|Ahead of print publication
Anticancer potential of ascorbic acid and inorganic selenium on human breast cancer cell line MCF-7 and colon carcinoma HCT-116
Magdah A Ganash
Department of Biology, Faculty of Science, King Abdulaziz University, Jeddah, Saudi Arabia
Magdah A Ganash,
Department of Biology, Faculty of Science, King Abdulaziz University, Jeddah
Source of Support: None, Conflict of Interest: None
Background and Objectives: Till now, cancer is a major health problem and one of the main causes of mortality worldwide. Ascorbic acid and selenium are the two most popular dietary supplements used to prevent cancer proliferative, therefore, the work aims to study the antitumor effect of ascorbic acid and selenium on HCT116 and MCF7 cell lines.
Materials and Methods: In the present study, the cytotoxic effect of different concentrations of ascorbic acid and selenium on human breast cancer cell line (MCF7 cells) and human colon carcinoma (HCT116) was studied.
]Results: Viability % of HCT116 cell line and MCF7 cell line decreased with increasing ascorbic acid concentrations (1–4 mM). The 50% inhibitory concentration (IC50) of five dilutions of each concentration of ascorbic acid was evaluated in the current study. IC50 was 0.18, 0.17, 0.16, and 0.16 mM for HCT116 cell line and was 0.86, 1.34, 1.74, and 0.47 mM for MCF7 cell line at 1, 2, 3, and 4 mM, respectively. Cell viability decreased depending on the selenium concentrations ranging from 20 to 100 mM. Selenium effect showed less cytotoxicity on MCF7 compared to HCT116 cells at all tested concentrations where the cell viability at 20, 40, 60, 80, and 100 mM selenium was 33.74, 29.48, 26.08, 54.53, and 20.89 for HCT116 cell and was 79.53, 76.01, 59.42, 54.53, and 51.98 for MCF7 cell, respectively. Ascorbic acid induced apoptosis by promoting the release of lactate dehydrogenase (LDH) in HCT116 and MCF7 cells, but reduced release of LDH was observed in selenium treatment but increased when it added to ascorbic acid because of a possible synergistic action that may produce an enhanced anticarcinogenic effect.
Conclusion: The present study documented that a combination of ascorbic acid and selenium produces an additive chemopreventive effect on carcinogenesis.
Keywords: Ascorbic acid, colon cancer cell line, human breast cancer, selenium
| > Introduction|| |
Cancer is characterized as a complex disease that includes the change of gene expression, sustains cell survival and proliferation, and can be modified by genomic and epigenomic factors. Cancer disease is a major health problem and considered one of the main causes of mortality around the world. After lung cancer, colon cancer is the second most prevalent cause of cancer death in men and women. Further, among cancers, breast cancer is known as the most common cancer and the second leading cause of death from cancer in women worldwide.,, A high level of oxidative stress is very suitable conditions for cancer cell operation due to oncogenic transformation, high baseline levels of reactive oxygen species (ROS), and metabolic reprogramming. In 2012, according to the Brazilian National Cancer Institute (INCA, in the Portuguese acronym), there were 14.1 million women and men suffering from cancer disease in the world, with a total of 8.2 million deaths from the disease.
Selenium (Se) as a trace element is essential and unique micronutrient for humans and animals health, and it has been shown to play an important role as an anticancer agent in animal and clinical applications. The mechanism for the toxic effects of Se has been proposed to occur attributable to its pro-oxidant ability to accelerate the oxidation of ROS, which can disrupt components of cells through lipid peroxidation. Numerous studies, reported that Se is an important dietary element for animals as well as humans, and there is increasing evidence for the efficacy of certain forms of Se as cancer-chemopreventive compounds. According to the reports of Zeng and Combs, selenium appears to have a defensive effect at various stages of carcinogenesis involving all stages (early and later) of cancer progression. Se deficiency has been reported to be positively correlated with tumor occurrence., Selenium inhibited the proliferation and invasion of tumor cells in tumor models; however, the concrete mechanism remains controversial.
Impacts of Se compounds as chemopreventive agents in vivo are correlated with their abilities to effect the regulation of the cell cycle, to induce apoptosis, and to inhibit tumor cell migration and invasion in vitro. Brozmanová et al. explained the broad interest to exploit the positive effects of Se on human health and cancer therapy and had studied the negative effects such as toxicity and DNA damage induction, resulting from high Se intake. Selenium exerts its antioxidant effects through selenoproteins, including glutathione peroxidase and thioredoxin reductases., Daily supplementation with 200 μg of Se has been reported to minimize the recurrence of different types of cancers.
