Elsevier

Free Radical Biology and Medicine

Volume 53, Issue 8, 15 October 2012, Pages 1607-1615
Free Radical Biology and Medicine

Original Contribution
Enhanced antitumor activity of vitamin C via p53 in Cancer cells

https://doi.org/10.1016/j.freeradbiomed.2012.07.079 Get rights and content

Abstract

Ascorbate is an important natural antioxidant that can selectively kill cancer cells at pharmacological concentrations. Despite its benefit, it is quite difficult to predict the antitumor effects of ascorbate, because the relative cytotoxicity of ascorbate differs between cancer cell lines. Therefore, it is essential to examine the basis for this fundamental disagreement. Because p53 is activated by DNA-damaging stress and then regulates various cellular conditions, we hypothesized that p53 can sensitize cancer cells to ascorbate. Using isogenic cancer cells, we observed that the presence of p53 can affect ascorbate cytotoxicity, and also reactivation of p53 can make cancer cells sensitive to ascorbate. p53-dependent enhancement of ascorbate cytotoxicity is caused by increased reactive oxygen species generation via a differentially regulated p53 transcriptional network. We also found that transcriptionally activated p53 was derived from MDM2 ubiquitination by ascorbate and subsequently its signaling network renders cancer cells more susceptible to oxidative stress. Similar to the p53 effect on in vitro ascorbate cytotoxicity, inhibition of tumor growth is also stronger in p53-expressing tumors than in p53-deficient ones in vivo. This is the first observation that ascorbate cytotoxicity is positively related to p53 expression, activating its transcriptional network to worsen intracellular oxidative stress and consequently enhancing its cytotoxicity. Based on our study, reactivation of p53 may help to achieve more consistent cytotoxic effects of ascorbate in cancer therapies.

Highlights

► One of the reasons for differing ascorbate cytotoxicity may be the presence of p53. ► The modification of p53 expression changed ascorbate cytotoxicity. ► Ascorbate overcame the threshold level of oxidative stress, causing cell death via p53. ► p53 and MDM2 are differentially regulated by ascorbate. ► In vivo antitumor activity is enhanced by the presence of p53.

Introduction

Ascorbate (also known as vitamin C), which is generally regarded as an antioxidant, has been known as an essential micronutrient and as an unorthodox therapy for cancer treatment. Initial ascorbate treatment of cancer patients conducted by Pauling and Cameron [1] showed conflicting results with regard to efficacy [2], [3]. Despite these uncertainties, ascorbate recently has been reevaluated as a potential cancer treatment. For effective cytotoxicity, the ascorbate concentration must reach a pharmacological level that varies depending on the cancer cell line. Emerging evidence indicates that high concentrations of ascorbate can be easily achieved by intravenous or intraperitoneal injection but not by oral dosing [3], [4]. Furthermore, pharmacological concentrations of ascorbate selectively kill cancer cells but not normal cells, a characteristic of an ideal cancer drug [5].

It is still questionable, however, to apply ascorbate for clinical uses, because it has been reported that ascorbate has quite different anticancer effects on various cancer cell lines [4], [6], [7], [8]. These data demonstrate that the concentration of ascorbate that can kill one cancer cell line does not have same effect on others. This relatively different cytotoxicity of ascorbate may have caused inconsistent therapeutic outcomes. Although a major cell death pattern can be shown, it has been reported that ascorbate-mediated cell death shows a mixed pattern between apoptosis, necrosis, and cell cycle arrest [9], [10], [11]. Because ascorbate cytotoxicity differs among cancer cell lines, the cell death mechanism can be also different among cell lines. This can make it difficult for researchers or clinics to analyze or anticipate results.

According to many studies, ascorbate at pharmacological concentrations can act as a DNA-damaging stress and pro-oxidant on cancer cells [9], [12], [13]. Based on these studies, we propose that a factor(s) that regulates DNA-damaging stress and oxidative stress might be extensively involved in ascorbate cytotoxicity. Because it was shown that ascorbate exerts its cytotoxicity via hydrogen peroxide (H2O2) generation [9], an element that can modify the balance between oxidants and antioxidants in cancer cells may be very helpful in explaining the differences in ascorbate cytotoxicity. So we focused on p53, which is a major tumor suppressor and transcription factor that responds to various DNA-damaging stresses and orchestrates the transcription of genes involved in cell cycle arrest, senescence, and apoptosis [14]. It has been shown that cellular oxidative stress can be related to one of several p53-related functions that are oxidative stress-generating phenomena [15]. However, it is still debated as to what mechanisms are involved in influencing p53 to promote intracellular oxidative stress or vice versa.

An ideal anticancer agent should be toxic to malignant cells but only minimally toxic to normal cells. In this regard, ascorbate might be a good cancer therapeutic agent. However, more basic work should be done before using it as a common anticancer drug, because the mechanism of action is not fully understood. Therefore, our study has focused on the conditions providing for a more consistent therapeutic effect of ascorbate. We have examined the role of p53 as a sensitizer for ascorbate cytotoxicity and hypothesize that p53 renders cells more sensitive to ascorbate-induced oxidative stress. We found that the presence of p53 can influence ascorbate cytotoxicity, and reactivation of p53 in cancer cells enhances anticancer activity of ascorbate by increasing reactive oxygen species (ROS) generation via the p53 transcriptional network. Furthermore, ascorbate-mediated p53 activation was achieved by ubiquitination of MDM2 and activation by p38MAPK. Finally, we identified that ascorbate had similar preferential cytotoxicity on wild-type p53-bearing tumors over p53-deficient tumors in vivo, which result was similar to the in vitro p53 effect on ascorbate cytotoxicity.

