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Table 4 Global molecular profiling studies investigating VitC in the cancer context

From: High-dose intravenous vitamin C, a promising multi-targeting agent in the treatment of cancer

Cancer type(s) Model system Methodology Treatment(s) Type of combination therapy VitC dosea Aim Omics results Ref.
 Colorectal DiFi (RS and XM Difi) cell lines SILAC-based MS (LC–ESI–MS-MS) 4 h and 24 h treatments with 1 mM VitCC and/or 50 μg/mL cetuximab Targeted high Hypothesis that VitC in combination with cetuximab could restrain the emergence of secondary resistance to EGFR blockade in CRC RAS/BRAF wild-type models - Identification of 4147 proteins
Switch from glycolysis to oxidative phosphorylation in cetuximab and combo-treated cells at 4 h
- downregulation of LDHA/LDHB
- upregulation of PDHA1/PDHB and respiratory enzymes
Perturbation of iron metabolism in VitC and combo-treated cells at 24 h
- downregulation of TFRC
- upregulation of FT
 Breast MDA-MB-231 cell line Biotin switch approach (enrichment of proteins containing oxidized thiols) followed by LC-MS/MS 30 min treatment with 10 mM ascorbic acid high Identify early alterations of the redoxome in cellular response to AA that might be linked to AA-induced cell death - Identification of 2910 cysteine-containing proteins
Oxidized targets upon AA treatment:
- antioxidant enzymes (eg. PRDX1)
- glycolysis and gluconeogenesis pathway (eg. PGK1)
- tricarboxylic acid cycle (eg. ACOT7)
- DNA, RNA and protein metabolism
Cell cycle arrest and translation inhibition associated with AA-induced cytotoxicity. PRDX1 expression levels correlated with AA differential cytotoxicity
 Breast MCF7 cell line LC-MS/MS 24 h treatment with 2 mM VitC high Effect of VitC in itself at different concentration levels on MCF-7 breast cancer cell line - Identification of 1694 proteins with differential regulation
Processes impacted by VitC treatment included
- unfolded protein response and inhibition of the cell translation (eIF2α, PKR/PKR pThr-446)
- apoptotic process
 Neuroblastoma SH-SY5Y cell line SUMO-1 IP followed by ESI-FT ICR MS 30 min treatment with 100 μM ascorbate (or 100 μM hydrogen peroxide) low Identify redox sensitive proteins of the conjugation machinery for SUMOylation. Oxidative stress (hydrogen peroxide), antioxidant (ascorbate) or control conditions were tested - Identification of 169 proteins
- Great overlap between all treatments
- Proteins identified only in the ascorbate sample included DTD2 and MGAT5B
- Proteins without predicted SUMOylation site indentified in both ascorabte and hydrogen peroxide treatments included TUBB4A, TUBB1, HNRNPH3, POLG2 and BUB3
 Gastric AGS cell line MALDI-TOF MS 24 h treatment with 300 μg/mL (~ 1.7 mM) VitC high Investigate the molecular mechanism of the inhibitory effect of VitC on AGS cell growth, and protein profiles in AGS cells after exposure to VitC treatment - 20 differential proteins identified
- downregulation eg. of TPM3 and TPM4
- upregulation of PRDX4 and TXND5
- Identified proteins are mainly involved in cell mobility, antioxidant and detoxification, signal transduction and protein metabolism
 Leukemia NB4 cell line MALDI–TOF 30 min treatment with 0.5 mM LAA (ascorbic acid) medium Identification of early protein targets of LAA in leukemia cells - 9 differential proteins identified
- changes in pI as a result of phosphorylation of a TPM isoform)
- downregulation eg of of SUPT6H and HSPA8
- upregulation eg. of MATN4 and NONO
 Sarcoma BALB/C mice implanted with S-180 cancer cells MALDI TOF-MS/MS Treatment with 1.5 mg/g body weight ascorbate every three days high Identify proteins involved in the ascorbic acid-mediated inhibition of tumor progression - 11 differential proteins identified
- upregulastion of RKIP and ANXA5
 Colorectal BALB/C mice implanted with CT-26 cancer cells MALDI TOF-MS/MS Treatment with 1.5 mg/g body weight ascorbate every three days high Proteome changes of tumor tissue were investigated after intraperitoneal administration of a high concentration of ascorbic acid - 18 differential proteins identified
- upregulation eg. of EIF3I, NPM1 and VIM
- regulation of cytoskeleton remodeling
 Breast MCF7 cell line LC-MS/MS 18 h treatment with 1 μM DOX (doxorubicin) or DOX + 200 μM of VitC Chemo medium Describe the changes in protein expression and proliferation of the MCF-7 cells induced by the VitC applied with doxorubicin - Identification of 229 proteins
- Downregulation of cytoskeletal (FLNA), ribosomal (eg. RPL27A), transcriptional (eg. HNRNPH1), immune system and antioxidant (HSP90AA1, SOD1) proteins in DOX + VitC-treated cells
- Upregulation of GAPDH, GPI and ACTA1
 Leukemia HL-60 cell line LC-MS/MS 48 h treatment with 10 μM As2O3 (arsenic trioxide) or As2O3 + 100 μM L-AA (ascorbic acid) + 50 μM α-TOC (α-tocopherol) Chemo + Dietary suppl. low Evaluate the synergistic mechanism of action of vitamins, such as L-ascorbic acid (L-AA) and a-tocopherol (a-TOC) in As2O3 chemotherapy - Number of identified proteins n.s.
