Effect of glutathione depletion on inhibition of cell cycle progression and induction of apoptosis by melphalan (L-phenylalanine mustard) in human colorectal cancer cells

Intracellular levels of glutathione have been shown to affect the sensitivi ty of cells to cell death-inducing stimuli, as well as the mode of cell dea th. We found in five human colorectal cancer cell lines (HT-29, LS-180, LOV O, SW837, and SW1116) that GSH depletion by L-buthionine-[S, R]-sulfoximine (BSO) below 20% of control values increased L-phenylalanine mustard (L-PAM ; Melphalan) cytotoxicity 2- to 3-fold. Effects on kinetics of both cell cy cle progression and cell death were further investigated in the HT-29 cell line. BSO treatment alone had no effect on cell cycle kinetics, but did enh ance the inhibition of S phase progression as induced by L-PAM; at high con centration of of L-PAM, BSO pretreatment resulted in blockage in all phases of the cell cycle. Yet, BSO pretreatment did not affect the intracellular L-PAM content. L-PAM induced apoptosis in both normal and GSH-depleted cell s. A combination of annexin V labeling and propidium iodide staining reveal ed that even the higher concentration oil-PAM (420 mu M) did not induce apo ptosis until 48 hr after treatment, but that induction of cell death was ma rkedly accelerated as a result of GSH depletion: 48 hours after L-PAM (420 mu M) treatment, GSH-depleted cells showed a dr fold increase in DNA fragme ntation and a 7-fold increase in the fraction of apoptotic (annexin V-posit ive) cells as compared to cells with normal GSH levels. Various antioxidant treatment modalities could not prevent this potentiating effect of GSH dep letion on L-PAM cytotoxicity, suggesting that reactive oxygen species do no t play a role. These data show that after BSO treatment the mode of L-PAM-i nduced cell death does not necessarily switch from apoptosis to necrosis. ( C) 1999 Elsevier Science Inc.