Search

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 surfa

A major problem with anti-cancer drug treatment is the development of acquired multidrug resistance (MDR) of the tumor cells. Verotoxin-1 (VT-1) exerts its cytotoxicity by targeting the globotriaosylceramide membrane receptor (Gb3), a glycolipid associated with multidrug resistance. Gb3 is overexpressed in many human tumors and tumor cell lines with inherent or acquired MDR. Gb3 is co-expressed and interplays with the membrane efflux transporter P-gp encoded by the MDR1 gene. P-gp could act as a lipid flippase and stimulate Gb3 induction when tumor cells are exposed to cancer chemotherapy. Recent work has shown that apoptosis and inherent or acquired multidrug resistance in Gb3-expressing tumors could be affected by VT-1 holotoxin, a sub-toxic concentration of the holotoxin concomitant with chemotherapy or its Gb3-binding B-subunit coupled to cytotoxic or immunomodulatory drug, as well as chemical manipulation of Gb3 expression. The interplay between Gb3 and P-gp thus gives a possible physiological approach to augment the chemotherapeutic effect in multidrug resistant tumors.

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.

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.

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.

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.

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.

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.