Section snippets

Cell lines

Human cancer cells HCT116+/+, MCF7, HeLa, HCT116−/−, and SKOV3 were grown in Dulbecco's modified Eagle's medium (WelGene, Seoul, South Korea), and A549 and H1299 cells were maintained in RPMI 1640 medium containing 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and 2 mM glutamine. All media were supplemented with 10% fetal bovine serum, 100 μg/ml penicillin, and 100 U/ml streptomycin. Cultures were grown at 37 °C in a 5% CO2 atmosphere. HCT116+/+ and HCT116−/− cells were a kind gift from

The presence of p53 influences the pharmacological concentration of ascorbate cytotoxicity

We initially examined ascorbate cytotoxicity by dose–response assays in seven cancer cell lines. The cancer cell lines we used can be classified into two groups, those expressing wild-type p53 (HCT116+/+, MCF7, A549, and HeLa) and those that are p53-deficient (HCT116−/−, H1299, and SKOV3). Our results suggest a spectrum of effective cytotoxic doses of ascorbate in the cancer cell lines with differing p53 status (Fig. 1A and Supplementary Table 1). To examine the question of p53 influence on

Discussion

A purpose of this study was to investigate a factor for improving the consistent cytotoxicity of ascorbate. Ascorbate can influence various signaling pathways that regulate the cancer microenvironment [26], [27], which can complicate comparisons that use cell lines with different genetic backgrounds to examine the mechanisms of ascorbate cytotoxicity. As already reported in many studies, p53 has been used as one of targets for effective cancer therapy [28], [29], [30]. It has been shown that

Acknowledgment

This work was supported by a National Research Foundation of Korea grant funded by the Korean government (MEST, 2011-0001381 and 2011–0030701).

References (37)

  • E. Cameron et al.

    Supplemental ascorbate in the supportive treatment of cancer: prolongation of survival times in terminal human cancer

    Proc. Natl. Acad. Sci. USA

    (1976)
  • E.T. Creagan et al.

    Failure of high-dose vitamin C (ascorbic acid) therapy to benefit patients with advanced cancer: a controlled trial

    N. Engl. J. Med.

    (1979)
  • S.J. Padayatty et al.

    Vitamin C pharmacokinetics: implications for oral and intravenous use

    Ann. Intern. Med.

    (2004)
  • Q. Chen et al.

    Ascorbate in pharmacologic concentrations selectively generates ascorbate radical and hydrogen peroxide in extracellular fluid in vivo

    Proc. Natl. Acad. Sci. USA

    (2007)
  • Q. Chen et al.

    Pharmacologic doses of ascorbate act as a pro-oxidant and decrease growth of aggressive tumor xenograft in mice

    Proc. Natl. Acad. Sci. USA

    (2008)
  • Q. Chen 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. USA

    (2005)
  • K.A. Naidu et al.

    p53 enhances ascorbyl stearate-induced G2/M arrest of human ovarian cancer cells

    Anticancer Res.

    (2007)
  • F.W. Cheung et al.

    Sodium 5,6-benzylidene-L-ascorbate induces oxidative stress, autophagy, and growth arrest in human colon cancer HT-29 cells

    J. Cell. Biochem.

    (2010)
  • Cited by (34)

    • Vitamin C-induced competitive binding of HIF-1α and p53 to ubiquitin E3 ligase CBL contributes to anti-breast cancer progression through p53 deacetylation

      2022, Food and Chemical Toxicology
      Citation Excerpt :

      VC functions as a cofactor for prolyl-hydroxylase, which is responsible for HIF-1α hydroxylation and degradation (Gan et al., 2019; Li et al., 2010; Nytko et al., 2011; Fischer and Miles, 2017). In addition, VC promotes cancer cell apoptosis by regulating p53 (Lee et al., 2015; Gong et al., 2016; Rubis et al., 2019; Kim et al., 2012). In the current study, we identified that Casitas B Cell lymphoma (CBL), as an E3 ligase of HIF-1α and p53, could balance the two genes under VC treatment.

    • The potential role of vitamin C in empowering cancer immunotherapy

      2022, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      Another study has also reported that PD-L1 and p53 protein levels are correlated in patients with lung adenocarcinoma [107]. It has been shown that vitamin C treatment induces an up-regulation of p53 [108]. The mechanisms by which vitamin C modulates cytokines expression is mostly unknown.

    • Ascorbate kills breast cancer cells by rewiring metabolism via redox imbalance and energy crisis

      2021, Free Radical Biology and Medicine
      Citation Excerpt :

      Several recent studies probed the molecular and cellular mechanisms, through which high levels of ascorbate damage cancer cells [22,25–27]. It has been shown that ascorbate induces oxidative stress selectively in cancer cells while normal cells are spared due to higher reductive capacity and more robust redox homeostasis [22,25,28]. Multiple reports also showed that the ascorbate-associated oxidative stress resulted in inhibited glucose uptake and glycolysis [22,29,30].

    • Antitumor and antiviral activities of 4-substituted 1,2,3-triazolyl-2,3-dibenzyl-L-ascorbic acid derivatives

      2019, European Journal of Medicinal Chemistry
      Citation Excerpt :

      Even though l-ascorbic acid (L-ASA) cancer therapy has a controversial history [1], interest in L-ASA has been renewed due to its recently demonstrated ability to selectively kill cancer cells [2–5].

    View all citing articles on Scopus
    View full text