The key to the anti-tumour effect of ascorbic acid is the production of cytotoxic hydrogen peroxide [10
]. In this study a panel of 11 human cancer cell lines was tested for their susceptibility to ascorbic acid. Three glioblastoma cell lines and the 3 breast carcinoma cell lines demonstrated EC50
mmol/L and were obviously susceptible to ascorbic acid mediated cytoxtoxicity. Five of 11 carcinoma cells lines with EC50
mmol/L were only marginally influenced in their viability by elevated ascorbic acid concentrations (Figure ).
In accordance with previous studies [9
], we found a toxic effect of ascorbic acid based on the local production of hydrogen peroxide. Cell lines, e.g. BT-20, 23132/87, SKOV-3, with a natural resistance to the incubation with ascorbic acid, also demonstrated a natural resistance to toxic effects mediated by hydrogen peroxide (Table ). In contrast, cell lines, e.g. U-251 and U-87, susceptible to the incubation with ascorbic acid, were also more susceptible to the incubation with hydrogen peroxide. In addition, ascorbic acid resistant cancer cell lines are more able to protect themselves with increased catalase enzymatic activity, in contrast to ascorbic acid susceptible cancer cell lines (Figure ). Catalase-silencing sensitizes BT-20 breast carcinoma cells to ascorbic acid mediated cell death. In addition to catalase, enzymes of the peroxidase family, e.g. glutathione peroxidase, are also important for cell protection. In the present study, expression of glutathione peroxidase was also proofed for all tested cancer cell lines, but the level of protein and enzymatic activity did not strongly correlate with the resistance of cancer cell lines to the ascorbic acid-mediated cytotoxic effect (not shown). The catalase knock-down in BT-20-KD-CAT cells did not influence glutathione peroxidase activity (Additional file 1
: Figure S1), suggesting that glutathione peroxidase may not play a major role in protecting cancer cells against cytotoxic hydrogen peroxide.
Ascorbic acid is able to act as a strong electron donator by reducing iron ions (Fe3+
). These ions may exist alone or bound on matrix metal proteins [12
]. Other metal ions like Cu2+
can also be used as an electron carrier. These ions can be oxidized and donate their electrons on oxygen by generating a superoxide anion (O2-
). Superoxide dismutase catalyses the reaction of O2-
to hydrogen peroxide that can induce apoptosis in different ways: blocking the activity of a plasma membrane Na+
exchange system leading to reduced cytosolic pH values or attacking DNA, usually by its conversion into DNA-damaging hydroxyl ion (OH·
]. In the present study we found that extracellular catalase prevented the cell toxic effect of ascorbic acid and supported cell viability of ascorbic acid susceptible cancer cell lines (Figure ). Catalase catabolizes hydrogen peroxide to water and oxygen and helps to protect aerobic organisms against excessive hydrogen peroxide production. The cytotoxic effect of extracellular ascorbic acid is finally mediated by the development of extracellular hydrogen peroxide which is membrane permeable [18
]. In addition, it is well known that ascorbic acid enters directly into the cell with sodium-dependent vitamin C transporter (SVCT1 and SVCT2) and in its oxidized form dehydro-ascorbic acid can be internalized by hexose transporters GLUT 1, GLUT 3, and GLUT 4 [19
]. Both ascorbic acid and its oxidized form are in extra- and intracellular balance, depending on their pH-value. The extracellular amount of ascorbic acid was identified as the more important one, because ascorbic acid has toxic effects on cells even if there is only little expression of those transporters [9
It seems that many cancers demonstrate substantially lower catalase activity than normal tissues, allowing cancers to generate a moderate intracellular level of oxidative stress to aid their proliferation and survival [15
]. It is known that expression of catalase is regulated at message, protein and activity levels [22
]. We could show that the tumour cell lines used in the present study are different in their catalase activity. Szatrowski described that rapidly proliferating cells such as cancer cells generate abnormally high hydrogen peroxide levels. This and other factors increased oxidative stress during neoplastic transformation and may promote the selection of cells with modified (increased or decreased) catalase activity. The modified catalase expression in cancer cells remains puzzling but it seems that prolonged exposure to reactive oxygen species (ROS) downregulates catalase expression via hypermethylation of the catalase promoter and, in addition, transcription factors seem to be involved [23
]. Catalase is also down-regulated in healthy cells transformed with T-antigen of SV40 or Ras, although the underlying mechanisms of this down-regulation are still unknown [25
]. Interestingly, it also has been observed that catalase levels are modified in cancer cell lines resistant to some chemotherapeutic agents or hydrogen peroxide [26
]. In summary, catalase expression is regulated in a wide array of cellular processes.
The use of ascorbic acid in tumour therapy is a matter of some controversy [28
]. Nevertheless, ascorbic acid is used in tumour therapy, especially when evidence based medicine or supportive therapy fail [32
]. Many conventional and novel anti-cancer drugs have been reevaluated for their association with ROS production. For instance, doxorubicin is a redoxcycling anthracycline that generates ROS. Biologics can also induce apoptosis through the generation of ROS. Rituximab, an anti-CD20 monoclonal antibody approved for the treatment of non-Hodgkin’s lymphoma, induces a rapid and intense production of ROS in human lymphoma cells [34
]. Another aspect of ROS is that they are able to provoke uncontrolled cell growth by overstimulation of MAP Kinases signal transduction pathways [35
]. Furthermore, ROS can activate hypoxia induced factor 1 (HIF-1) that stimulates the cells to gain energy from glucose under hypoxic conditions. HIF-1 increases the expression of glycolysis enzymes and additionally stimulates the development of new blood vessels (neovascularisation) by increasing the expression of angiogenic factors (e.g. VEGF) to enhance oxygen supply [39
]. Increased levels of ROS, however, damage cell structure and function [40
On the basis of our data, we were able to show a correlation between catalase activity and resistance of cancer cell lines to the ascorbic acid induced cytotoxic effect. Moreover, catalase is significant for cell protection against hydrogen peroxide. The ascorbic acid resistant cell line BT-20 became more susceptible to ascorbic acid after sh-RNA mediated catalase knock-down and the rate of apoptosis increased in these cells.