- Downregulation of cell cycle and translation in cells treated with As2O3, L-AA, and a-TOC compared to As2O3-only
- Identification of numerous proteins associated with apoptosis and cell stress in combination treatment
 Breast, Lung A549 and MDA-MB-231 cell lines SILAC-based MS (LC-MS/MS) untreated (A549 cell line resistant to 1 mM AUF (auranofin) + 2.5 mM VitC, MDA-MB-231 cell line sensitive) Anti-inflammatory untreated Decipher the underlying mechanisms for differential response of lung and breast cancer cell models to redox-modulating molecule auranofin (AUF) and to combinations of AUF and VitC - Identification of f 4131 proteins common to both cell lines
- proteins involved in GSH synthesis and reduction, the pentose phosphate pathway and those belonging to other metabolic pathways (eg PGDH and PTGR1) more abundant in A549 (resistant) cells
 Melanoma A2058 cell line RNA-seq 48 h treatment with 0.1 mM VitC low Examined the possible mechanisms that could reveal how VitC suppresses cell migration and anchorage-independent growth of A2058 cells - 66 genes differentially expressed
- alterations predominantly in genes involved in extracellular matrix remodeling.
- ARGHAP30, TRIM63 and PTPN7 among 10 most differential genes
 Melanoma A2058 cell line RNA-seq 7 days treatment with 100 μM ascorbate low To elucidate potential mechanism of ascorbate in inducing apoptosis in A2058 cells. Re-analyse data of Gustafson et al., 2015 using updated algorithms - 344 genes including 20 non-coding RNAs (ncRNA) differentially expressed
- expression of CLU gene one of the most downregulated genes
 Breast MDA-MB-231 cell line RNA-seq 3 days treatment with 100 μM VitC low Analysis of transcriptomic changes associated with increased 5hmC generation following exposure to VitC - 778 differentially expressed genes
- TNFSF10, TFRC and PGK1 among 10 most differential genes
 Renal Cell 786-O cell line RNA-seq Treatment for 10 passages with 100 μM AsANa (sodium L-ascorbate; VitC) or 100 μM APM (oxidation-resistant VitC derivative) low Examine ccRCC phenotype changes at the global transcriptome level after treatment of VitC for 10 passages - 81 differentially expressed genes
- most notable genes positively enriched in VitC-treated cells belong to multiple metabolic pathways, such as peroxisome and pentose phosphate pathways
- most notable gene sets negatively enriched in VitC-treated cells include DNA replication and mismatch repair genes
 Bladder T24 cell line RNA-seq 0.25 mM VitC, time n.s. medium Explore the role of 5hmC in bladder cancer and the therapeutic efficacy of VitC in increasing the 5hmC pattern - 1172 differentially expressed genes were identified
- differential genes mainly associated with focal adhesion, DNA replication, cell cycle, and several cancer-related pathways.
