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Vitamin C shifts the transcriptome of breast cancer cells. (a) The heatmap showing the relative transcript levels of the differential genes in MDA-MB-231 cells treated with 100 μM vitamin C or no vitamin C shows that the expression pattern changes after treatment. (b) Differentially expressed genes are identified by edgeR (905 genes) and DESeq. 2 (1,424 genes). 778 genes were identified by the two methods.
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Genomic loss of 5-hydroxymethylcytosine (5hmC) accompanies malignant cellular transformation in breast cancer. Vitamin C serves as a cofactor for TET methylcytosine dioxygenases to increase 5hmC generation. Here we show that the transcription of SVCT2, a major vitamin C transporter, was decreased in human breast cancers (113 cases) compared to norm...
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... quantifying transcript levels, and to display alternative splice variants and non-coding RNA species. MDA-MB-231 cells were treated with vitamin C at 100 μM for 3 days. DNA and RNA were simultaneously extracted from MDA-MB-231 cells cultured in the same wells (n = 3 per group). Vitamin C-induced 5hmC was confirmed by dot-blot assay (data not shown) in samples submitted for sequencing. We observed a shift in the MDA-MB-231 transcriptome after vitamin C treatment as shown by heatmap (Fig. 3a). 905 genes were deter- mined to be differentially expressed by edgeR and 1,424 genes were determined to be differentially expressed by DESeq. 2. Of these, 778 genes were significantly and differentially expressed using both methods (Fig. 3b). Of the 778 genes, 363 genes were downregulated while 415 genes were up-regulated. The top 10 upregulated genes (TNFSF10, CYP1B1) and downregulated genes (TFRC, PGK1, BNIP3, NDRG1, BNIP3L, ADM, PDK1, HK2) were chosen for validation based on fold changes. The expression changes of all 10 genes were verified by qRT-PCR (Supplementary Table ...
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... quantifying transcript levels, and to display alternative splice variants and non-coding RNA species. MDA-MB-231 cells were treated with vitamin C at 100 μM for 3 days. DNA and RNA were simultaneously extracted from MDA-MB-231 cells cultured in the same wells (n = 3 per group). Vitamin C-induced 5hmC was confirmed by dot-blot assay (data not shown) in samples submitted for sequencing. We observed a shift in the MDA-MB-231 transcriptome after vitamin C treatment as shown by heatmap (Fig. 3a). 905 genes were deter- mined to be differentially expressed by edgeR and 1,424 genes were determined to be differentially expressed by DESeq. 2. Of these, 778 genes were significantly and differentially expressed using both methods (Fig. 3b). Of the 778 genes, 363 genes were downregulated while 415 genes were up-regulated. The top 10 upregulated genes (TNFSF10, CYP1B1) and downregulated genes (TFRC, PGK1, BNIP3, NDRG1, BNIP3L, ADM, PDK1, HK2) were chosen for validation based on fold changes. The expression changes of all 10 genes were verified by qRT-PCR (Supplementary Table ...
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... next assayed whether levels of vitamin C that are physiologically achievable might exert an antitumor effect. As noted above, vitamin C at 100 μM increases global 5hmC as efficiently as a pharmacological concentra- tion (500 μM) in breast cancer cells in vitro. We reasoned that by upregulating TRAIL, vitamin C might activate apoptosis. Treatment with vitamin C at 100 μM for 3 days, but not at lower concentrations, induced apoptosis in MDA-MB-231 cells measured by colorimetric TUNEL assay (Fig. 5d). The induction of apoptosis was then ver- ified in two other breast cancer cell lines BT549 and HCC1937 (Fig. 5e,f). In contrast, glutathione (GSH), a gen- eral antioxidant, had no effect on 5hmC generation and did not affect apoptosis of breast cancer cells. To further confirm the induction of apoptosis by vitamin C, breast cancer cell lines including MDA-MB-231, BT549, and HCC1937 were probed for active caspase using poly caspase immunofluorescence staining on live cells. Caspase stained cells increased after 3 days treatment with vitamin C at 100 μM, but treatment at lower concentrations increased apoptosis only minimally ( Fig. 6a-d). These results suggest that treatment of breast cells with vitamin C at a physiological concentration induces apoptosis and involves caspase activation. To test if apoptosis induction is TRAIL dependent, MDA-MB-231 cells were treated with an inhibitory monoclonal antibody against TRAIL in MDA-MB-231 cells during vitamin C treatment (100 μM) (Fig. 6e,f). The anti-TRAIL antibody at concentra- tions of 0.1 and 1 μg/mL significantly reduced apoptosis (P = 0.01 and 0.007) in vitamin C treated MDA-MB-231 cells where control mouse IgG did not (Fig. 6e). Furthermore, addition of TRAIL protein (0.1 μg/mL) to the media induced apoptosis in ~75% of MDA-MB-231 cells, but this effect was also largely abolished by addition of anti-TRAIL antibody to the media (P = 0.0001, Supplementary Figure 3). These results suggest the induction of apoptosis by vitamin C is primarily mediated by ...