It is necessary for the normal functioning of the immune system; tumors can escape and relapse under immune pressure by recruiting immunosuppressive cells into the tumor microenvironment. Henson et al. stated that Vitamin C (L,3-ketothreohexuronic acid lactone) is a water-soluble vitamin with antioxidant properties. An active reducing agent included in various biological effects. Reports by Cameron and Pauling have suggested that high dose of Vitamin C has anticancer effects. The suppressing effect of ascorbic acid toward a number of mutagens/carcinogens was shown by numerous studies in humans and animals.,, According to Takahashi et al., high-dose Vitamin C induces pro-oxidant effects and is more cytotoxic to cancer than it is to normal cells. Authors report that in vitro studies of neuroblastoma, pancreatic cancer, bladder cancer, and other tumor types showed the cytotoxic effect of Vitamin C, while in vivo studies supported this anticancer effects of the Vitamin C. Vitamin C at a dose of 10 mM induced apoptosis in neuroblastoma and melanoma cells and was shown to be an important modulator for the growth of mouse myeloma cells in an in vitro colony assay. In addition, the previous studies showed that Vitamin C treatment-induced cytotoxicity by depletion of adenosine triphosphate in certain cancer cells., Therefore, Vitamin C-induced H2O2 may alter intracellular metabolism in cancer cells by disrupting the redox balance. Knowledge of ascorbic acid in biology and treatment of cancer has increased at a rapid rate, especially during last two decades. However, the possible health benefits of the combined administration of dietary ascorbic acid and selenium have not been studied extensively. Therefore, the work aims to study the antitumor effect of ascorbic acid, selenium, and their combination on HCT-116 cell line and MCF-7 cell line.
| > Materials and Methods|| |
Sodium selenite (Na2 SeO3) and ascorbic acid were purchased from Sigma-Aldrich. Stock solutions of chemicals were prepared in distilled and sterilized water; the filtrate was sterilized and stored at 4°C till use. Dimethyl sulfoxide (DMSO), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) and trypan blue dye were purchased from Sigma (St. Louis, Mo., USA). Fetal bovine serum, DMEM, RPMI-1640, HEPES buffer solution, L-glutamine, gentamycin, and 0.25% trypsin-ethylenediaminetetraacetic acid were purchased from Lonza (Belgium).
Mammalian cell lines
Human breast cancer cell (MCF-7 cells) and human colon carcinoma (HCT-116) were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA).
Cell line propagation
The cells were grown on RPMI-1640 medium amended with 10% inactivated fetal calf serum and 50 μg/ml gentamycin. The cells were maintained at 37°C in a humidified atmosphere with 5% CO2 and were subcultured two to three times a week.
Cytotoxicity evaluation using viability assay
For antitumor assays, the tumor cell lines were suspended in the medium at concentration 5 × 104 cell/well in Corning® 96-well tissue culture plates and then incubated for 24 h. The tested compounds (Na2 SeO3 and ascorbic acid) were then added into 96-well plates (three replicates) to achieve six dilutions for each compound. Six vehicle controls with media or 0.5% DMSO were run for each 96-well plate as a control. After incubating for 24 h, the numbers of viable cells were determined by the MTT test. Briefly, the media were removed from the 96-well plate and replaced with 100 μl of fresh culture RPMI 1640 medium without phenol red; then, 10 μl of the 12 mM MTT stock solution (5 mg of MTT in 1 mL of phosphate buffered saline) was added to each well including the untreated controls. The 96-well plates were then incubated at 37°C and 5% CO2 for 4 h. An 85 μl aliquot of the media was removed from the wells, and 50 μl of DMSO was added to each well and mixed thoroughly with the pipette and incubated at 37°C for 10 min. Then, the absorbance was measured in triplicates at 490 nm using ELISA Reader System (SunRise TECAN, Inc., USA) to determine the number of viable cells and the % of viability was calculated by the following equation: (1 − [ODt/ODc]) ×100%, where ODt is the mean optical density of wells treated with the tested sample and ODc is the mean optical density of untreated cells. The relation between surviving cells and treatments concentration is plotted to get the survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory concentration (IC50), the concentration required to cause toxic effects in 50% of intact cells, was estimated from graphic plots of the dose–response curve for each concentration using GraphPad Prism software (San Diego, CA, USA).
