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. 2014 Feb;18(2):305-13.
doi: 10.1111/jcmm.12188. Epub 2013 Nov 25.

Vitamin C suppresses cell death in MCF-7 human breast cancer cells induced by tamoxifen

Tamilselvan Subramani  1 , Swee Keong Yeap, Wan Yang Ho, Chai Ling Ho, Abdul Rahman Omar, Suraini Abdul Aziz, Nik Mohd Afizan Nik Abd Rahman, Noorjahan Banu Alitheen
Affiliations

Vitamin C suppresses cell death in MCF-7 human breast cancer cells induced by tamoxifen

Tamilselvan Subramani et al. J Cell Mol Med. 2014 Feb.

Abstract

Vitamin C is generally thought to enhance immunity and is widely taken as a supplement especially during cancer treatment. Tamoxifen (TAM) has both cytostatic and cytotoxic properties for breast cancer. TAM engaged mitochondrial oestrogen receptor beta in MCF-7 cells and induces apoptosis by activation of pro-caspase-8 followed by downstream events, including an increase in reactive oxygen species and the release of pro-apoptotic factors from the mitochondria. In addition to that, TAM binds with high affinity to the microsomal anti-oestrogen-binding site and inhibits cholesterol esterification at therapeutic doses. This study aimed to investigate the role of vitamin C in TAM-mediated apoptosis. Cells were loaded with vitamin C by exposure to dehydroascorbic acid, thereby circumventing in vitro artefacts associated with the poor transport and pro-oxidant effects of ascorbic acid. Pre-treatment with vitamin C caused a dose-dependent attenuation of cytotoxicity, as measured by acridine-orange/propidium iodide (AO/PI) and Annexin V assay after treatment with TAM. Vitamin C dose-dependently protected cancer cells against lipid peroxidation caused by TAM treatment. By real-time PCR analysis, an impressive increase in FasL and tumour necrosis factor-α (TNF-α) mRNA was detected after TAM treatment. In addition, a decrease in mitochondrial transmembrane potential was observed. These results support the hypothesis that vitamin C supplementation during cancer treatment may detrimentally affect therapeutic response.

Keywords: ROS; apoptosis; breast cancer; free radical; lipid peroxidation; tamoxifen; vitamin C.

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Figures

Fig 1
Fig 1
(A) Accumulation of vitamin C in MCF-7 cells after exposure to 50 μM and 500 μM dehydroxy ascorbic acid (DHA). (B) Time-and dose-dependent uptake after exposure to DHA. Results represent means ± SD of at least six independent experiments.
Fig 2
Fig 2
Vitamin C attenuates the cytotoxicity of tamoxifen (TAM) in MCF-7 cells. (A) MCF-7 cells were loaded with dehydroxy ascorbic acid (DHA) to varying concentration of intracellular vitamin C. The cells were washed and treated with TAM for 24 hrs. Morphological assessment of MCF-7 cells stained with AO/PI. The population of viable, apoptotic and necrotic cells was plotted after treatment. (B) MCF-7 cells loaded with various concentration of vitamin C and apoptosis were measured by annexin V/PI staining. Three separate experiments were conducted in triplicate and the results shown represent the mean values and SDs of single representative experiment.
Fig 3
Fig 3
Effect of tamoxifen (TAM) on FasL and TNF-α mRNA expression. The highest amount of FasL and TNF-α mRNA expression were detected after 24 hrs. The influence of different concentration of vitamin C on FasL and TNF-α mRNA expression was analysed. Results represent means ± SD of at least five independent experiments. **P < 0.001 versus control; #P < 0.01 versus TAM.
Fig 4
Fig 4
Effect of vitamin C on (A) lipid peroxidation (B) synthesis of superoxide dismutase (SOD). Both the processes were significantly modulated by vitamin C. Values were assessed 24 hrs after tamoxifen treatment. Results represent means ± SD of at least six independent experiments. **P < 0.05.
Fig 5
Fig 5
Effect of vitamin C on mitochondrial transmembrane potential was assessed after tamoxifen (TAM) treatment. The Δψm levels of cells, exposed as units of mean fluorescence intensity, were calculated as percentage of control. Mean ± SD of at least six independent experiments. **P < 0.01 versus TAM.
Fig 6
Fig 6
Effect of vitamin C on caspase-7,-8. MCF-7 cells were loaded with various concentrations of vitamin C prior to treatment of tamoxifen (TAM) were lysed and the activity of caspase-7,-8 in the cell lysate were determined. The data represented the mean ± SD of three identical experiments made in the five replicates. **P < 0.01 versus TAM.
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