Marcus C. Neuffer and Dr. Byron K. Murray, Microbiology
Recent studies have suggested that vitamins and vitamin derivatives can enhance the efficacy of chemotherapeutic drugs (2,3). In this study the combination of 13-cis-Retinoic Acid (RA), a Vitamin A derivative, and Vitamin E Succinate (VES) are used with the chemotherapeutic agent 5-Fluorouracil (5FU). Previous studies have suggested that the RA and VES combination have a synergistic effect on inhibiting the growth of the HT-29 human colon adenocarcinoma cell line (1). The RA and VES combination also caused the cells to go through morphological and biochemical changes indicative of apoptosis. The purpose of this study was to treat the HT-29 cell line with the RA and VES combination joined with nontoxic levels of 5FU. The hypothesis was that the RA and VES combination with the chemotherapeutic agent would have a greater effect on inhibiting the cancer cell line than the agents would have alone, and that the combination of the agents would kill the cell line through apoptosis (programmed cell death).
The nontoxic levels of 5FU were first determined by seeding 24 well plates with 800,000 cells per well, and then treating the cells with different concentrations of 5FU ranging from 0.1 ug/ml to 1.0 ug/ml. The wells were seeded at a high concentration so that they would be confluent the following day. On the following day the medium was changed and new 5FU was added to the wells. The same procedure was followed and the cells were enumerated every other day for a week. The experiment was repeated at least three times and it was determined that nontoxic levels of 5FU were below 0.3 ug/ml.
The next procedure was to combine nontoxic levels of 5FU with a RA and VES combination. Previous studies showed that the most effective concentration of RA and VES was at 7.5 ug/ml (1). This concentration was used with concentrations of 0.025 ug/ml, 0.05 ug/ml, 0.1 ug/ml, and 0.2 ug/ml of 5FU. 24 well plates were once again seeded with 800, 000 cells per well and were treated every other day with either the 5FU concentrations alone, or the 5FU concentrations joined with the RA and VES combination. The experiment was repeated three times and data were compiled and graphed. The data in the graph below represents an average of three experiments. The standard deviations range from 0 to 3.19E+05.
The results show that the RA and VES combination with the 0.2 ug/ml of 5FU was effective in inhibiting the growth of the HT-29 cell line. There is even a slight decrease in cells from day 6 to day 7. However, it can also be seen that the 0.2 ug/ml of 5FU alone has close to the same effect as the 0.2 ug/ml of 5FU with the RA and VES combination. The RA and VES combination used with the other concentrations of 5FU also inhibited the cell line, but not as well as the RA and VES combination with the 0.2 ug/ml of 5FU.
To determine if cell death is occurring through necrosis or apoptosis Hoescht staining, caspase assays, and Scanning Electron Microscopy still need to be performed. Also, to determine if the cell population will continue to drop or plateau the experiment needs to be extended to day 10. These tests and experiments have not been performed yet because of the amount of time it has taken to grow and treat the cells
In addition to the experiments done using RA and VES with 5FU on a confluent monolayer of HT-29 cells, there have also been experiments performed using the same variables on preconfluent HT-29 cells (Moffit, L. Personal Communication). The experiments suggest that RA and VES with 5FU have a greater inhibition on the HT-29 cell line when it is non-confluent than when it is confluent. Experiments still need to be performed to determine if it is necrotic or apoptotic death.
References
- Brown, B., Garvin, K. R., Hughes, B. G., O’Neil, K. L., Murray, B. K. 2000. Induction of Apoptosis in HT-29 Human Colon Adenocarcinoma Cells by 13-cis-Retinoic Acid and Vitamin E Succinate. (submitted) Cancer Research.
- Chinery, R., Brockman, J.A. Peeler, M.O, Shyr, Y. Beauchamp, R.D., and Coffey, R.J. 1997. Antioxidants enhance the cytotoxicity of chemotherapeutic agents in the colorectal cancer: A p-53 independent induction of p21 (WAF1/CIP1) via c/EBPß. Nature Med., 3:1233-1241.
- Prasad, K.N., Kumar, A., Kochupillai, V., and Cole, W.C. 1999. High doses of multiple antioxidant vitamins: essential ingredients in improving the efficacy of standard cancer therapy. J. Am. Coll. Nutr., 18: 13-25