Author of

• 12418

  +

  P3.05 - Poster Session 3 - Preclinical Models of Therapeutics/Imaging (ID 159)

  • Event: WCLC 2013
  • Type: Poster Session
  • Track: Biology
  • Presentations: 2
  • Moderators:
  • Coordinates: 10/30/2013, 09:30 - 16:30, Exhibit Hall, Ground Level

  • 12422

    +

    P3.05-004 - Targeting glucosylceramide synthase induction of cell surface globotriaosylceramide (Gb3) co-expressed with multidrug resistance 1 (MDR1) protein as new treatment approach in acquired cisplatin-resistance of malignant pleural mesothelioma and non-small cell lung cancercisplatin-resistance in a malignant mesothelioma cell line (ID 990)

    09:30 - 09:30  |  Author(s): K. Grankvist
    • Abstract

    Background
    Background: Development of acquired resistance to cisplatin treatment is a major problem when treating patients with malignant pleural mesothelioma (MPM) and non-small cell lung cancer (NSCLC). Chemotherapy leads to tumor cell stress activation of glucosylceramide synthase (GCS) to eliminate ceramide by glycosylation and formation of glycosphingolipids (GSLs) such as globotriaosylceramide (Gb3), the functional receptor of verotoxin-1. Ceramide elimination leads to stimulated cell proliferation and blocked apoptosis, thus stimulating tumor progression. GSLs also transactivate multidrug resistance 1/P-Glycoprotein (MDR1) and possibly multidrug resistance protein 1 (MRP1) expression which confers tumour cell resistance by further preventing ceramide accumulation and stimulating drug efflux. We investigated if Gb3, MDR1 and MRP1 are co-expressed and co-localized in MPM and NSCLC cells with acquired cisplatin resistance and if GSC activity inhibitors DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) and cyclosporin A would reduce their expression and relieve cisplatin-resistance.

    Methods
    Methods: Cell surface as well as intracellular expression of Gb3, MDR1 and MRP1 was analysed by flow cytometry and confocal microscopy using specific protein antibodies on P31 MPM and H1299 NSCLC cell lines and corresponding sub-lines (P31res, H1299res) with acquired cisplatin resistance.

    Results
    Results: Gb3 and MDR1, but not MRP1 were co-expressed and partly co-localized on the cell surface, and Gb3 and MDR1 as well as MRP1 intracellular co-expressed but not co-localized in P31res and H1299res cells. P31 cells expressed minute cell surface Gb3 and the non-resistant cells had less cell surface and but similar intracellular expression of Gb3 and MDR1. Glycosphingolipid synthesis inhibitors PPMP and cyclosporin A radically decreased intracellular Gb3, MDR1 and MRP1-expression in all cell sub-lines whereas cell surface Gb and MDR1 expression was decreased only by PPMP but not by cyclosporin A.

    Conclusion
    conclusion: These results indicate that cell surface Gb3 that is co-expressed and co-localised with MDR1 is a likely tumour biomarker for acquired cisplatin resistance in MPM and NSCLC and that therapy with GCS activity inhibitors or Gb3 blockers affecting ceramide metabolism may overcome or substantially reduce acquired cisplatin drug resistance. Targeting the functional interplay between Gb3 and MDR1 might aid in the development of new drug therapies against acquired drug resistance in MPM and lung cancer.

  • 12423

    +

    P3.05-005 - Deregulation of Bcl-2 family protein expression and preserved post-target apoptosis resistance to the BH3-mimetic GX15-070 on acquisition of cisplatin-resistance in a malignant mesothelioma cell line (ID 1047)

    09:30 - 09:30  |  Author(s): K. Grankvist
    • Abstract

    Background
    Background: Platinum-based drugs, such as cisplatin, are the standard treatment for aggressive malignant pleural mesothelioma (MPM) and non-small-cell lung cancer (NSCLC), but inherent as well as acquired resistance are major clinical problems leading to therapy failure and low median survival after diagnosis. Cisplatin exposure initiates the mitochondrial signaling pathway of apoptosis, by activation of BH3-only proteins i.e. pro-apoptotic members of the Bcl-2 family of proteins. Therapy failure may be the result of decreased apoptosis due to caspase-9-deactivation or over-expression of the anti-apoptotic proteins Bcl-X~L~ and Mcl-1. Affecting cisplatin resistance by targeting post- and off-target apoptosis signalling proteins with pro-apoptotic BH3-mimetics, would possible sensitize cancer cells to cisplatin treatment.

    Methods
    Methods: We investigated the expression of Bcl-2 family and other proteins involved in apoptosis signal transduction and the difference between the response to equiapoptotic cisplatin concentrations as well as the response to the pro-apoptotic BH3-mimetics ABT-737 and GX15-070, alone or in combination. To separate mitochondrial-dependent from –independent signalling we compared initiator-caspase-dependent parental P31 MPM cells with its acquired cisplatin-resistant (P31res) sub-line which has initiator-caspase-independent caspase-3-activated apoptosis,

    Results
    Results: On acquisition of cisplatin-resistance, the expression of the pro-apoptotic and anti-apoptotic Bcl-2-family proteins was either not changed or slightly decreased in P31res cell compared to parental P31 cells. A 6-h exposure to equiapoptotic concentrations of cisplatin, on the other hand, increased the expression of potent pro-apoptotic BH3-only proteins as well as the anti-apoptotic Bcl-x protein in P31 but not P31res cells whereas Bcl-x expression was almost annihilated in P31res cells. TUNEL results showed a synergic effect on apoptosis when cisplatin was combined with the BH3-mimetic GX15-070 in P31res, but only an additive effect in P31. The BH3-mimetic ABT-737 did not augment cytotoxicity or apoptosis either per se or when combined with cisplatin and/or GX15-070. GX15-070 efficiently inhibited anti-apoptotic Bcl-2-family-, inhibitors of apoptosis (IAP) - and heat shock protein (HSP) - family protein expression both with and without cisplatin in P31 cells, whereas preserved protein expression was noted in P31res cells after 6 h incubation with cisplatin. Sub-toxic concentrations of the IAP-inhibitor AT-406 and the HSP90-inhibitor 17-AAG with GX15-070 markedly potentiated cisplatin cytotoxicity in P31res cells.

    Conclusion
    Conclusion: P31 malignant mesothelioma cell acquisition of cisplatin-resistance led to deregulated Bcl-2 family protein expression and induced post-target apoptosis resistance to the BH3-mimetic GX15-070. GX15-070 had a synergistic effect on cisplatin-induced apoptosis in P31res cells. The synergy was due to efficient GX15-070 inhibition of expression of the anti-apoptotic Bcl-x protein despite apoptosis resistance by preserved IAP and HSP protein expression. Cisplatin therapy combined with GX15-070 in acquired cisplatin resistance was even more efficacious when combined also with inhibitors of IAP- and HSP- apoptosis signalling pathways.