Jeffery Sivert Tessem and Dr. Byron K. Murray, Microbiology
In recent years in both clinical and experimental research the benefits of vitamins as anti-cancer agents have been described. Derivatives of vitamin C have been shown to “diminish the tumor mass in inoperable tumor patients” as well as cause degeneration of hepatocellular carcinoma in rats due to direct cytotoxicity to the tumor cells (1). Vitamin K3 has also been shown to induces DNA damage, reduced expression of c-myc, and caused apoptosis in human T-lymphocyte Jurkat cells, and other effects have also been seen in other cell lines (1). Studies have been done using these two vitamins in conjunction on human bladder and prostate cell lines, with effects similar to apoptosis called autoschizis (2,3). These effects are believed to be due to a redox cycle between vitamin C and vitamin K3 that produces oxygen radical, with vitamin C fostering the reduction of vitamin K3 by way of a single-electron reduction, which will increase the rate of redox cycling as well as increasing oxidative stress and cytotoxicity (1). In this study we show the synergistic effects of vitamin C and vitamin K3 on the WiDR human colon adenocarcinoma cell line. These data suggest that the synergistic effects of vitamin combinations may be an effective means for induction of apoptosis in human cancer cells.
We have shown that concentrations of vitamin C greater than 3.1 mM are needed for 50% of the population to die (CD50), while concentrations of vitamin K3 greater than 11μM are needed for a CD50. The most dramatic effects were noticeable between 900/9 μM and 225/2.5 μM VC/VK3. It was observed that when vitamin C and vitamin K3 where used alone at these concentrations that the only effect was a slight increase in cell growth, however when used in combination a large decrease in cell growth occurred. These effects were obtained after 24 hours treatment by doing cell counts using the trypan blue cell exclusion protocol, with subsequent days following the same trend. The effects of the combinations were then investigated within the first 24 hours, with cell counts every 6 hours post-treatment to determine when the greatest change in cell proliferation occurred. This data indicated that the greatest effects did indeed occur in the first 2- 3 hours, which is consistent with the theory of redox cycling.
The mitochondrial activity was assayed by using the alamarBlue assay using a Dynes MRX microplate reader, with our subsequent data indicating that the vitamin combinations do affect the mitochondrial activity, with the greatest decrease in activity corresponding with the most elevated concentrations of vitamin combinational treatment.
In order to verify the common physical changes that occur in apoptosis we used two different visualization techniques. After exposing the cells to the vitamin concentrations for 24 hours we used a novel Hoescht/Propidium Iodide staining technique that allowed for differentiation between normal, necrotic, and apoptotic cells show a brighter compact blue that corresponds with DNA condensation and blebbing (Fig. 1). We also used electron microscopy to verify the presence of blebbing that is common with apoptotic cells (Fig. 2). Our data showed that VC/VK3 was effective in inducing apoptosis in WiDR cells, and we demonstrated that apoptosis did occur at concentrations as low as 250/2.5 μM, however it did not occur with cells treated only with VC or VK3. This was evidenced by chromatin condensation and blebbing seen through the Hoescht/Propidium Iodide staining and also blebbing, loss of microvillia, and cell shrinkage seen by way of electron microscopy seen with the vitamin combination but not with the vitamins alone.
To determine if any blockage in the cell cycle was occurring (another hallmark of apoptosis) cell cycle analysis was performed by treating the cells for 24 hours, fixing in ice cold ethanol, and staining with Propidium iodide and using a Coulter Epics XL flow cytometer. This data suggested that the combination of vitamins block the cell cycle in G0/G1, as compared to controls, and that VC and VK3 alone did not have the same effect. To determine if a transposition of membrane phosphatidylserine was shown, a annexin-V stain was employed, however no transposition was shown. Another hallmark of apoptosis that we looked for was chromatin fragmentation. We did this by flow cytometry and looking for a sub G0 peak, which was not evident. The comet assay was also employed to determine if fragmentation was occurring however no results have become evident yet. In previous experiments using the same vitamin system in other cell lines showed no DNA fragmentation, so these results do follow the previous data.
We theorize that apoptosis induced by VC/VK3 resulted from a synergistic effect of the two vitamins. Each vitamin alone at the given concentrations had no adverse effects on WiDR cells. However, with the two vitamins in combination, the molar amount needed of each to induce changes was decreased drastically. It is proposed that the form: Q + AscH- → Q· – + Asc· – + H+; and Q· – + O2 →Q + O2 · – with VK3 as the quinone that is passed the electron from VC, and that finally passes the electron to oxygen, by a one electron transfer (6). The mechanism of the oxygen radicals inducing apoptosis is not known, and will need further studies to investigate that mechanism.
References
- Sakagami, H., Satoh, K., Hakeda, Y., and Kumegawa, M. 2000. Apoptosis-Inducing Activity of Vitamin C and Vitamin K. Cell mol. Biol., 46:129-143.
- Gilloteaux, J., Jamison, J.M., Arnold, D., Ervin, E., Eckroat, L., Docherty J. J., Neal, D., and Summers, J.L. 1998. Cancer Cell Necrosis by Autoschizis: Synergism of Antitumor Activity of Vitamin C: Vitamin K3 on Human Bladder Carcinoma T24 Cells. Scanning, 20:564-575.
- Venugopal, M., Jamison, J.M., Gilloteaux, J., Koch, J.A., Summers, M., Hoke, J., Sowick, C., Summers, J.L. 1996. Synergistic Antitumour Activity of Vitamins C and K3 Against Human Prostate Carcinoma Cell Lines. Cell Biology International, 20:787-797.