The present study examined the possibility to enhance lung cancer cell cytotoxicity and apoptosis... more The present study examined the possibility to enhance lung cancer cell cytotoxicity and apoptosis of the anticancer drug cisplatin by exposure with adenylate cyclase (AC) toxin from Bordetella pertussis. A malignant mesothelioma cell line (P31) and a small-cell lung cancer cell line (U1690) were exposed to increasing concentrations of cisplatin and AC toxin, alone or in combination. Cytotoxicity was determined by a fluorescein-based assay and apoptosis by flow cytometry quantification of annexin V binding. Caspase-3, -8, and -9 activities were measured by enzyme activity assays. The cytotoxicity of AC toxin was time and dose dependent with an LD50 value at 72 h of 3 and 7 mg/L for P31 cells and U1690 cells, respectively. Cisplatin showed a similar time- and dose-dependent cytotoxicity, which was increased in the presence of a low toxic concentration (1 mg/L) of AC toxin. Furthermore, cisplatin caused a dose-dependent increase of annexin V binding cells of both cell lines after 24-h incubation, which was also enhanced in combination with AC toxin. AC toxin (1 mg/L) increased cisplatin-induced caspase-3, -8, and -9 activities in U1690 cells. Only minor increases of caspase-8 and -9 were noted for P31 cells. The present results, together with the knowledge that bacterial toxins decrease side effects of traditional cancer treatment, suggest a possibility to use them to enhance the therapeutic effect of cancer chemotherapy with reduced clinical adverse effects.
PDF file - 490K, Supplementary Table S1. Systematic comparison of the effects of CDDP and PARP in... more PDF file - 490K, Supplementary Table S1. Systematic comparison of the effects of CDDP and PARP inhibitors on cancer cell lines used in this study. Supplementary Figure S1. Generation and characterization of CDDP-resistant human NSCLC cell clones. Supplementary Figure S2. Effects of CDDP and PARP inhibitors on the intracellular levels of poly(ADPribosyl) ated proteins in WT and CDDP-resistant NSCLC cells. Supplementary Figure S3. DNA damage as induced by PARP inhibitors is not a consequence of apoptotic caspase activation. Supplementary Figure S4. Markers of homologous recombination (HR) in CDDP-sensitive and CDDP-resistant A549 cells responding to PARP inhibitors. Supplementary Figure S5. DNA repair activity of A549 cell extracts towards dA�T- and THF�T-containing substrates. Supplementary Figure S6. DNA glycosylase activity of A549 cell extracts towards Hx�T- and U�G-containing substrates. Supplementary Figure S7. DNA repair activity of A549 cell extracts towards 5-OHC�T-and 8oxoG�...
Inhibition of Na+, K+, ATPase pump activity induces apoptosis in cisplatin-resistant pleural meso... more Inhibition of Na+, K+, ATPase pump activity induces apoptosis in cisplatin-resistant pleural mesothelioma cells via Bak expression and caspase-3 activity
Background Cisplatin is used for treatment of malignant pleural mesothelioma (MPM) and non-small ... more 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 resi...
Globotriaosylceramide (Gb3) and heat-shock protein 70 (HSP70) are often co-localized on the cell ... more 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 targeti ...
Background: A major obstacle when treating cancer patients with cisplatin is that cancer often ac... more 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 ...
Acquired cisplatin resistance is a common feature of tumours following cancer treatment with cisp... more Acquired cisplatin resistance is a common feature of tumours following cancer treatment with cisplatin and also of non-small cell lung cancer (H1299) and mesothelioma (P31) cell lines exposed to cisplatin. To elucidate the cellular basis of acquired cisplatin resistance, a comprehensive bioenergetic analysis was undertaken. We demonstrate that cellular oxygen consumption was significantly decreased in cisplatin resistant cells and that the reduction was primarily due to reduced mitochondrial activity as a result of reduced mitochondrial abundance. The differential mitochondrial abundance was supported by data showing reduced sirtuin 1 (SIRT1), peroxisome-proliferator activator receptor-γ co-activator 1-alpha (PGC1α), sirtuin 3 (SIRT3) and mitochondrial transcription factor A (TFAM) protein expression in resistant cells. Consistent with these data we observed increased reactive oxygen species (ROS) production and increased hypoxia inducible factor 1-alpha (HIF1α) stabilization in cis...
