title: In vivo and in vitro sensitization of leukemic cells to adriamycin-induced apoptosis by pentoxifylline. Involvement of caspase cascades and IkappaBalpha phosphorylation.
abstract: The aim of this work was to investigate whether in vivo and in vitro pentoxifylline (PTX) sensitizes hematological tumor cells to adriamycin (ADM)-induced apoptosis, and to investigate the involvement of caspase cascades and phosphorylated forms of IkappaBalpha. Balb/c mice inoculated intraperitoneally with L5178-Y murine lymphoma cells were used for in vivo experiments and for survival studies. The U937 human monocytic cell line was used for in vitro experiments. Both cell lines were treated under similar experimental conditions with PTX and/or ADM to assess their effects on apoptosis. Apoptosis was evaluated by fluorescence microscopy with ethidium bromide and acridine orange staining and confirmed by electrophoretic DNA analysis. Caspase inhibitors Z-VAD-fmk, Z-DEVD-fmk, and Z-LEHD-fmk were used to investigate the involvement of caspase cascades. C-terminally and Ser32 phosphorylated forms of IkappaBalpha were evaluated in cytoplasmic extracts in the absence or presence of TNFalpha.
  
  
  RESULTS
  In vivo, PTX (50 mg/kg) with ADM (5 mg/kg) increased the apoptotic index relative to PTX or ADM administered alone, time- and dose-dependently. DNA laddering appeared in lymphoma cells treated with PTX+ADM at 24 h, whereas neither untreated control, PTX-, nor ADM-treated cells showed DNA fragmentation. All (100%) tumor-bearing mice treated with PTX (25 mg/kg)+ADM (2.5 mg/kg) survived for 1 year, whereas the mortality rates of mice treated with either PTX or ADM alone at the same doses were similar to that of untreated tumor-bearing mice (28+/-3 days). Caspase inhibitors inhibited apoptosis more efficiently in PTX- or ADM-treated cultures than in PTX+ADM-treated cultures. Pretreatment with TNFalpha (10 ng/mL) increased apoptosis in PTX- or ADM-treated U937 cells. However, the apoptotic index of PTX+ADM-treated cultures was significantly reduced and the expression of C-terminally and Ser32 phosphorylated IkappaBalpha was reduced. PTX sensitizes hematological malignancies to ADR-induced apoptosis. An independent caspase pathway is involved in PTX+ADM-induced apoptosis. The phosphorylation status of IkappaBalpha is closely related via TNFalpha to the possible mechanisms of drug resistance.
authors.affiliation: Centro de Investigación Biomédica de Occidente, IMSS, División de Inmunología, Sierra Mojada 800, Col. Independencia, 44340 Guadalajara, Jalisco, México.
authors.forename: José Manuel
authors.initials: JM
authors.lastname: Lerma-Díaz
authors.forename: Georgina
authors.initials: G
authors.lastname: Hernández-Flores
authors.forename: Jorge R
authors.initials: JR
authors.lastname: Domínguez-Rodríguez
authors.forename: Pablo C
authors.initials: PC
authors.lastname: Ortíz-Lazareno
authors.forename: Piedad
authors.initials: P
authors.lastname: Gómez-Contreras
authors.forename: Ramón
authors.initials: R
authors.lastname: Cervantes-Munguía
authors.forename: Daniel
authors.initials: D
authors.lastname: Scott-Algara
authors.forename: Adriana
authors.initials: A
authors.lastname: Aguilar-Lemarroy
authors.forename: Luis F
authors.initials: LF
authors.lastname: Jave-Suárez
authors.forename: Alejandro
authors.initials: A
authors.lastname: Bravo-Cuellar
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D015153:Blotting, Western; D020169:Caspases / Q000502:physiology*; D004317:Doxorubicin / Q000494:pharmacology; D006801:Humans; D020672:I-kappa B Proteins / Q000378:metabolism*; D066298:In Vitro Techniques; D007938:Leukemia / Q000188:drug therapy*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D000072000:NF-KappaB Inhibitor alpha; D010431:Pentoxifylline / Q000494:pharmacology*; D010766:Phosphorylation; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology; D020298:U937 Cells
chemical_list: D000970:Antineoplastic Agents; D020672:I-kappa B Proteins; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; D014409:Tumor Necrosis Factor-alpha; D000072000:NF-KappaB Inhibitor alpha; D004317:Doxorubicin; D020169:Caspases; D010431:Pentoxifylline