 Hepatocellular Huh-7 cell line xenograft tumour mouse model Microarray 3 days treatment of mice with IP injection of 4.0 g/kg or 2.0 g/kg ascorbate high Assess effects of high-dose ascorbate on hepatoma - 192 genes/ncRNAs uniquely differentially expressed in HCC tumour tissue obtained from mice treated specifically with high-dose ascorbate (4.0 g/kg/3 days)
- deregulated genes were involved in insulin receptor signalling, metabolism and mitochondrial respiration
 Lymphoma JLPS and JLPR cell lines (sensitive/resistant to ascorbate) Microarray untreated (JLPR cell line resistant to VitC (incubation of JLPS cells with increasing ascorbate concentrations from 100 μM to 1 mM over 6 month), JLPRS cell line sensitive) untreated Identify possible mechanisms of ascorbate resistance - Acquired ascorbate resistance associated with downregulation of eg. HMGB1 and MYC and upregulation of eg. ATF5 [173]
 Leukemia HL60 and MOLM13 cell lines RNA-seq 12 or 72 h treatment with 250 μM L-AA (ascorbic acid) medium Analyse expression of genes upregulated by Tet2 restoration in cKit+ cells in HL60 and MOLM13 cells treated with L-AA - 14/50 genes upregulated by Tet2 restoration in mouse cKit+ cells also induced in both human leukemia lines after 12 h of VitC treatment, including genes involved in apoptotic and death receptor signaling (eg. BAX) and NOTCH signaling
- Of the top genes downregulated by Tet2-restoration, 34/50 were downregulated in both leukemia lines after 12 h of VitC
- Hence, VitC treatment can enhance TET2 function in human leukemia cells in a manner similar to the effects of Tet2 restoration in mouse HSPCs
 Breast MCF-7 cell line Microarray 3 days treatment with 100 nM RA (retinoic acid) and/or 1 mM AA (ascorbic acid) Chemo high Elucidate the mechanism by which RA + AA inhibits breast carcinoma proliferation - 29 genes were up-regulated and 38 genes were down-regulated after RA + AA treatment
- up-regulation of antioxidant enzymes (eg. GPX2) and proteins involved in apoptosis (eg. CDK11B), cell cycle regulation (eg. EDN1) and DNA repair (eg. RAD51C)
- RA or AA on their own failed to upregulate antioxidant genes
 Breast, Colorectal MCF-7, MDA-MB231 and HT29 cell lines LC-MS 4 h treatment with 3 mM ascorbate high Gain insight into the cellular effects of high doses of ascorbate - Metabolic shift, reversal of Warburg effect, disruption of redox homeostasis
- Cell death dependent on ascorbate-induced oxidative stress and accumulation of ROS, DNA damage, and depletion of essential intracellular co-factors including NAD+/NADH
- disruption of glycolysis, rapid drop in ATP levels
- inhibition the TCA cycle and increased oxygen consumption
 Breast, Colorectal MCF7 and HT29 cell lines CE-TOF MS 1 h treatment with VitC (0.2 mM, 1 mM or 10 mM) high Understand anticancer mechanisms of VitC - Levels of upstream metabolites in the glycolysis pathway and TCA cycle were increased in both cell lines following treatment with VitC
- ATP levels decreased concentration-dependently
- VitC inhibited energy metabolism through NAD depletion, thereby inducing cancer cell death
 Colorectal HCT116 and VACO432 cell lines LC-MS/MS 2 mM VitC for 30 min to 2 h high Clarify the mechanism by which VitC kills cancer cells while sparing normal cells. Profile metabolic changes following VitC treatment - Glycolytic intermediates upstream of GAPDH accumulated while those downstream were depleted suggesting that GAPDH was inhibited
- Oxidative PPP metabolites increased, indicating that the blockage may shift glycolytic flux into the oxidative PPP
- Cysteine, the major limiting precursor for GSH biosynthesis, was also dramatically depleted following VitC treatment
- As expected, VitC treatment induced a substantial increase in endogenous ROS in KRAS and BRAF mutant cells
 Hepatocellular SMMC-7721 cell line NMR spectroscopy 48 h treatment with 50 μmol/L OXA (oxaliplatin) and/or 1 mmol/L VitC Chemo high Assess the global metabolic changes in HCC cells following VitC treatment - VitC treatment led to inhibition of energy metabolism via NAD+ depletion and amino acid deprivation
- OXA caused significant perturbation in phospholipid biosynthesis and phosphatidylcholine biosynthesis pathways
- Glutathione metabolism and pathways related to succinate and choline may play central roles in conferring the combined effect between OXA and VitC
  1. Twenty-four studies were retrieved from PubMed using search terms (vitamin c OR ascorbate OR ascorbic acid) AND (proteomics OR mass-spectrometry OR metabolomics OR transcriptomics OR RNA-seq OR RNA sequencing OR microarray OR genomics OR DNA sequencing OR WES) AND (cancer). a high dose ≥ 1 mM or 1 g/kg, low dose ≤ 0.1 mM