The Na+, K+, ATPase inhibitor ouabain sensitizes resistant human malignant pleural mesothelioma c... more The Na+, K+, ATPase inhibitor ouabain sensitizes resistant human malignant pleural mesothelioma cells to cisplatin by increased Bak expression
Volume regulation is essential for cellular functions, including cell death, such as apoptosis. F... more Volume regulation is essential for cellular functions, including cell death, such as apoptosis. Flow cytometry is standard for nonadherent cells, such as blood cells. Our aim was to explore image analysis methods to study adherent cancer cells of a solid tumor. P31 mesothelioma cells were perifused (40 min) and studied by phase-contrast microscopy. A noise reduction of the cell contour was tested to more accurately yield the cell shape factor (SF). The optical halo around the cell was analyzed for information about membrane blebbing. The projected cell area (PCA) slowly increased under control perfusion, the halo outside more than the halo inside. Cisplatin (apoptosis) caused an immediate increase in the PCA-halo outside (5.9 +/- 1.2 %, P < 0.01, 1-5 min) and the SF indicated decreased roundness (P < 0.05). The SF-halo inside became more irregular than the outside, which was different from the control cells. The morphology reflected instant blebbing, and the cell bodies showed fragmentation after about 20 min. Ouabain resulted in only small changes in PCA and SF, significantly different from both control and cisplatin conditions. Image analysis (PCA and SF) on perifused adherent cancer cells may serve as a tool to follow the sensitivity of cancer chemotherapy and to study cell death patterns.
Acquired resistance to cisplatin treatment is a caveat when treating patients with non-small cell... more 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-resi...
The present study examined the possibility to enhance lung cancer cell cytotoxicity and apoptosis... more The present study examined the possibility to enhance lung cancer cell cytotoxicity and apoptosis of the anticancer drug cisplatin by exposure with adenylate cyclase (AC) toxin from Bordetella pertussis. A malignant mesothelioma cell line (P31) and a small-cell lung cancer cell line (U1690) were exposed to increasing concentrations of cisplatin and AC toxin, alone or in combination. Cytotoxicity was determined by a fluorescein-based assay and apoptosis by flow cytometry quantification of annexin V binding. Caspase-3, -8, and -9 activities were measured by enzyme activity assays. The cytotoxicity of AC toxin was time and dose dependent with an LD50 value at 72 h of 3 and 7 mg/L for P31 cells and U1690 cells, respectively. Cisplatin showed a similar time- and dose-dependent cytotoxicity, which was increased in the presence of a low toxic concentration (1 mg/L) of AC toxin. Furthermore, cisplatin caused a dose-dependent increase of annexin V binding cells of both cell lines after 24-h incubation, which was also enhanced in combination with AC toxin. AC toxin (1 mg/L) increased cisplatin-induced caspase-3, -8, and -9 activities in U1690 cells. Only minor increases of caspase-8 and -9 were noted for P31 cells. The present results, together with the knowledge that bacterial toxins decrease side effects of traditional cancer treatment, suggest a possibility to use them to enhance the therapeutic effect of cancer chemotherapy with reduced clinical adverse effects.
