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Acquired cisplatin resistance in malignant pleural mesothelioma cells is reversed by both BH3-mimetic obatoclax and IAP-inhibitor AT-406
Klinisk Kemi. (Kjell Grankvist)ORCID iD: 0000-0002-4968-6192
Umeå University, Faculty of Medicine, Department of Odontology, School of Dentistry.
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Treatment of malignant pleural mesothelioma (MPM) with cisplatin often leads to acquired resistance with ensuing therapy failure, which may be the consequence of decreased apoptosis due overexpression of anti-apoptotic Bcl-2 family proteins or inhibitor of apoptosis (IAP) family proteins. Pro-apoptotic BH3-mimetics that antagonize the anti-apoptotic Bcl-2 protein family members and IAP inhibitors, which target the IAP family, could re-sensitize resistant MPM cells to cisplatin. We studied the effects of cisplatin, IAP inhibitor AT-406 and the BH3-mimetics ABT-737 and obatoclax on apoptosis and cytotoxicity in a cisplatin-resistant subline of MPM (P31res) and its parental cell line (P31). We used protein arrays and Western blot to study the differences between P31 and P31res cells in apoptosis signal transduction as well as the effects of cisplatin and obatoclax . P31res cells displayed changes in the Bcl-2 family protein expression in response to cisplatin and a massive inhibition of Bcl-x expression by obatoclax. The IAP-binding proteins Smac/Diablo and Htra2 were downregulated in P31res cells and cisplatin further downregulated Htra2. This suggested that Bcl-2 family proteins and IAP-related proteins may play a role in cisplatin resistance in the studied cell lines. Obatoclax decreased IAP protein expression in both P31 and P31res subline cells but addition of cisplatin abolished this effect in P31res cells. The IAP inhibitor AT-406 and BH3-mimetic obatoclax increased cisplatin cytotoxicity and apoptosis. Combined use of obatoclax with IAP inhibition only had a slight additive effect. This warrants further studies of targeting anti-apoptotic Bcl-2 family proteins and IAPs in malignant pleural mesothelioma. 

Keyword [en]
Obatoclax, ABT-737, cisplatin, acquired drug resistance, inhibitors of apoptosis, malignant mesothelioma
National Category
Cell and Molecular Biology
Research subject
cellforskning; Medical Cell Biology; Oncology
Identifiers
URN: urn:nbn:se:umu:diva-119774OAI: oai:DiVA.org:umu-119774DiVA: diva2:923685
Funder
Swedish Cancer Society
Available from: 2016-04-27 Created: 2016-04-26 Last updated: 2016-04-29Bibliographically approved
In thesis
1. Targeting Gb3 and apoptosis-related proteins to overcome cisplatin resistance
Open this publication in new window or tab >>Targeting Gb3 and apoptosis-related proteins to overcome cisplatin resistance
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Gb3 och apoptos-relaterade proteiner som måltavla för att bryta cisplatinresistens
Abstract [en]

Background Cisplatin is used for treatment of malignant pleural mesothelioma (MPM) and non-small cell lung cancer (NSCLC) but treatment with cisplatin often leads to acquired resistance to cisplatin, resulting in poor patient survival. Globotriaosylceramide (Gb3) and multidrug resistance protein 1 (MDR1) have been associated with cisplatin resistance. Gb3 serves as a receptor for verotoxin-1 (VT-1), which induces apoptosis, and has been shown to have a functional dependency to MDR1 and heat shock protein 70 (HSP7o). The Bcl-2 family of proteins and inhibitors of apoptosis (IAPs) are key regulators of apoptosis. BH3-mimetics mimic pro-apoptotic BH3-only proteins, while Smac mimetics mimic the IAP-binding protein Smac/Diablo. These drugs have shown great promise in reversing cisplatin resistance. Exosomes are small bio-nanoparticles secreted and taken up by both cancer cells and normal cells. They have the ability to transfer properties between cells and have been shown to confer resistance to cisplatin.

Methods In this thesis, NSCLC cell line H1299 and MPM cell line P31 were studied using western blot, flow cytometry, proteome profilers, confocal microscopy and gene expression arrays to investigate changes in protein and gene expression after acquisition of cisplatin resistance (P31res and H1299res) or after incubation with exosomes or drugs that target these. The cytotoxic and apoptotic effects were studied using fluorometric cytotoxicity assay (FMCA) and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay.

Results This thesis confirms that Gb3 is a potential target for cisplatin resistance reversal. Incubation with glycosphingolipid production inhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) and VT-1 led to reduced Gb3 cell surface expression and increased cytotoxic effect of cisplatin in all cell lines. Gb3 and MDR1 was not co-localized in any studied cell line, but Gb3 and HSP70 were co-localized on the cell surface and PPMP and VT-1 led to a decrease of both Gb3 and HSP70. Both BH3-mimetic obatoclax and Smac mimetic AT-406 had an additive effect on cisplatin-induced cytotoxicity and apoptosis in P31 and a synergistic effect in P31res. Results indicate that exosomes from cisplatin-resistant cell lines can transfer HSP70 to the surface of cells.

Conclusion Cell surface Gb3 and HSP70, the Bcl-2/IAP-family proteins and exosomal transfer of cisplatin resistance characteristics are potential targets in combatting cisplatin resistance that show therapeutic promise and warrant further research.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2016. 48 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1807
Keyword
Cisplatin resistance, exosomes, gb3, HSP70, the Bcl-2 family
National Category
Cell and Molecular Biology
Research subject
cellforskning; Medical Cell Biology; Oncology
Identifiers
urn:nbn:se:umu:diva-119778 (URN)978-91-7601-475-2 (ISBN)
Public defence
2016-05-23, E04, By 6E, Nod R, Norrlands universitetssjukhus, 901 85 Umeå, Umeå, 13:00 (English)
Opponent
Supervisors
Funder
Swedish Cancer Society, CAN 2011/599
Available from: 2016-05-02 Created: 2016-04-26 Last updated: 2016-05-26Bibliographically approved

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