Depletion of extracellular cysteine with hydroxocobalamin and ascorbate in experimental murine cancer chemotherapy

Treatment of mice bearing P388 lymphocytic leukemia with combined hydroxocobalamin (0.1 mmol/kg) and sodium ascorbate (1.0 mmol/kg) for 10 consecutive days resulted in 70% increase in survival. Treatment with dehydroascorbate (1.0 mmol/kg) similarly increased survival. The extended treatment for 15 days posttumor inoculation failed to improve survival as did lowering the tumor inocula from 10(6) to 10(4) cells in similarly treated mice. P388 cells in primary culture with cystine as a source of cysteine require exogenous thiols or disulfides for growth and also grow with cysteine but only when trace copper is sequestered with bathocuproine sulfonate. This is due to the copper catalyzed oxidation of cysteine to cystine, which these cells cannot utilize. Cells grew in medium preincubated for 8 h with cysteine and bathocuproine sulfonate, but lysed when the preincubated medium included hydroxocobalamin (10 microM) and sodium ascorbate (100 microM). This defect was corrected by resupplementation with cysteine or cystine plus bishydroxyethyl disulfide after addition of the cells. Further support for cysteine depletion as a factor in therapy is obtained by therapeutic interference with 4-thiamethionine (25 mumol/kg). This mixed disulfide of cysteine and methyl mercaptan enters cells as a methionine analogue, is reduced to supply cysteine, and thus withstands the oxidative environment that limits the utilization of cysteine as a nutrient. These results suggest that dehydroascorbate is generated from combined hydroxocobalamin and ascorbate and accounts for the thiol-prive activity of combined hydroxocobalamin and ascorbate therapy. The ultimate failure of therapy by thiol oxidation may be due to the increased availability of cysteine produced by host tissues, possibly infiltrating macrophages, that occurs independently of extended treatment and the size of the tumor inoculum.