PDF file - 490K, Supplementary Table S1. Systematic comparison of the effects of CDDP and PARP in... more PDF file - 490K, Supplementary Table S1. Systematic comparison of the effects of CDDP and PARP inhibitors on cancer cell lines used in this study. Supplementary Figure S1. Generation and characterization of CDDP-resistant human NSCLC cell clones. Supplementary Figure S2. Effects of CDDP and PARP inhibitors on the intracellular levels of poly(ADPribosyl) ated proteins in WT and CDDP-resistant NSCLC cells. Supplementary Figure S3. DNA damage as induced by PARP inhibitors is not a consequence of apoptotic caspase activation. Supplementary Figure S4. Markers of homologous recombination (HR) in CDDP-sensitive and CDDP-resistant A549 cells responding to PARP inhibitors. Supplementary Figure S5. DNA repair activity of A549 cell extracts towards dA�T- and THF�T-containing substrates. Supplementary Figure S6. DNA glycosylase activity of A549 cell extracts towards Hx�T- and U�G-containing substrates. Supplementary Figure S7. DNA repair activity of A549 cell extracts towards 5-OHC�T-and 8oxoG�...
Inhibition of Na+, K+, ATPase pump activity induces apoptosis in cisplatin-resistant pleural meso... more Inhibition of Na+, K+, ATPase pump activity induces apoptosis in cisplatin-resistant pleural mesothelioma cells via Bak expression and caspase-3 activity
Background Cisplatin is used for treatment of malignant pleural mesothelioma (MPM) and non-small ... more 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 resi...
Globotriaosylceramide (Gb3) and heat-shock protein 70 (HSP70) are often co-localized on the cell ... more 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 targeti ...
Background: A major obstacle when treating cancer patients with cisplatin is that cancer often ac... more 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 ...
Acquired cisplatin resistance is a common feature of tumours following cancer treatment with cisp... more Acquired cisplatin resistance is a common feature of tumours following cancer treatment with cisplatin and also of non-small cell lung cancer (H1299) and mesothelioma (P31) cell lines exposed to cisplatin. To elucidate the cellular basis of acquired cisplatin resistance, a comprehensive bioenergetic analysis was undertaken. We demonstrate that cellular oxygen consumption was significantly decreased in cisplatin resistant cells and that the reduction was primarily due to reduced mitochondrial activity as a result of reduced mitochondrial abundance. The differential mitochondrial abundance was supported by data showing reduced sirtuin 1 (SIRT1), peroxisome-proliferator activator receptor-γ co-activator 1-alpha (PGC1α), sirtuin 3 (SIRT3) and mitochondrial transcription factor A (TFAM) protein expression in resistant cells. Consistent with these data we observed increased reactive oxygen species (ROS) production and increased hypoxia inducible factor 1-alpha (HIF1α) stabilization in cis...
The Na+, K+, ATPase inhibitor ouabain sensitizes resistant human malignant pleural mesothelioma c... more The Na+, K+, ATPase inhibitor ouabain sensitizes resistant human malignant pleural mesothelioma cells to cisplatin by increased Bak expression
Volume regulation is essential for cellular functions, including cell death, such as apoptosis. F... more Volume regulation is essential for cellular functions, including cell death, such as apoptosis. Flow cytometry is standard for nonadherent cells, such as blood cells. Our aim was to explore image analysis methods to study adherent cancer cells of a solid tumor. P31 mesothelioma cells were perifused (40 min) and studied by phase-contrast microscopy. A noise reduction of the cell contour was tested to more accurately yield the cell shape factor (SF). The optical halo around the cell was analyzed for information about membrane blebbing. The projected cell area (PCA) slowly increased under control perfusion, the halo outside more than the halo inside. Cisplatin (apoptosis) caused an immediate increase in the PCA-halo outside (5.9 +/- 1.2 %, P < 0.01, 1-5 min) and the SF indicated decreased roundness (P < 0.05). The SF-halo inside became more irregular than the outside, which was different from the control cells. The morphology reflected instant blebbing, and the cell bodies showed fragmentation after about 20 min. Ouabain resulted in only small changes in PCA and SF, significantly different from both control and cisplatin conditions. Image analysis (PCA and SF) on perifused adherent cancer cells may serve as a tool to follow the sensitivity of cancer chemotherapy and to study cell death patterns.
Acquired resistance to cisplatin treatment is a caveat when treating patients with non-small cell... more 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-resi...
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