ASSESSING THE SURVIVAL OF MRC-5 AND A549 CELL LINES UPON EXPOSURE TO ASCORBIC ACID AND SODIUM ASCORBATE Ibrahim O. Farah, Veshell L. Lewis, Wellington K. Ayensu and Ousman Mahmud Department of Biology, Jackson State University, Jackson, MS 39217, USA ABSTRACT Lung cancer is among the highly prevalent and deadly cancers in the United States and worldwide. Cells that are cancerous exhibit higher rates of glycolysis as compared to normal cells. In an attempt to exploit this uniquely enhanced glucose-dependent ATP generation phenomenon, the authors hypothesize that exposure of cancer cells to normal organic metabolites that are capable of inhibiting glycolysis would have a negative impact on survival by altering growth and viability characteristics vastly through decline in ATP build up essentially leading to collapse in energy supply; normal lung cells will not demonstrate such changes. The human lung fibroblast cell line MRC-5 and the cancerous human lung alveolar epithelial cell line A549 were utilized in this study as in vitro models of normal and cancerous lung cell lines respectively. Using standard methods, both cell lines were maintained in culture and exposed to ascorbic acid and sodium ascorbate reagents at concentration levels ranging from 31.3-2,000 µg/ml. Cell survival measurements using MTT andt4 Cellometric assays monitored with phasecontrast photo-imaging were carried out in quadruplicates. Results indicate that exposure characteristics to these metabolites followed concentration-dependent cell mortality/survival curves by the cancerous versus normal cell lines respectively. Ascorbic acid and sodium ascorbate showed statistically significant (p<0.05) differential negative effects on the cancerous A549 cell line in comparison to unexposed controls as well as to effects measured with the normal lung MRC-5 cell line; this is highly indicative of a promising therapeutic potential. Keywords: Warburg effect, glycolysis inhibition, lung cancer, natural organics, T4 cellometer, bioenergetics, biotherapeutics INTRODUCTION Cancerous cells exhibit higher glycolytic rates than normal cells. This has partly been attributed to injury to mitochondrial respiratory mechanisms [1, 2, and 3] that result in an increased ATP generation through the glycolytic pathway. Increased ATP synthesis via the glycolytic pathway in any cell would normally run into deficiency in coenzymes (NAD+) necessary to keep pace with the reduced environment in the cytosol. Mitochondrial respiration can be negatively affected by mutations within the mitochondrial DNA (mtdna) that are known to occur in various malignancies including those of the breast, prostate, and pancreatic cancers [5, 6, 7, and 8]. In attempting to exploit this unique phenomenon of enhanced glycolytic-dependent ATP generation, we envisaged a situation in which exposure of cancerous cells to organic metabolites as possible inhibitors of this abnormal glycolysis, would lead to overuse of these substrates by cancerous cells for faster energy supplies; the process was visualized as leading to ATP depletion and cancer cell demise [9, 10, 11 and 12]. We therefore hypothesized that normal lung cells are capable of escaping the cytotoxic effect due to their slow normal glycolysis and aerobic ATP generation, while cancerous cells will be killed off at concentrations that are non injurious to normal cells: this can afford computation of therapeutic indexes to eliminate the abnormal cancerous cells. Thus delivery of normal organic metabolites presented at optimized dose can afford a means of selectively inducing cancer cells demise while normal cells will survive the onslaught [13, 14]. Using standard procedure, both cell lines were maintained in culture and exposed at concentrations ranging from 31.3-2,000 µg/ml. We studied the effects of two normal organic metabolites (ascorbic acid and sodium ascorbate) on the survival of human cancerous lung alveolar epithelial cell line (A549) and normal human lung fibroblast cell line (MRC-5) as in vitro model cell lines.