Lactate dehydrogenase release assay
According to Nakagawa et al., the apoptotic effect of ascorbic acid and was examined by measurement of lactate dehydrogenase (LDH) release where 1.5 × 105 cells per well were plated in 24-well cell culture plate and then incubated for overnight. The collected supernatant added to a black 96-well culture plate (200 μL per well) after cells was exposed to different concentrations of ascorbic acid and selenium for 48 h. LDH cytotoxicity assay kit as per the manufacturer's instructions was used to measure LDH release. Absorbance at a wavelength of 450 nm in each well was detected using an enzyme-linked immunoassay instrument.
| > Results and Discussion|| |
The results showed varying sensitivities of HCT-116 cell line and MCF-7 cell line to different concentrations of Vitamin C [Table 1] and [Figure 1]. This phenomenon may be regulated by multiple factors, such as redox system, transporter expression, and hypoxia condition. MCF-7 cell line was more resistance to ascorbic acid toxicity than HCT-116 cell line. Viability % of HCT-116 cell line and MCF-7 cell line decreased with increasing ascorbic acid concentrations 1–4 mM [Table 1]. According to Chen et al., the high-dose [Table 1] of Vitamin C has significant anticancer effects in vitro and in vivo. In contrast, recently, Unlu et al. reviewed that there is no clear evidence that high dose of Vitamin C (10 g/infusion) is beneficial in the treatment of cancer. Uetaki et al. reported that the metabolomic profiles of MCF7 human breast adenocarcinoma and HCT29 human colon cancer cells were dramatically altered after exposure to cytotoxic concentrations of ascorbic acid. Cytotoxicity and IC50 of five dilutions of each concentration of ascorbic acid were evaluated in the current study. IC50 was 0.18, 0.17, 0.16, and 0.16 mM for HCT-116 cell line and was 0.86, 1.34, 1.74, and 0.47 mM for MCF-7 cell line at 1, 2, 3, and 4 mM, respectively. Ascorbic acid represses the proliferation of tumors through numerous mechanisms as reported previously, where Tsao and Miyashita stated that large amounts of ascorbic acid intake can change the levels of certain amino acids in body fluids and may deplete the bioavailability of lysine, glutamine, and cysteine, two amino acids which are required for rapidly growing tumors. Furthermore, Uetaki et al. suggest that ascorbic acid inhibited energy metabolism through NAD depletion, thereby inducing cancer cell death.
|Table 1: Cytotoxicity of different concentrations of ascorbic acid against HCT-116 cell line and MCF-7 cell line|
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|Figure 1: Cytotoxicity of different concentrations of ascorbic acid against HCT-116 cell line and MCF-7 cell line|
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In this study, the effect of Se on MCF7and HCT-116 cells was evaluated, where the cell viability decreased depending on the selenium concentrations ranging from 20 to 100 mM [Table 2] and [Figure 2]. Numerous researchers have shown that minerals may play important roles in inhibiting the growth of cancer cells. Among these elements is selenium., Furthermore, studies on colon cancer have demonstrated that arsenic trioxide and selenium are cytotoxic in human colon cancer cell lines., Selenium effect showed less cytotoxicity on MCF7 compared to HCT-116 cells at all tested concentration where the cell viability at 20, 40, 60, 80, and 100 mM selenium was 33.74, 29.48, 26.08, 54.53, and 20.89 for HCT-116 cell and 79.53, 76.01, 59.42, 54.53, and 51.98 for MCF7 cell, respectively [Table 2]. Anticancer mechanisms of selenium were reported in numerous studies, where Whanger found that selenium compounds inhibit the development of cancer cells by reducing cell. Previous studies have also reported that selenium acts as a protective factor against cancer correlates with their abilities to affect the regulation or to control the cell cycle, to stimulate or increase apoptosis, and to inhibit tumor cell migration and invasion in vitro. Although the anticancer activity of selenium is not completely known, it has been suggested that various mechanisms, such as antioxidant protection by seleno-enzymes, inhibit the development of tumor cells, regulate and modulate the cell cycle, inhibit apoptosis, and repair DNA. Recent studies have indicated that selenium not only has the potential to prevent cancer but could also be used to treat cancer and enhance the effectiveness of cancer therapy in combination with other anticancer drugs or radiation. In the current study, although the positive impact of Se on human breast cancer cell line and human colon carcinoma was obtained, reports from other studies suggested that Se supplementation exhibited beneficial effects on lung, bladder, colorectal, esophageal, and gastric cardia.,, Some mechanisms of selenite's anticancer effect were reported earlier and include activation of natural killer cells and apoptosis., In addition, it is hypothesized that selenite can oxidize polythiols associated with cancer membrane-bound proteins which appear under the reducing conditions of hypoxic tumor tissue.