Citations
... The apoptosis-inducing factor initiates its translocation to the cell nucleus, where it instigates chromatin condensation and DNA fragmentation. Notably, vitamin C levels have been correlated with the upregulation of TRAIL, a known apoptosis inducer [67]. ...
Breast cancer is one of the most common cancers worldwide, at the same time being one of the most prevalent causes of women's death. Many factors such as alcohol, weight fluctuations, or hormonal replacement therapy can potentially contribute to breast cancer development and progression. Another important factor in breast cancer onset includes micronutrient status. In this narrative review, we analyzed 23 micronutrients and their possible influence on breast cancer onset and progression. Further, the aim of this study was to investigate the impact of micronutrient status on the prevention of breast cancer and its possible influence on various therapeutic pathways. We researched meta-analyses, systemic and narrative reviews, retrospective studies, as well as original studies on human and animal models. The results of these studies indicate a possible correlation between the different levels of micronutrients and a decreased risk of breast cancer as well as a better survival rate. However, further studies are necessary to establish adequate doses of supplementation of the chosen micronutrients and the exact mechanisms of micronutrient impact on breast cancer therapy.
... One of the most interesting observations in our study was significantly higher expression of TET3 in luminal B breast cancer patients in comparison to non-luminal HER2 positive breast cancer patients as well as in healthy subjects. Previously, one in vitro study revealed that hormone -dependent MCF-7 cell line expressed a higher level of TET3 in comparison to control cells 65 . Our observation can be linked with potential role of estrogens in the modulation of TET3 expression. ...
The active DNA demethylation process, which involves TET proteins, can affect DNA methylation pattern. TET dependent demethylation results in DNA hypomethylation by oxidation 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) and its derivatives. Moreover, TETs’ activity may be upregulated by ascorbate. Given that aberrant DNA methylation of genes implicated in breast carcinogenesis may be involved in tumor progression, we wanted to determine whether breast cancer patients exert changes in the active DNA demethylation process. The study included blood samples from breast cancer patients (n = 74) and healthy subjects (n = 71). We analyzed the expression of genes involved in the active demethylation process (qRT-PCR), and 5–mC and its derivatives level (2D-UPLC MS/MS). The ascorbate level was determined using UPLC-MS. Breast cancer patients had significantly higher TET3 expression level, lower 5-mC and 5-hmC DNA levels. TET3 was significantly increased in luminal B breast cancer patients with expression of hormone receptors. Moreover, the ascorbate level in the plasma of breast cancer patients was decreased with the accompanying increase of sodium-dependent vitamin C transporters (SLC23A1 and SLC23A2). The presented study indicates the role of TET3 in DNA demethylation in breast carcinogenesis.
... Vitamin C is a co-factor of TET enzymes that increases 5hmC levels and promotes apoptosis mediated by Tumor Necrosis Factor Ligand Superfamily Member 10 (TRAIL) expression in MDA-MB-231 BC cells [88]. Conversely, vitamin C decreases the Mesenchymalto-Epithelial Transition (MET), and apoptosis and increases cell proliferation and invasion through down-regulation expression in MDA-MB-231 BC cells with miR-302/363 cluster overexpression, which acts as tumor suppressor miRNAs [89]. ...
