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Targeting Gb3 and apoptosis-related proteins to overcome cisplatin resistance
Umeå University, Faculty of Medicine, Department of Medical Biosciences, Clinical chemistry. (Kjell Grankvist)ORCID iD: 0000-0002-4968-6192
2016 (English)Doctoral thesis, comprehensive summary (Other academic)Alternative title
Gb3 och apoptos-relaterade proteiner som måltavla för att bryta cisplatinresistens (Swedish)
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 [en]
Cisplatin resistance, exosomes, gb3, HSP70, the Bcl-2 family
National Category
Cell and Molecular Biology
Research subject
cellforskning; Medical Cell Biology; Oncology
Identifiers
URN: urn:nbn:se:umu:diva-119778ISBN: 978-91-7601-475-2 (print)OAI: oai:DiVA.org:umu-119778DiVA: diva2:923698
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
List of papers
1. Targeting glucosylceramide synthase induction of cell surface globotriaosylceramide (Gb3) in acquired cisplatin-resistance of lung cancer and malignant pleural mesothelioma cells
Open this publication in new window or tab >>Targeting glucosylceramide synthase induction of cell surface globotriaosylceramide (Gb3) in acquired cisplatin-resistance of lung cancer and malignant pleural mesothelioma cells
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2015 (English)In: Experimental Cell Research, ISSN 0014-4827, E-ISSN 1090-2422, Vol. 336, no 1, 23-32 p.Article in journal (Refereed) Published
Abstract [en]

BACKGROUND: Acquired resistance to cisplatin treatment is a caveat when treating patients with non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM). Ceramide increases in response to chemotherapy, leading to proliferation arrest and apoptosis. However, a tumour stress activation of glucosylceramide synthase (GCS) follows to eliminate ceramide by formation of glycosphingolipids (GSLs) such as globotriaosylceramide (Gb3), the functional receptor of verotoxin-1. Ceramide elimination enhances cell proliferation and apoptosis blockade, thus stimulating tumor progression. GSLs transactivate multidrug resistance 1/P-glycoprotein (MDR1) and multidrug resistance-associated protein 1 (MRP1) expression which further prevents ceramide accumulation and stimulates drug efflux. We investigated the expression of Gb3, MDR1 and MRP1 in NSCLC and MPM cells with acquired cisplatin resistance, and if GCS activity or MDR1 pump inhibitors would reduce their expression and reverse cisplatin-resistance.

METHODS: Cell surface expression of Gb3, MDR1 and MRP1 and intracellular expression of MDR1 and MRP1 was analysed by flow cytometry and confocal microscopy on P31 MPM and H1299 NSCLC cells and subline cells with acquired cisplatin resistance. The effect of GCS inhibitor PPMP and MDR1 pump inhibitor cyclosporin A for 72h on expression and cisplatin cytotoxicity was tested.

RESULTS: The cisplatin-resistant cells expressed increased cell surface Gb3. Cell surface Gb3 expression of resistant cells was annihilated by PPMP whereas cyclosporin A decreased Gb3 and MDR1 expression in H1299 cells. No decrease of MDR1 by PPMP was noted in using flow cytometry, whereas a decrease of MDR1 in H1299 and H1299res was indicated with confocal microscopy. No certain co-localization of Gb3 and MDR1 was noted. PPMP, but not cyclosporin A, potentiated cisplatin cytotoxicity in all cells.

CONCLUSIONS: Cell surface Gb3 expression is a likely tumour biomarker for acquired cisplatin resistance of NSCLC and MPM cells. Tumour cell resistance to MDR1 inhibitors of cell surface MDR1 and Gb3 could explain the aggressiveness of NSCLC and MPM. Therapy with GCS activity inhibitors or toxin targeting of the Gb3 receptor may substantially reduce acquired cisplatin drug resistance of NSCLC and MPM cells.

