Modulatory Effects of Vitamin B12 on Doxorubicin Accumulation and Sensitivity in Human HepG2 Cells
The most complex naturally occurring molecule, vitamin B12 or cobalamin (Cbl), is promising in the treatment of various cancers. Previous research has shown that cobalamin is involved in the downregulation of multi-drug resistance protein 1 (MDR-1), a gene encoding the efflux transport protein P-glycoprotein (P-gp), a transporter heavily implicated in the phenomenon of multi-drug resistance. As such we initially investigated the time dependence of Cbl on the functionality on another efflux transport protein, multidrug resistance protein 2 (MRP-2) by measuring its effects on cellular accumulation of 5(6)-carboxy-2´,7´dichlorofluorescein (CDCF), a well-defined fluorescent substrate of MRP-2. We then tested the effects of different Cbl concentrations on the efficacy of oxaliplatin, a prominent platinum based anti-cancer drug, as well on accumulation and efficacy of doxorubicin, an anthracycline, in a HepG2 cancer cell line. We hypothesised that the addition of Cbl would lead to the increased accumulation of CDCF, and that increasing concentrations of Cbl would lead to consequent increases in the cytotoxic effects of both oxaliplatin and doxorubicin as well as accumulation of doxorubicin. To assess CDCF accumulation, cells were pre-treated with 100nM cobalamin for 24, 48 and 72 hours, then tested using cellular accumulation protocols using a MoFlo XDP flow cytometer. The mean cellular accumulation of CDCF is significantly increased by 136% and 72% in HepG2 cells pre-treated with Cbl 100 nM for 24 and 48 hr, respectively; however, pretreatment with Cbl for 72 hr does not result in a statistically significant increase (by 27%) of CDCF accumulation in HepG2 cells. For testing the effects of Cbl on oxaliplatin cytotoxicity, cells pre-treated with Cbl concentrations of 200, 100 and 50 nM were assessed for oxaliplatin-induced cytotoxicity using MTT cell viability assays. Our results suggest IC50 values of cells treated with 100 nM and 200 nM to be approximately half of those of the control values, but there are no statistically significant differences between control and treated cells. HepG2 cells pre-treated with 1000, 500 and 100 nM Cbl were incubated with doxorubicin for 2 hours and analysed using a MoFlo XDP flow cytometer. Interesting results were obtained, where 500 nM but not 1,000 nM Cbl induced the greatest doxorubicin accumulation, suggesting a bell-shaped dose response curve. Doxorubicin cytotoxicity assays were carried out using the same concentrations as in the doxorubicin accumulation studies, but instead treated directly in 96-well plates rather than pre-treated. The effects of cobalamin on cytotoxicity of doxorubicin was then tested using MTT cell viability assays. This yielded results consistent with the doxorubicin accumulation studies, where 500 nM Cbl caused the greatest increase in cytotoxicity from doxorubicin, exhibiting a significantly lower IC50 value than for other Cbl treatment concentrations. The effects on CDCF accumulation and oxaliplatin cytotoxicity may be attributed to indirectly to Cbl, as Cbl involvement in human methionine synthase activity is associated with the production of folate, a molecule which effluxes via MRP-2, thus showing the potential of cobalamin to produce a molecule which acts as a competitive inhibitor of MRP-2 in the efflux of oxaliplatin. The increases in the accumulation and cytotoxicity of doxorubicin in Cbl treated cells may be largely attributed to the down-regulation of MDR-1 and/or ABCC2, and thus P-gp/MRP-2 expression. However, the unusual bell-shaped dose response curve of Cbl may arise from the methionine dependency in cancer cells, where the anti-cancer effects of Cbl could be outweighed by the pro-cancer effects of increased methionine induced by Cbl at an extremely high concentration. In conclusion, the results presented in this thesis may implicate the concentration of cobalamin as a major determining factor in its potential applicability in a clinical setting. As such further research may be warranted to more thoroughly investigate the effectiveness of cobalamin as an anti-cancer agent as well as the potential mechanisms behind both its anti-cancer and pro-cancer effects.