METHODS Reagents: F-12K, Dulbecco s Modified Eagle s (DMEM) culture media, Phosphate- buffered saline (PBS), Trypsin EDTA, Fetal bovine serum (FBS), Penicillin-Streptomycin, A549 human lung cancer cell line, and MRC-5 human normal lung cells line were each purchased from American Type Culture Collection (ATCC) located in Manassas, VA, USA. The analytical grade chemical reagents, ascorbic acid and sodium ascorbate were purchased from Sigma-Aldrich, Inc in St. Louis, MO, USA. Cell Culture: A549, human lung cancer cell line, was purchased from ATCC and were cultured in F- 12k media. The F-12k medium was a mixture of 85% F-12k media, 10% FBS, and 5% penicillin. Cell culture was started with a mixture of 4ml of the F-12k media and 1 ml of A-549 cells in a 5ml flask (Corning Company). MRC-5, a normal human lung cell line, was purchased from ATCC and was cultured in DMEM media composed of a mixture of 85% DMEM media, 10% FBS, and 5% penicillin. The cells were maintained at 37 o C and 5% CO 2 in a humidified incubator. Once each cell line became confluent cells were trypsinized (Trypsin EDTA) and counted by the use of a hemocytometer. From the cell count appropriate dilution were made for specific plating necessary to deliver constant number of cells for exposure studies (C 1 V 1 =C 2 V 2 ). MTT assay: The purpose of the MTT assay was to measure cellular metabolic viability. The yellow tetrazolium MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5- diphenyltetrazolium bromide) is reduced by metabolically active cells to generate the reduced agent. The purple formazan that results within the cell was solubilized in DMSO and measured by spectrophotometric methods (Thermo Scientific, Multiskan Ascent from Waltham, MA, USA). After incubation for 72 hours in the 96 wells plate, the cells were examined by using a phase-contrast microscope for cell morphology and growth/damage parameters. This was followed by the MTT assay. Briefly, the chemical mixture was removed from the 96 well plates and 10 µl of MTT reagent were added to each well. The 96 well plates were incubated at 37 o C and 5% CO 2 for 2 to 4 hours until the purple precipitate was visible. Hundred (100) µl of detergent reagent (DMSO) were added to each of the 96 well plate and set for 10 minutes before proceeding to the next step where the 96 well plates were positioned into the spectrophotometer and the absorbance was recorded at 570 nm. Several concentrations of sodium ascorbate and ascorbic acid were evaluated ranging from 0 (control) to 2000 µg/ml. Tests were done in quadruplets and experiments were repeated three times (12 data points per concentration). The T4 Cell Analyzer: Cell Analysis by High Content Cell Analyzer (HCCA); Nexcelom, Bioscience in Lawrence, MA, USA: Once cell growth became maximal at confluence cells were exposed to each organic chemical mixture at 2,000 µg/ml concentration for 48 hours in an incubator at 37 o C and 5% CO 2. After 48 hours incubation period, cells were typsinized, centrifuged, and resuspended in fresh media of 2 ml volumes for counting. Lastly, 20µl of the cell-containing media and 20 µl of trypan blue were mixed and injected into a slide and read by the high content cell analyze, which will automatically calculate the % of viable cells in 1 ml of sample. Chemically exposed cells were compared to control cells in at least duplicate samples/chemical exposure for and in between cell lines. Statistical Analysis: The viability parameters of both the A549 and MRC-5 were determined by using one way ANOVA (SAS Software) and Student s T-test (EXCEL; at the level of 0.05 or <0.05 for statistical significance), for treated A549 compared to MRC-5 cell line and their respective controls. Data was also normalized through the use of percentages and tested for statistical significance based on comparisons of differences in the computed means, standard deviations/errors, and p-values. RESULTS Sodium Ascorbate: This study examined the in-vitro responses of the A549 and MRC-5 cell lines upon their exposure to sodium ascorbate. Results of the MTT assay are presented in fig. I A and I B and table