Keyword
Cisplatin,  Glucosylceramide synthase (GCS),  Globotriaosylceramide (Gb3),  Lung cancer,  Multidrug resistance 1/P-glycoprotein (MDR1), Multidrug resistance-associated protein 1 (MRP1),  Malignant pleural mesothelioma (MPM),  DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP),  Cyclosporin A,  Acquired drug resistance
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:umu:diva-103786 (URN)10.1016/j.yexcr.2015.05.012 (DOI)000358821700003 ()26004871 (PubMedID)
Funder
Swedish Cancer Society
Available from: 2015-05-30 Created: 2015-05-30 Last updated: 2017-12-04Bibliographically approved
2. Cell membrane expression of HSP70 in cisplatin resistant tumour cells may be targeted through the co-localized glycosphingolipid Gb3
Open this publication in new window or tab >>Cell membrane expression of HSP70 in cisplatin resistant tumour cells may be targeted through the co-localized glycosphingolipid Gb3
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Globotriaosylceramide (Gb3) and heat-shock protein 70 (HSP70) are often co-localized on the cell surface of tumours and facilitates metastasis and cisplatin resistance. We hypothesized that targeting Gb3 could also inhibit HSP70 expression in non-small cell lung cancer (NSCLC) and malignant pleural mesothelioma (MPM).

Gb3 and HSP70 were co-localized on the cell surface. The glucosylceramide synthaseinhibitor DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP), Gb3 synthase siRNA and VT-1 reduced Gb3 and HSP70 expression, while HSP90-inhibitor 17-N-allylamino-17-demethoxygeldanamycin (17-AAG) increased Gb3 and HSP70. The combination of 17-AAG and VT-1 had a synergistic cytotoxic effect.

Cell membrane HSP70 expression in cisplatin-resistant tumour cells may be targeted through co-localized Gb3. 

Keyword
Globotriaosylceramide, heat shock protein 70, cisplatin, verotoxin-1, glycosphingolipid, glucosylceramide synthase
National Category
Cell and Molecular Biology
Research subject
cellforskning; Medical Cell Biology; Oncology
Identifiers
urn:nbn:se:umu:diva-119786 (URN)
Funder
Swedish Cancer Society, CAN 2011/599
Available from: 2016-04-27 Created: 2016-04-27 Last updated: 2016-04-29Bibliographically approved
3. Acquired cisplatin resistance in malignant pleural mesothelioma cells is reversed by both BH3-mimetic obatoclax and IAP-inhibitor AT-406
Open this publication in new window or tab >>Acquired cisplatin resistance in malignant pleural mesothelioma cells is reversed by both BH3-mimetic obatoclax and IAP-inhibitor AT-406
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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
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:nbn:se:umu:diva-119774 (URN)
Funder
Swedish Cancer Society
Available from: 2016-04-27 Created: 2016-04-26 Last updated: 2016-04-29Bibliographically approved
4. The role of exosomes in transferal of acquired cisplatin resistance
Open this publication in new window or tab >>The role of exosomes in transferal of acquired cisplatin resistance
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background: A major obstacle when treating cancer patients with cisplatin is that cancer often acquires cisplatin resistance during treatment, resulting in poor patient survival time. One important mechanism of cisplatin resistance is thought to be transferal of pro-survival characteristics, such as increased expression of the anti-apoptotic heat shock protein 70 (HSP70) through release and uptake of exosomes, small bioparticles that contain proteins, messenger RNA (mRNA) or micro RNA (miR).

Methods: We studied the morphology and size distribution of exosomes released by malignant pleural mesothelioma (MPM) and non-small cell lung cancer cells through confocal microscopy and nanoparticle tracking analysis. We assessed whether exosomes extracted from cells of the studied cell lines could fuse with the plasma membrane and introduce HSP70 to the cell surface through confocal microscopy.

Results: Exosomes secreted from cisplatin-resistant P31res cells and H1299res cells were able to fuse with the plasma membrane and present HSP70 on the surface of cells from the corresponding cell line (P31 and H1299) as well as the cells they originated from.

Conclusions: Cisplatin resistance transferal through exosomes may lead to better future treatment alternatives.

Keyword
Cisplatin, acquired drug resistance, exosomes, globotriaosylceramide (Gb3), heat shock protein 70 (HSP70)
National Category
Cell and Molecular Biology
Research subject
cellforskning; Medical Cell Biology; Oncology
Identifiers
urn:nbn:se:umu:diva-119775 (URN)
Funder
Swedish Cancer Society, CAN 2011/599
Available from: 2016-04-27 Created: 2016-04-26 Last updated: 2016-04-29Bibliographically approved

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