Effect of High-dose Vitamin C Combined With Anti-cancer Treatment on Breast Cancer Cells

Materials and Methods

Cell lines. The breast epithelial cell line MCF10A and breast cancer cell lines MDA-MB-231, MCF-7, and SK-BR-3 were purchased from the American Type Culture Collection (ATCC). MCF10A was maintained in a Dulbecco’s Modified Eagle’s Media (DMEM)/F-12 (1:1) medium (Lonza, Walkersville, MD, USA) supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA), 10 ng/ml epidermal growth factor, 0.5 μg/ml hydrocortisone, 100 ng/ml cholera toxin, and 10 μg/ml insulin. MDA-MB-231, MCF-7 and SK-BR-3 were all grown in DMEM (Gibco) supplemented with 10% FBS. The tamoxifen-resistant MCF-7 cells (TRAM-R) and long-term estrogen-deprived MCF-7 cells (LTED) were provided by Dr. Richard J. Santen (University of Virginia, USA). The TRAM-R cells were continuously maintained in DMEM containing 10% FBS and 10-7mol/l tamoxifen, while the LTED cells were maintained in phenol-red-free DMEM containing 5% charcoal-dextran-stripped FBS.

Materials. Tamoxifen and Faslodex (ICI 182,780) were purchased from Sigma-Aldrich (St. Louis, MI, USA) and Abcam plc (Cambridge CB4 0FL, UK), respectively. Eribulin mesylate (Halaven®, Eisai Inc., Tokyo, Japan), ascorbic acid (Bio Chemical R&D, Gyeounggi-do, Korea), trastuzumab (Herceptin®, Roche Diagnostics, Basel, Switzerland), paclitaxel (Genexol®, Samyang Biopharm, Daejeon, Korea), and doxorubicin hydrochloride (A.D.mycin®, Boryung co., Gyeonggi-do, Korea) were all kindly provided.

Cell apoptosis assay. The cells were seeded in 48-well plates at a density of 2×104 cells per well. After culture for 24 h, cells were washed with PBS, medium was changed and cells were exposed to vitamin C for 4 h. Next, plates were centrifuged, the medium was removed and apoptotic cell death was determined using Cell Death Detection ELISAPLUS kit (Roche, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, after lysis and centrifugation, cell lysates were incubated with biotin-labelled anti-histone and with peroxidase-conjugated antibodies to DNA in a streptavidin-coated microtiter plate for 2 h at room temperature. After incubation, peroxidase substrate was added, the plate was incubated at room temperature for about 15 min and the peroxidase activity was determined by ELISA reader at a detection wavelength of 405 nm and 490 nm.

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Detection of catalase activity. It has been suggested that the anti-cancer effect of vitamin C is due to the accumulation of H2O2 due to the insufficiency of catalase in cancer cells (14). Therefore, the catalase activity in each breast cancer cell line was measured using a Catalase Assay Kit (Cayman, MI, USA) following the manufacturer’s instructions and compared with the catalase activity in the normal breast epithelial cell line (MCF10A). The catalase activity was calculated by normalization with the total protein amount in the lysates. The protein concentration was estimated using a Bradford Protein Assay Kit (Bio-Rad, CA, USA). The experiment was repeated three times and the data presented are the means±S.D.

Cell viability assay. The high-dose vitamin C-induced cell death in the breast cell lines was quantified using a cell viability assay. The cells were plated in 48-well plates at a cell density of 2×104 per well. After culturing overnight, the cells were exposed to vitamin C for 2 h, washed with phosphate buffered saline (PBS), and cultured for an additional 24 h in a growth medium with ascorbic acid.

To explore the combined effect of anti-cancer agents and vitamin C, the cells were plated 2×104 per well in 48-well tissue culture plates and allowed to attach overnight. The cells were then treated with an anti-cancer agent for 24 h, treated with vitamin C for 2 h, washed and cultured for additional 24 h, or treated with vitamin C for 2 h on consecutive days, washed and cultured for additional 24 h after each exposure.

Cell proliferation was estimated using an MTT assay. Briefly, 200 μl of MTT solution (2 mg/ml) were added to cells for 4 h. After removing the MTT solution, 400 μl of dimethyl sulfoxide (DMSO) were added to cells. The optical density was then measured at 540 nm using an absorbance reader (BioTek Instruments Inc., Winooski, VT, USA).

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Statistical analysis. Data was expressed as the mean±standard deviation (SD) of three or more independent experiments. Differences between two groups were evaluated using the Student’s t test. Results were considered significant at p<0.05.

References

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About the Author: Tung Chi