Breast Cancer (BC) was the most common female cancer in incidence and mortality worldwide in 2020. Similarly, BC was the top female cancer in the USA in 2022. Risk factors include earlier age at menarche, oral contraceptive use, hormone replacement therapy, high body mass index, and mutations in BRCA1/2 genes, among others. BC is classified into Luminal A, Luminal B, HER2-like, and Basal-like subtypes. These BC subtypes present differences in gene expression signatures, which can impact clinical behavior, treatment response, aggressiveness, metastasis, and survival of patients. Therefore, it is necessary to understand the epigenetic molecular mechanism of transcriptional regulation in BC, such as DNA demethylation. Ten-Eleven Translocation (TET) enzymes catalyze the oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) on DNA, which in turn inhibits or promotes the gene expression. Interestingly, the expression of TET enzymes as well as the levels of the 5hmC epigenetic mark are altered in several types of human cancers, including BC. Several studies have demonstrated that TET enzymes and 5hmC play a key role in the regulation of gene expression in BC, directly (dependent or independent of DNA de-methylation) or indirectly (via interaction with other proteins such as transcription factors). In this review, we describe our recent understanding of the regulatory and physiological function of the TET enzymes, as well as their potential role as biomarkers in BC biology.
... 27,28 Additionally, AA has been reported to promote apoptosis in breast cancer cells. 29 In this regard, MCF-7 cells were treated for 24 h using increasing concentrations (1.25, 2.5, 5, 10, 20, 40, 80, 160, and 320 μg/mL) of free AA, free GO, or AA and GO loaded in AA/NSs, GO/NSs, or AA-GO/NSs, in addition to plain niosomes and DOX. The cell viability assay was evaluated using the MTT assay. ...
Geranium oil (GO) has antiproliferative, antiangiogenic, and anti-inflammatory properties. Ascorbic acid (AA) is reported to inhibit the formation of reactive oxygen species, sensitize cancer cells, and induce apoptosis. In this context, AA, GO, and AA-GO were loaded into niosomal nanovesicles to ameliorate the physicochemical properties of GO and improve its cytotoxic effects using the thin-film hydration technique. The prepared nanovesicles had a spherical shape with average diameters ranging from 200 to 300 nm and exhibited outstanding surface negative charges, high entrapment efficiencies, and a controlled sustained release over 72 h. Entrapping AA and GO in niosomes resulted in a lower IC 50 value than free AA and GO when tested on MCF-7 breast cancer cells. In addition, flow cytometry analysis showed higher apoptotic cells in the late apoptotic stage upon treating the MCF-7 breast cancer cells with AA-GO niosomal vesicles compared to treatments with free AA, free GO, and AA or GO loaded into niosomal nanovesicles. Assessing the antioxidant effect of the free drugs and loaded niosomal nanovesicles showed enhanced antioxidant activity of AA-GO niosomal vesicles. These findings suggest the AA-GO niosomal vesicles as a potential treatment strategy against breast cancer, possibly through scavenging free radicals.
... For example, ascorbate can globally increase the 5hmC content in mouse embryonic stem cells [100,129,130]. Similar effects were observed in colon, kidney, bladder, breast, and lung cancer cells as well as in human leukemia and hematopoietic stem cell (HSC) [131][132][133][134][135][136][137]. In line, ascorbate deprivation in L-gulonolactone oxidase-deficient mice impaired TET function and expanded the HSC compartment [100]. ...
Vitamin C is unbeatable - at least when it comes to sales. Of all the vitamin preparations, those containing vitamin C sell best. This is surprising because vitamin C deficiency is extremely rare. Nevertheless, there is still controversy about whether the additional intake of vitamin C supplements is essential for our health. In this context, the possible additional benefit is in most cases merely reduced to the known effect as an antioxidant. However, new findings in recent years on the mechanisms of oxygen-sensing and epigenetic control underpin the multifaceted role of vitamin C in a biological context and have therefore renewed interest in it. In the present article, therefore, known facts are linked to these new key data. In addition, available clinical data on vitamin C use of cancer therapy are summarized.
... Severe deficiencies of TET2 and TET3 in HSPC lead to a loss of 5hmC of over 90%, leading to the spontaneous accumulation of DSB and further leading to the rapid progression of leukemia [122]. In addition, in human colon cancer cells, hematopoietic stem and progenitor cells (HSPCs), and leukemia cell lines, vitamin C treatment also leads to the induction of 5hmC levels and an increase in DNA hypomethylation throughout the entire genome [118,[123][124][125][126][127][128]. ...
