor xenografts in mice. Proc Natl Acad Sci. 2008 Aug 12;105(32):11105–9.
9. Serrano OK, Parrow NL, Violet PC, Yang J, Zornjak J, Basseville A, et al. Antitumor effect of pharmacologic ascorbate in the B16 murine melanoma model. Free Radic Biol Med. 2015 Oct 1;87:193–203.
10. Xia J, Xu H, Zhang X, Allamargot C, Coleman KL, Nessler R, et al. Multiple Myeloma Tumor Cells are Selectively Killed by Pharmacologically-dosed Ascorbic Acid. EBioMedicine. 2017 Apr 1;18:41–9.
11. 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 Feb 5;6(222):222ra18.
12. Schoenfeld JD, Sibenaller ZA, Mapuskar KA, Wagner BA, Cramer-Morales KL, Furqan M, et al. O2⋅- and H2O2-Mediated Disruption of Fe Metabolism Causes the Differential Susceptibility of NSCLC and GBM Cancer Cells to Pharmacological Ascorbate. Cancer Cell. 2017 Apr 10;31(4):487-500.e8.
13. Shenoy N, Bhagat T, Nieves E, Stenson M, Lawson J, Choudhary GS, et al. Upregulation of TET activity with ascorbic acid induces epigenetic modulation of lymphoma cells. Blood Cancer J. 2017 Jul;7(7):e587–e587.
14. Gerecke C, Schumacher F, Edlich A, Wetzel A, Yealland G, Neubert LK, et al. Vitamin C promotes decitabine or azacytidine induced DNA hydroxymethylation and subsequent reactivation of the epigenetically silenced tumour suppressor CDKN1A in colon cancer cells. Oncotarget. 2018 Aug 28;9(67):32822–40.
15. Evans RM, Currie L, Campbell A. The distribution of ascorbic acid between various cellular components of blood, in normal individuals, and its relation to the plasma concentration. Br J Nutr. 1982 May;47(3):473–82.
16. Omaye ST, Schaus EE, Kutnink MA, Hawkes WC. Measurement of vitamin C in blood components by high-performance liquid chromatography. Implication in assessing vitamin C status. Ann N Y Acad Sci. 1987 Jan 1;498:389–401.
17. Kennes B, Dumont I, Brohee D, Hubert C, Neve P. Effect of Vitamin C Supplements on Cell-Mediated Immunity in Old People. Gerontology. 1983;29(5):305–10.
18. Young JI, Züchner S, Wang G. Regulation of the Epigenome by Vitamin C. Annu Rev Nutr. 2015 Jul 17;35:545–64.
19. Bienert GP, Schjoerring JK, Jahn TP. Membrane transport of hydrogen peroxide. Biochim Biophys Acta. 2006 Aug;1758(8):994–1003.
20. Oliveira-Marques V, Marinho HS, Cyrne L, Antunes F. Role of hydrogen peroxide in NF-kappaB activation: from inducer to modulator. Antioxid Redox Signal. 2009 Sep;11(9):2223–43.
21. Devadas S, Zaritskaya L, Rhee SG, Oberley L, Williams MS. Discrete generation of superoxide and hydrogen peroxide by T cell receptor stimulation: selective regulation of mitogen-activated protein kinase activation and fas ligand expression. J Exp Med. 2002 Jan 7;195(1):59–70.
22. Jackson SH, Devadas S, Kwon J, Pinto LA, Williams MS. T cells express a phagocyte-type NADPH oxidase that is activated after T cell receptor stimulation. Nat Immunol. 2004 Aug;5(8):818–27.
23. Magrì A, Germano G, Lorenzato A, Lamba S, Chilà R, Montone M, et al. High-dose vitamin C enhances cancer immunotherapy. Sci Transl Med. 2020 Feb 26;12(532):eaay8707.
24. Xu YP, Lv L, Liu Y, Smith MD, Li WC, Tan XM, et al. Tumor suppressor TET2 promotes cancer immunity and immunotherapy efficacy. J Clin Invest. 2019 Jul 16;129(10):4316–31.
25. Wu CY, Zhang B, Kim H, Anderson SK, Miller JS, Cichocki F. Ascorbic Acid Promotes KIR Demethylation during Early NK Cell Differentiation. J Immunol Baltim Md 1950. 2020 Sep 15;205(6):1513–23.
26. Luchtel RA, Bhagat T, Pradhan K, Jacobs WR, Levine M, Verma A, et al. High-dose ascorbic acid synergizes with anti-PD1 in a lymphoma mouse model. Proc Natl Acad Sci U S A. 2020 Jan 21;117(3):1666–77.
27. Lee C, Jeong H, Bae Y, Shin K, Kang S, Kim H, et al. Targeting of M2-like tumor-associated macrophages with a melittin-based pro-apoptotic peptide. J Immunother Cancer. 2019 Jun 7;7(1):147.
28. Xu Y, Guo X, Wang G, Zhou C. Vitamin C Inhibits Metastasis of Peritoneal Tumors By Preventing Spheroid Formation in ID8 Murine Epithelial Peritoneal Cancer Model. Front Pharmacol [Internet]. 2020 [cited 2022 Oct 17];11. Available from: https://www.frontiersin.org/articles/10.3389/fphar.2020.00645
29. Ang AD, Vissers MCM, Burgess ER, Currie MJ, Dachs GU. Gene and Protein Expression Is Altered by Ascorbate Availability in Murine Macrophages Cultured under Tumour-Like Conditions. Antioxidants. 2021 Mar;10(3):430.
30. Someya K, Nakatsukasa H, Ito M, Kondo T, Tateda KI, Akanuma T, et al. Improvement of Foxp3 stability through CNS2 demethylation by TET enzyme induction and activation. Int Immunol. 2017 Aug 1;29(8):365–75.