|Table 2: Cytotoxicity of different concentrations of selenium against HCT-116 cell line and MCF-7 cell line|
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|Figure 2: Cytotoxicity of different concentrations of selenium against HCT-116 cell line and MCF-7 cell line|
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Although the antitumor roles of Se and ascorbic acid alone have been recognized for many years, little is known about the antitumor activity of combined treatment of Se and ascorbic acid. The combination of ascorbic acid with Se elucidates changes in viability of HCT-116 cell line and MCF-7 cell line compared with using these treatments alone. Numerous studies have suggested that antioxidants inhibit tumor development.,,, Therefore, the current study investigated whether ascorbic acid and selenium could inhibit tumor growth. In this study, the obtained data indicate that HCT-116 cell line and MCF-7 cell line viability are altered after their treatment by ascorbic acid and Se [Table 3] and [Figure 3], where the percentage of cell viability was 21.25%, 18.37%, 16.82%, and 12.54% for HCT-116 cell line and was 39.84%, 23.52%, 21.44%, and 18.68% for MCF-7 cell line at 1, 2, 3, and 4 mM of ascorbic acid supplemented with 20 mM selenium, respectively [Table 3], compared with treatment with ascorbic acid alone [Table 1] or with Se alone at 20 mM [Table 2]. This result is strongly supported by Novotny and Milner, who concluded that ascorbic acid (50 or 125 μmol/L) accentuated the in vitro growth inhibition of canine mammary tumor cell line 13 induced by Na2 SeO3 (12.6 μmol/L). Previous studies have also demonstrated the synergistic effects of several antioxidants such as ascorbic acid and selenium., Furthermore, Thompson et al. found that the combination of ascorbic acid and Se increased the tumor incidence (83%) even higher than in controls (64%).
|Table 3: Cytotoxicity of different concentrations of ascorbic acid with 20 (mM) selenium against HCT-116 cell line and MCF-7 cell line|
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|Figure 3: Cytotoxicity of different concentrations of ascorbic acid with 20mM selenium against HCT-116 cell line and MCF-7 cell line|
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Increase the release of LDH is an indicator for cell apoptosis triggered by anticancer compounds. In this study, ascorbic acid induced apoptosis by promoting the release of LDH [Figure 4]. One recent study by Fernandes et al. reported that at higher concentrations, ascorbic acid induced apoptosis in bone cancer cells. Sanmartín et al. demonstrated that induction of apoptosis is considered an important cellular event that can account for the cancer preventive effects of Se. Although reduced release of LDH was observed in selenium treatment [Figure 5] but increased when it added to ascorbic acid [Figure 4], because of a possible synergistic action that may produce an enhanced anticarcinogenic effect. These results clearly indicated that ascorbic acid particularly when added to selenium induced apoptosis in HCT-116 and MCF-7 cells.
|Figure 4: Lactate dehydrogenase activity of HCT-116 cell line and MCF-7 cell line treated with ascorbic acid combined with selenium|
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|Figure 5: Lactate dehydrogenase activity of HCT-116 cell line and MCF-7 cell line treated with selenium|
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| > Conclusion|| |
Overall, the present study shows the antiproliferative effects of ascorbic acid and selenium, especially on HCT-116 cell line. Therefore, these results suggest that ascorbic acid and selenium may indeed have applications as a potential anticancer therapeutic agent. The present study also documented that a combination of ascorbic acid and selenium produces an additive chemopreventive effect on carcinogenesis. However, further investigation is required to further elucidate the molecular mechanisms underpinning ascorbic acid and selenium utility in the regulation of cancers.
I would like to acknowledge King Abdulaziz University.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2], [Table 3]