... As plasma vitamin C levels decreased significantly in patients receiving chemotherapy, vitamin C treatment combined with standard chemotherapy could increase the effectiveness of chemotherapy [136]. The function of vitamin C in DNA methylation and tumor suppression has also been validated in subsequent studies using cancer cells in the kidneys, bladder, lungs, colon, and breast [127,128,137]. A reduction in 5hmC was associated with poor survival in renal cell carcinoma and vitamin C restored 5hmC levels via TET activation, significantly impeding tumor growth in vitro and in vivo [138,139]. ...
Epigenetic modifications are crucial regulators of gene expression that critically impact cell lineage differentiation, survival, and proliferation, and dysregulations are commonly observed in various cancers. The aberrantly modified epigenome confers unique features on tumor cells, including sustained proliferative potential, resistance to growth-suppressive or cell death signals, augmented replicative immortality, invasion, and metastasis. As a result, epigenetic abnormalities exhibit significant impacts on all stages of oncogenesis from its onset to progression to metastasis. Among various epigenetic mechanisms in mammals, DNA cytosine methylation–demethylation is recurrently disrupted in cancers. Due to its inherent reversibility, targeting DNA methylation dynamics has gained tremendous attention as a promising therapeutic option that can ameliorate the effects of cancer-specific epigenetic abnormalities by restoring normal conditions. Various small molecules targeting DNA (de)methylation regulators have been developed as potential cancer therapeutics, some of which are approved for usage in clinics. Clinical trials of many other molecules are underway for both hematological malignancies and solid tumors. In this review, we discuss the DNA methylation/demethylation pathway as a promising target for therapeutic intervention in cancer and highlight the development of various epigenetic drugs targeting DNA-modifying enzymes such as DNA methyltransferases (DNMTs) and ten-eleven translocation (TET) enzymes.
... Therefore, TRAIL has received widespread attention as a core component of cancer treatment [33]. Previous studies have revealed that stronger cell proliferation ability and lower drug-induced cell apoptosis are correlated with tamoxifen resistance [34], and that upregulating the expression could inhibit the progression of breast cancer and hepatocellular carcinoma [35]. Here, we used a series of gain-and loss-of-function experiments to evaluate whether TRAIL is related to tamoxifen resistance in breast cancer. ...
Simple Summary
While the prognosis of hormone receptor-positive (HR+) breast cancer has been significantly improved, tamoxifen resistance remains a challenge in the treatment of HR+ breast cancer. This study identified that tRF-16-K8J7K1B, a novel small ncRNA derived from the 3′-end of tRNAAla-TGC, was highly expressed in tamoxifen-resistant cells compared to parental cells. Moreover, extracellular tRF-16-K8J7K1B confers tamoxifen resistance via incorporation into exosomes and then degrades the expression of apoptosis-related proteins, reducing the proportion of drug-induced cell apoptosis. Therefore, we propose that exosomal tRF-16-K8J7K1B could be a potential predictive biomarker and therapeutic target for overcoming tamoxifen resistance.
Abstract
Tamoxifen resistance remains a challenge in hormone receptor-positive (HR+) breast cancer. Recent evidence suggests that transfer ribonucleic acid (tRNA)-derived fragments play pivotal roles in the occurrence and development of various tumors. However, the relationship between tRNA-derived fragments and tamoxifen resistance remains unclear. In this study, we found that the expression of tRF-16-K8J7K1B was upregulated in tamoxifen-resistant cells in comparison with tamoxifen-sensitive cells. Higher levels of tRF-16-K8J7K1B were associated with shorter disease-free survival in HR+ breast cancer. Overexpression of tRF-16-K8J7K1B promotes tamoxifen resistance. Moreover, extracellular tRF-16-K8J7K1B could be packaged into exosomes and could disseminate tamoxifen resistance to recipient cells. Mechanistically, exosomal tRF-16-K8J7K1B downregulates the expression of apoptosis-related proteins, such as caspase 3 and poly (ADP-ribose) polymerase, by targeting tumor necrosis factor-related apoptosis-inducing ligand in receptor cells, thereby reducing drug-induced cell apoptosis. Therapeutically, the inhibition of exosomal tRF-16-K8J7K1B increases the sensitivity of breast cancer cells to tamoxifen in vivo. These data demonstrate that exosomal tRF-16-K8J7K1B may be a novel therapeutic target to overcome tamoxifen resistance in HR+ breast cancer.