1 and 2. Sodium ascorbate boosted the metabolic activity of both the A549 and MRC-5 cell lines (fig. I and II). Responses for MRC-5 and A549 cell lines showed differential statistical differences excluding when the treated A549 cell line was compared to its control (tables 1 and 2). Figures IIA and IIB show the phase-contrast photo-display of the effects of sodium ascorbate on the A549 cell line compared to its control. The cell morphology and confluency for the exposed A549 are different; cells are smaller and their numbers were lower compared to the control cell display. The results of the T4 cell analysis showed statistically significant differences between the MRC- 5 and the A549 cell lines. Viability of the MRC-5 was at 100% for the control and 86.60 for treated cells. Viability of the A549 showed 100% versus 77.48% for the control and treated cells respectively. When both treated cell lines were compared for viability, there was no statistically significant difference between the two contrasting the findings with the MTT assay as compared to the differential response for the T4 data of these cell lines to sodium ascorbate (table 3). Figure 1A: Response Trends of Sod Ascorbate on the A549 and MRC-5 Cell Lines Figure 1B: Response Dynamics of sod Ascorbate on A549 and MRC-5 Figure IIA: Response Trends of Ascorbic Acid on the A549 and MRC-5 Cell Lines Figure IIB: Response Dynamics of Ascorbic Acid on A549 and MRC-5 Figure IIIA (ascorbate) Figure IIIB Figure IVA (acid) Figure IVB Table 1: ANOVA Mean Comparisons, Statistical Significance, and P-values of A549 cell line* using MTT assay data for 0-2,000 µg/ml of chemical at 48 hrs of incubation Chemical Mean of Control Mean of test p-value Statistical significance (p=0.05) Sodium Ascorbate 0.691 2.551 0.0169 Significant metabolic boost Ascorbic Acid 0.837 1.144 0.1816 Non significant metabolic boost The MRC-5 test group had a highly significant metabolic boost. Table 2: ANOVA Mean Comparisons, Statistical Significance, and -values of MRC-5 cell line using the MTT normalized data. Chemical Mean of A549 Mean of MRC-5 p-value Statistical significance (p=0.05) Sodium Ascorbate 88.96* 244.75 0.0007 Extreme significant metabolic injury to the A549 line Ascorbic Acid 156.36 117.94 0.0554 No statistical metabolic boost for both lines *= metabolic injury.
Table 3: T4 Cellometer per cent (%) Data** and T-test Statistical Analysis of Exposure of A549 and MRC- 5 Cells to Sodium ascorbate and Ascorbic acid Chemical Control Treated p-value Control Treated p-value Treated Treated p-value A549 A549 MRC-5 MRC-5 A549 MRC-5 Sodium 100.00 77.48 0.1182 100.00 86.60 0.3212 77.48 86.60 0.2788 Ascorbate Ascorbic Acid 100.00 61.98 0.0334* 100.00 92.05 0.3369 61.98 92.05 0.0412* **Data represent exposure to 2,000 µg/ml of chemical for 48 hrs. Ascorbic Acid: This investigation analyzed the in-vitro responses of the A549 and MRC-5 cell lines upon their exposure to ascorbic acid. Results of the MTT assay are presented in fig. IIA and IIB, and tables 1 and 2; ascorbic acid was ineffective in reducing the metabolic activity due to the erratic responses recorded for both the A549 and MRC-5 cell lines. Responses for MRC-5 and A549 cell lines showed no statistically significant differences excluding when the treated the MRC-5 cell line was compared to its control (tables 1 and 2). Figures IVA and IVB show results of the phase- contrast photo-display for the effects of ascorbic acid on the A549 cell lines comparing the control to exposed cells. The cell morphology and confluency are different; cells are smaller and their numbers were lower compared to the control cell display. The results of the T4 cell analysis showed statistically significant differences between the MRC- 5 and the A549 cell lines. Viability of the MRC-5 was at 100% for the control and 92.05% for treated cells. Viability of the A549 showed 100% versus 61.98% for the control and treated cell respectively. When both treated cell lines were compared for viability, there was statistically significant difference between the two contrasting the findings with the MTT assay as to the differential response of these cell lines to ascorbic acid (tables 1, 2 and 3). DISCUSSION Lung cancer is one of the most prevalent and deadly cancers in the United States. This research evaluated the metabolic as well as actual viability of the A549 and MRC-5 cell lines upon exposure to ascorbic acid and sodium ascorbate aiming at inhibiting glycolysis in cancer cells. Glycolysis was closely examined in light of research of others that have proven that cancer cells exhibit a higher glycolytic rate than normal cells [1, 2, 3, 4 and 5]. This major influx of glycolysis is caused by deregulated metabolism within the cell with special reference to impaired mitochondria as a hallmark of the cancer phenotype. The root of the deregulated metabolism could in general stem from damage and impairment of genetic stability, as well as the presence of hypoxic environments (6, 7, and 8]. Deregulated metabolism results in uncontrollable cell proliferations that lead to the development of the cancer phenotype. So, during this endeavor, A549 and MRC-5 cell lines were exposed to ascorbic acid and sodium ascorbate for three different times and assessed for the impairment of metabolic viability by the MTT assay, High Content Cell Analyzer (HCCA) for cell death was applied to the A549 and MRC- 5 cell lines as based on related findings to assess the role of these two chemicals on the viability and survival parameters [12 and 13]. Sodium Ascorbate: The results from figures IA and IB of MTT assay showed that sodium ascorbate aided the A549 and MRC-5 in boosting their metabolic viability which was instigated at 31.25 μg/ml; the smallest concentration of sodium ascorbate. Both cell lines maintained a level of metabolic viability that was superior to those of their controls. The A549 cell line boost remained stabilized around a
