Jennetta Watson and Dr. Gary Booth, Integrative Biology
Caffeine and ephedrine are found together as constituents in a variety of dietary supplements promising to increase metabolism, raise energy levels, and promote weight loss. Much research has been done in this area; however, there is little to no information in the literature about studies focused on the toxicity and anti-proliferative properties of this same caffeine/ephedrine combination occurring at the cellular level. Caffeine’s toxicity against many cell types has been well documented for years.1 In addition, a number of studies have shown that using caffeine or its metabolites in combination with other chemical or physical reagents increases the overall toxic effect, specifically in regards to cancerous or highly proliferating cells.2 Ephedrine and ephedrine type alkaloids have also been shown to have toxic properties.3
In light of the literature’s data describing caffeine’s potential to form synergistic interactions with other reagents and ephedrine’s own cytotoxicity, it was logical to look at the possibilities of a similar synergism occurring between caffeine and ephedrine or their metabolites that would also be selective towards cancerous cells. To test for this synergistic drug interaction, I tested caffeine and ephedrine drug combinations on HeLa and 3T3 cells and analyzed the percentage of cells surviving in relation to a control. HeLa cells are a human cervical cancer cell line and were used as a general representative of cancer cells. 3T3 cells are a mouse fibroblast cell line or in other words, normal healthy cells. The mouse fibroblasts were chosen because they have been recommended as a valid substitution for human fibroblasts in pharmacological testing4 and because of accessibility.
The first step in determining synergism was to determine the individual dose curves of caffeine and ephedrine, as well as for the caffeine metabolites (theophylline, theobromine, 1,7- methylxanthine, and 1-methylxanthine) and ephedrine type alkaloids (norephedrine and pseudoephedrine). The dose curves ranged in concentration from 0.125 mM to 10 mM and were done on both HeLa and 3T3 cells. This range was sufficient to establish curves for each compound from 100% cell viability to10% or less. The dose curves for caffeine and ephedrine were comparable to those reported in other studies. The dose curves for the metabolites and ephedrine type alkaloids were also established in order to be used to analyze the combination results.
In determining the dose curves, I had two particular problems to overcome. First, the cell culture assay I used can have lots of nature variability, as the cells (especially 3T3 cells) can be more sensitive on some days than others. Therefore, results had to be analyzed in relation to how the cells were doing each day. Many times, the assays had to be redone because of cell problems. The other main problem was in getting three of the caffeine metabolites to dissolve at a high enough concentration to carry out the dose curve and the subsequent experiments. Several alternative solvents were tried, but most of these solvents proved to inhibit cell growth or kill the cells as compared to the cell media normally used to dissolve the drugs in. To avoid this problem suspensions rather than completely dissolved solutions were used for theobromine, 1,7- methylxanthine, and 1-methylxanthine.
After determining the dose curves, combinations of caffeine and ephedrine were put on the cells in ratios ranging from 4:1, 3:2, 1:1, 2:3, and 1:4. This also gave a dose response curve that could be analyzed in comparison to the individual dose curves of caffeine and ephedrine. One of the first combination curves I completed showed promising synergistic results at a ratio of 2 mM caffeine to 3 mM ephedrine. These results proved not to be reproducible and were likely due to experimental error. After repeating the experiment several times, as well as changing ratios, it was determined that no synergistic drug interaction between caffeine and ephedrine takes place on HeLa or 3T3 cells. The combination drug was toxic to both cell lines but the relationship was additive. That is, the cell viability was a function of the toxicity of the caffeine dose and the ephedrine dose alone; thus, adding more caffeine or ephedrine individually would have the same effect as a combination of the two.
The caffeine metabolites were also tested in combination with ephedrine, and the ephedrine type alkaloids were tested in combination with caffeine to see if any synergism occurred between these compounds. Again the results were disappointing. No synergistic drug interaction existed.
Caffeine and ephedrine alone and together did exhibit a more detrimental effect on the cancer cell line, HeLa, than the fibroblast cell line, 3T3. This suggests that the drugs are more selective against cancer cells than normal cells. This effect may also be due to the nature of the cell lines and their individual response to the sulforhodamine B cell culture assay used. In the future, more research must be done looking for changes in cell morphology and assaying for death versus growth inhibition before a definitive conclusion can be reached.
Although my hypothesis did not prove to be correct, in the process of completing this project, I learned much about scientific research, particularly the assays and data analysis skills associated with my project. I gained experience in developing experiments to look at the caffeine, ephedrine, and metabolite combinations in many different ways in order to make sure my final conclusion of no synergism was correct. I was also able to overcome problems associated with the assay and my drug combinations. Finally, I gained knowledge to help my lab continue research on the specificity of drugs to HeLa and 3T3 cells in the future.
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1 Deplanque, D. et al. “Caffeine does not cause override of the G2/M block induced by Uvc or gamma radiation in normal human skin fibroblasts.” British Journal of Cancer. 2000, Vol. 83, pp. 346-353.
2 Belizario, J.E. et al. “Caffeine potentiates the lethality of tumour necrosis factor in Cancer Cells.” British Journal of Cancer. 1993, Vol. 67, pp. 1229-1235.
3 Lee, M.K. et al. “Cytotoxicity assessment of ma-huang (Ephedra) under different conditions of preparation.” Toxicological Sciences. 2000, Vol. 56, pp. 424-430.
4 Levi-Shaffer, et al. “Xanthines inhibit 3T3 fibroblast proliferation.” Skin Pharmacology. 1991. Vol. 4, pp. 286-90.