... However, impacts of the high vitamin C concentrations on other non-NETs processes of cell death (apoptosis and autophagy) of neutrophils during sepsis are still unknown. In contrast, the ability of high doses vitamin C on the induction of both apoptosis and autophagy in several malignant cells is mentioned [88][89][90]. Hence, it seems that the impacts of vitamin C on several cell deaths depend on the ascorbate concentrations which unfortunately too less data. ...
Vitamin C (ascorbic acid), a water-soluble essential vitamin, is well-known as an antioxidant and an essential substrate for several neutrophil functions. Because of (i) the importance of neutrophils in microbial control and (ii) the relatively low vitamin C level in neutrophils and in plasma during stress, vitamin C has been studied in sepsis (a life-threatening organ dysfunction from severe infection). Surprisingly, the supraphysiologic blood level of vitamin C (higher than 5 mM) after the high-dose intravenous vitamin C (HDIVC) for 4 days possibly induces the pro-oxidant effect in the extracellular space. As such, HDIVC demonstrates beneficial effects in sepsis which might be due to the impacts on an enhanced microbicidal activity through the improved activity indirectly via enhanced neutrophil functions and directly from the extracellular pro-oxidant effect on the organismal membrane. The concentration-related vitamin C properties are also observed in the neutrophil extracellular traps (NETs) formation as ascorbate inhibits NETs at 1 mM (or less) but facilitates NETs at 5 mM (or higher) concentration. The longer duration of HDIVC administration might be harmful in sepsis because NETs and pro-oxidants are partly responsible for sepsis-induced injuries, despite the possible microbicidal benefit. Despite the negative results in several randomized control trials, the short course HDIVC might be interesting to use in some selected groups, such as against anti-biotic resistant organisms. More studies on the proper use of vitamin C, a low-cost and widely available drug, in sepsis are warranted.
... A literature study has revealed that in breast cancer, wound healing fluid that originates from surgical sites increases the aggressiveness of cancer cells that last even after the surgery (Agresti et al., 2019). The negative regulation of programmed cell death is directly associated with cancer as it has been shown in the Dissecting big RNA-Seq cancer data using machine learning to find disease-associated genes literature that tumor cell growth results not only from abnormal cell proliferation but also from reduced apoptosis (Sant et al., 2018). ...
The recent advancements of machine learning and deep learning (DL) methods are making it possible to create systems that automatically mine patterns and learn from data. Applications of those methods in chemistry, in particular QSAR and drug discovery, are already available. While DL can be applied as a conventional way of learning from chemical descriptors, the potentialities of the method are far more. In particular the capabilities of DL to autonomously extract, through multiple transformations, the structural elements that are correlated with the property under investigation can help in discovering the link between a chemical and its biological/physical effects. After presenting the principal DL methods developed for chemical problems, the focus is on a study case in mutagenicity prediction that uses directly the chemical graph, either as SMILES, graphs, or images, and applies convolutional and recurrent networks. The knowledge extracted from the networks is analyzed and compared with the accepted structural alerts for mutagenicity. The next challenges and the future of DL for QSAR are finally discussed.
... A literature study has revealed that in breast cancer, wound healing fluid that originates from surgical sites increases the aggressiveness of cancer cells that last even after the surgery (Agresti et al., 2019). The negative regulation of programmed cell death is directly associated with cancer as it has been shown in the Dissecting big RNA-Seq cancer data using machine learning to find disease-associated genes literature that tumor cell growth results not only from abnormal cell proliferation but also from reduced apoptosis (Sant et al., 2018). ...
In this chapter, we have discussed a relatively advanced and successful hybrid machine learning workflow that may help to unravel causative agents of disease from high throughput RNA-Seq datasets. The method is then applied to a breast cancer dataset taken from the Gene Expression Omnibus repository, and disease genes associated with breast cancer are identified. Finally, using the PPI network analysis approach, we observed the significance that the detected disease genes possess a role in the causal mechanism of disease. This method discussed here is universal and can be applied to any RNA-Seq data independent of disease.