similar range close to its control; however, the MRC-5 cell line, at 125 μg/ml concentration, has even reached a level that was 3.5 folds of its control but took a sharp steady drop that still remained superior to the control. In table 3, Sodium ascorbate had no statistically significant effect on the A549 cells (p=0.118). As can be seen in figures IIIA and IIIB, the A549 cell line control had substantial cell viability and maintained its normal morphological structure however, when exposed to the highest concentration of sodium ascorbate the cell viability declined along with changes in morphological structure. In table 1, as a result of exposure to sodium ascorbate, the A549 compared to its control showed that its level of difference in metabolic viability was highly statistically significant (p= 0.0169). In table 2, it was determined that the results of comparing the A549 and MRC-5 cell line showed extreme statistical significance in metabolic viability/injury with a disadvantage to the A549 cell line (p=0.0007). In table 3, the HCCA determined that the level of difference in cell viability for the A549 cell line compared to its control was not statistically significant (p=0.1182) and the MRC-5 cell line compared to its control provided a level of difference that was also not statistically significant (p= 0.32120). The cell analyzer also showed that the MRC-5 cell line opposed to the A549 cell line provided a level of difference that was not statistically significant with a p-value of 0.2788. However, there was a clear decline in the viability of the A549 as compared to the MRC-5 cell line that needs further investigation. The discrepancy in the findings between the two methods for the MRC-5 could be attributed to the nature of the chemical as a metabolic booster and an electron acceptor that may confound the MTT assay results. The assessments performed in this research proved that sodium ascorbate is ineffective despite its ability to inhibit cell metabolism (static) and cause cell death, once it reached a certain level of metabolic boost. From the research of others it has been proven that the inhibition of metabolic proliferation has not taken place due to its inability to induce apoptosis of cells [ 3, 4, 5, 14, and 15]. Further, findings are needed to explain this discrepancy between metabolic viability and cellular survival. Ascorbic Acid: The results from figures IIA and IIB of MTT assay showed that ascorbic acid gave ineffective results due the erratic levels of metabolic viability recorded for both A549 and MRC-5 cell lines that constantly remained superior to their controls. In table 1, the analysis determined that the results of exposure to ascorbic acid by the A549 cell line compared to its control was not significant (p=0.1816). Nonetheless, figures IVA and IVB show that the A549 cell line control had substantial cell viability and maintained changes in normal morphological structure, however, when exposed to the highest concentration of ascorbic acid the cell viability declined along with its morphological structure. When the MRC-5 was compared to its control, it was shown that its level of difference was statistically significant (p=0.0183, boost; data not shown). In table 2, it was shown that for the comparison of A549 and MRC-5 cell line the findings were not statistically significant (p=0.0554; static vs. boost). In table 3, the HCCA analysis showed that the level of difference for the A549 cell line compared to its control was significant (p=0.0334; cell death) and the MRC-5 cell line compared to its control provided a level of difference that was not statistically significant (p=0.3369). The cell analyzer, however, showed that the MRC-5 cell line opposed to the A549 cell line provided a level of difference that was statistically significant (p=0.0412) due to the death of the A549 compared to the stable viability of the MRC-5 cells. The assessments performed in this research proved that ascorbic acid is effective in the treatment of lung cancer in vitro. From the research of others it has been established that ascorbic acid interferes in the functioning of the hypoxia Inducible Factor (HIF) in cancer [16, 17, 18, and 19].
