title: Enhancement of melphalan activity by buthionine sulfoximine and electroporation in melanoma cells.
abstract: Melphalan represents the reference drug for locoregional chemotherapy of melanoma; nevertheless, treatment failure may occur because of resistance to chemotherapy. Refractory melanoma cells show either an increased capability of drug inactivation, which is known to be associated with elevated intracellular levels of glutathione (GSH), or a decreased melphalan uptake. The aim of this study was to explore a biochemical and a biophysical strategy, and their combination, to overcome melphalan resistance in melanoma cells. The biochemical strategy was based on the treatment of melanoma cells with DL-buthionine (S,R)-sulfoximine (BSO) to deplete the GSH levels, thus reducing melphalan inactivation. In the biophysical strategy, cell membrane electroporation was used to increase melphalan uptake. The SK-MEL 28-resistant human melanoma cell line was pretreated with 50 μmol/l BSO for 24 h and then treated with increasing melphalan doses, with or without electroporation. Spectrophotometric quantification of cell viability was used to determine melphalan cytotoxicity. Intracellular total GSH was measured using a kinetic enzymatic assay. BSO induced 3.50-fold GSH depletion in untreated cells and a similar reduction was also maintained in melphalan-treated cells. BSO pretreatment produced a 2.46-fold increase in melphalan cytotoxicity. Electroporation increased melphalan cytotoxicity 1.42-fold. The combination of both BSO pretreatment with melphalan plus electroporation led to a 4.40-fold increase in melphalan cytotoxicity compared with melphalan alone. Pretreatment with BSO and cell membrane permeabilization by electroporation enhanced the cytotoxic activity of melphalan in melanoma cells. Their rational combination deserves further investigation and may improve the efficacy of locoregional chemotherapy of melanoma.
authors.affiliation: aDepartment of Morphology, Surgery and Experimental Medicine, University of Ferrara, Ferrara bLaboratory of Clinical Biophysics, IGEA S.p.A., Modena cSarcoma and Melanoma Unit, Veneto Institute of Oncology (IOV-IRCCS), Padova, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Ongaro
authors.forename: Agnese
authors.initials: A
authors.lastname: Pellati
authors.forename: Monica
authors.initials: M
authors.lastname: De Mattei
authors.forename: Francesca
authors.initials: F
authors.lastname: De Terlizzi
authors.forename: Carlo R
authors.initials: CR
authors.lastname: Rossi
authors.forename: Luca G
authors.initials: LG
authors.lastname: Campana
mesh_terms: D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D000971:Antineoplastic Combined Chemotherapy Protocols / Q000494:pharmacology; D019328:Buthionine Sulfoximine / Q000008:administration & dosage / Q000494:pharmacology; D045744:Cell Line, Tumor / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D019008:Drug Resistance, Neoplasm; D018274:Electroporation / Q000379:methods*; D005978:Glutathione / Q000378:metabolism; D006801:Humans; D008545:Melanoma / Q000188:drug therapy* / Q000473:pathology; D008558:Melphalan / Q000008:administration & dosage / Q000494:pharmacology*
chemical_list: D018906:Antineoplastic Agents, Alkylating; D019328:Buthionine Sulfoximine; D005978:Glutathione; D008558:Melphalan