Methionine Restriction Must Be Continuously Maintained to Selectively Inhibit Cancer Cells Co-cultured With Normal...

Methionine addiction (Hoffman effect) is a fundamental and general cancer hallmark targetable by methionine restriction, using methionine-depleted media or diet, or recombinant methioninase (rMETase). Our previous studies showed differential sensitivity of HCT-116 colon-cancer cells and Hs27 normal fibroblasts to rMETase in co-culture. The present study aimed to demonstrate the rescue conditions of cancer cells by methionine replenishment in the co-cultures of HCT-116 and Hs27 cells after rMETase treatment.

Equal numbers of HCT-116 colon-cancer cells and Hs27 normal fibroblasts were co-cultured in 6-well plates in Dulbecco's modified Eagle's medium (DMEM). Two days after seeding, co-cultures were treated with rMETase at the HCT-116 IC50 (0.46 U/ml) or left untreated as controls. Growth of each cell type in co-culture was evaluated by phase-contrast microscopy on days 2, 4, 6, 8, 10, and 12 after treatment to assess the response to rMETase. On day 12, the existing medium in all wells was replaced with fresh DMEM containing methionine (methionine replenishment). Regrowth of HCT-116 and Hs27 was then assessed by phase-contrast microscopy 3, 6, and 9 days later.

In the untreated control group, HCT-116 cancer cells rapidly proliferated, and progressively overtook the Hs27 fibroblasts and predominated by day 12. In the rMETase-treated group, viable HCT-116 cells progressively decreased and were almost undetectable by day 12, whereas Hs27 cells remained viable throughout the observation period. After day-12 replenishment of methionine, previously rMETase-treated co-cultures showed reappearance of viable HCT-116 cells by day 3 and dominance over Hs27 cells by day 9.

Continuous treatment with rMETase is necessary to maintain inhibition of cancer cells and normal-cell dominance in co-culture with cancer cells. These results have clinical implications indicating that methionine restriction must be continually maintained to inhibit cancer.