CONCLUSIONS It is concluded that treatment with 62.6-2,000 µg/ml of ascorbic acid or sodium ascorbate produced moderate effects and proved to be differential killing agents of the lung cancer cell line (A549), while in contrast they served as promoters of metabolism in the normal lung fibroblast cell line (MRC-5), providing a healthy outcome. The major findings from this study is that there is great potential for using natural organics in the differential destruction of lung cancer as based on the survival responses of the A549 and MRC-5 cell lines to these two natural organics. Further, studied are warranted to establish the ideal conditions for selective killing of other cancer cell lines in terms of organic concentrations, incubation time, and combination synergy of the effective products. ACKNOWLEDGMENTS This research is supported by NIH/NCRR RCMI grant # G12-RR 13459. REFERENCES 1. Warburg, Otto. (1956). On the Origin of Cancer Cells. Science. http://www.sciencemag.org., 2007. 2. H. Bucay The biological significance of cancer: Mitochondria as a cause of cancer and the inhibition of glycolysis with citrate as a cancer treatment Medical Hypotheses, Volume 69, Issue 4, Pages 826-828A, 1989. 3. R. Buc, S. Demaugre, M. Moncion and N. Leroux Metabolic consequences of pyruvate kinase inhibition by oxalate in intact heptocytes. Biochemie. 63(7): 595-602, 1981. 4. A. Carosio, V. Roberto; B. Zuccari, et al. Sodium Ascrobate induces apoptosis in neuroblastoma cell lines by interfering with iron uptake. Molecular Cancer. 6:55, 2007. 5. Carew, Jennifer and pen Huang Mitochondrial defects in cancer. Molecular Cancer. Vol. 1:9, 2002. 6. National Cancer Institute Understanding Cancer Series: Cancer. www.cancer.gov.2006. 7. M. Reimann Normal Growth and Cancer. Philadelphia and Montreal: Lippincott Company. p.295, 1963. 8. Safety (MSDS) data for ascorbic acid. Oxford University.10-09, 2005. 9. T. St. John With Every Breath: A Lung Cancer Guidebook. www.lungcancerguidebook.org., 2003. 10. Woodard, Gladys The effect of 2-Deoxy-D-glucose on glycolysis and respiration on tumor and normal tissues. Biochemical Research Foundation. Vol.14:599-605, 1954. 11. Xu, Rui-hua, Helene Pelicano, Yan Zhou, Jennifer S. Carew, Li Feng, Kapil N. Bhalla, Michael J. Keating, and Peng Huane Inhibition of Glycolysis in Cancer Cells: A Novel Strategy to Overcome Drug Resistance Associated with Mitochondrial Respiratory Defect and Hypoxia. Cancer Research. Vol.65: 613-615, 2005. 12.. J.B. Jacobs Characteristics of a human diploid cell designated MRC-5. Nature. 168-170, 1970. 13. S.A. Ahmed, R.M. Gogal, and J.E. Walsh A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H] thymidine incorporation assay. J Immunol. Methods. Vol.170 (2): 211-24, 1994. 14. M. Nociari, A. Shalev, P. Benias, and C. Russo A novel one-step, highly sensitive fluorometric assay to evaluate cell-mediated cytotoxicity. J Immunol Methods. Vol.78, 221-27, 1998. 15. L.A. Knowles, B.S. Raval, H.W. Harris, and R.M. Ratcliffe Effect of ascrobate on the activity of hypoxia-inducible factor in the cancer cells. Cancer Research United Kingdom Oncology Laboratory. Vol. 63(8): 17648, 2003. 16. H. Liu, S. Niramol, P. Waldemar and T. J. Lampidis Hypoxia increases tumor cell sensitivity to glycolytic inhibitors: a strategy for solid tumor therapy (Model C). Biochemical Pharmacology, Volume 64, Issue 12. Pages 1745-1751, 2002. 17. D.R. Lide Handbook of Data on Organic Compounds, 3 rd Ed; CRC Press: Boca Raton, FL. p.4386, 1994. 18. H. Liu, N. Savaraj, W. Priebe W, and T.J. Lampidis Hypoxia increases tumor cell sensitivity to glycolytic inhibitors: a strategy for solid tumor therapy (Model C). Biochem. Pharmacol.64(12): 1745-51, 2002. 19. Roudier, Bachelet, and Perrin Pyruvate reduces DNA damage during hypoxia and after reoxygenation in heptocellular carcinoma cells. Vol. 274 Issue 19 pp. 5188-98, 2007.