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 sensitivity of cells to cell death-inducing stimuli, as well as the mode of cell death. We found in five human colorectal cancer cell lines (HT-29, LS-180, LOVO, 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 cycle 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 enhance the inhibition of S phase progression as induced by L-PAM; at high concentration 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 cells. A combination of annexin V labeling and propidium iodide staining revealed that even the higher concentration of L-PAM (420 microM) did not induce apoptosis until 48 hr after treatment, but that induction of cell death was markedly accelerated as a result of GSH depletion: 48 hours after L-PAM (420 microM) treatment, GSH-depleted cells showed a 4-fold increase in DNA fragmentation and a 7-fold increase in the fraction of apoptotic (annexin V-positive) cells as compared to cells with normal GSH levels. Various antioxidant treatment modalities could not prevent this potentiating effect of GSH depletion on L-PAM cytotoxicity, suggesting that reactive oxygen species do not play a role. These data show that after BSO treatment the mode of L-PAM-induced cell death does not necessarily switch from apoptosis to necrosis.
Department of Surgery, Leiden University Medical Center, The Netherlands.
A L
AL
Vahrmeijer
J H
JH
van Dierendonck
J
J
Schutrups
C J
CJ
van de Velde
G J
GJ
Mulder
D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology*; D000975:Antioxidants / Q000494:pharmacology; D017209:Apoptosis / Q000502:physiology; D019328:Buthionine Sulfoximine / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D015179:Colorectal Neoplasms / Q000188:drug therapy* / Q000473:pathology; D005978:Glutathione / Q000502:physiology*; D019073:HT29 Cells; D006801:Humans; D008558:Melphalan / Q000494:pharmacology*; D014407:Tumor Cells, Cultured
D018906:Antineoplastic Agents, Alkylating; D000975:Antioxidants; D019328:Buthionine Sulfoximine; D005978:Glutathione; D008558:Melphalan