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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�C-containing substrates. Supplementary Figure S8. Expression levels and functional activity of XRCC1 and POL in CDDP-sensitive and CDDP-resistant cancer cells that overexpress PARP1 and contain high levels of poly(ADP-ribosyl)ated proteins.

// Fintan Geoghegan 1 , Robert J. Buckland 2 , Eric T. Rogers 1 , Karima Khalifa 1 , Emma B. O’Connor 1 , Mary F. Rooney 1 , Parviz Behnam-Motlagh 2 , Torbjorn K. Nilsson 2 , Kjell Grankvist 2 and Richard K. Porter 1 1 School of Biochemistry and Immunology, Trinity Biomedical Science Institute (TBSI), Trinity College Dublin, Dublin 2, Ireland 2 Dept of Medical Biosciences, Clinical Chemistry, Umea University, Umea, Sweden Correspondence to: Robert J. Buckland, email: robert.buckland@umu.se Keywords: cisplatin resistance, bioenergetics, SIRT3, non-small cell lung cancer, mesothelioma Abbreviations: OCR: oxygen consumption rate; ECAR: extracellular acidification rate Received: July 22, 2017 Accepted: September 21, 2017 Published: October 16, 2017 ABSTRACT 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 cisplatin resistant cells when compared to cisplatin sensitive controls. We also observed an increase in AMP kinase subunit α2 (AMPKα2) transcripts and protein expression in resistant H1299 cells. mRNA expression was also reduced for cisplatin resistant H1299 cells in these genes, however the pattern was not consistent in resistant P31 cells. There was very little change in DNA methylation of these genes, suggesting that the cells are not stably reprogrammed epigenetically. Taken together, our data demonstrate reduced oxidative metabolism, reduced mitochondrial abundance, potential for increased glycolytic flux and increased ROS production in acquired cisplatin resistant cells. This suggests that the metabolic changes are a result of reduced SIRT3 expression and increased HIF-1α stabilization.

The selection of human cancer cell lines in cis-diamminedichloroplatinum(II) (CDDP, best known as cisplatin) is accompanied by stereotyped alterations that contribute to the acquisition of a CDDP-resistant state. Thus, CDDP resistance often leads to the upregulation of the DNA repair enzyme poly (ADP-ribose) polymerase-1 (PARP1) with the consequent intracellular accumulation of poly (ADP-ribose) (PAR)-modified proteins. Here we report another frequent alteration accompanying CDDP resistance, namely upregulation of the antiapoptotic BCL-2 family protein MCL-1. Six out of 8 CDDP resistant cancer cell lines manifested an increase in MCL-1 protein expression level, while only a minority of cell lines overexpressed BCL-2 or BCL-XL. BCL-XL was decreased in six out of 8 cancer cell lines. Importantly, MCL-1 overexpressing, CDDP resistant cells appear to be 'addicted' to MCL-1 because they died upon depletion of MCL-1 by RNA interference or pharmacological inhibition of MCL-1 expression by the BH3 mimetic obatoclax. Knockdown of PARP1 did not succeed in reducing MCL-1 expression, while depletion or inhibition of MCL-1 failed to affect the activity of PARP1. Hence, the two resistance mechanisms are not linked to each other by a direct cause-effect relationship. Importantly, CDDP-resistant, MCL-1 overexpressing human non-small cell lung cancers responded to monotherapy with obatoclax in vivo, in xenotransplanted mice, underscoring the probable therapeutic relevance of these findings.

In order to obtain a comprehensive DNA methylation signature of HER2-positive breast cancer (HER2+ breast cancer), we performed a genome-wide methylation analysis on 17 HER2+ breast cancer and compared with ten normal breast tissue samples using the Illumina Infinium HumanMethylation450 BeadChip (450K). In HER2+ breast cancer, we found altered DNA methylation in genes involved in multicellular development, differentiation and transcription. Within these genes, we observed an overrepresentation of homeobox family genes, including several genes that have not been previously reported in relation to cancer (DBX1, NKX2–6, SIX6). Other affected genes included several belonging to the PI3K and Wnt signaling pathways. Notably, HER2, AKT3, HK1, and PFKP, genes for which altered methylation has not been previously reported, were also identified in this analysis. In total, we report 69 candidate biomarker genes with maximum differential methylation in HER2+ breast cancer. External validation of gene expression in a selected group of these genes (n = 13) revealed lowered mean gene expression in HER2+ breast cancer. We analyzed DNA methylation in six top candidate genes (AKR1B1, INA, FOXC2, NEUROD1, CDKL2, IRF4) using EpiTect Methyl II Custom PCR Array and confirmed the 450K array findings. Future clinical studies focusing on these genes, as well as on homeobox-containing genes and HER2, AKT3, HK1, and PFKP, are warranted which could provide further insights into the biology of HER2+ breast cancer.

Background: A major problem with cisplatin treatment is the development of acquired-drug resistance of the tumour cells. Verotoxin-1 (VT-1) exerts its cytotoxicity by targeting the membrane glycolipid globotriasosylceramide (Gb3), a molecule associated with drug resistance. Cisplatin- and VT-1-induced apoptosis involves mitogen-activated protein kinase (MAPK) activation, and deactivation of MAPKs is associated with cisplatin resistance. This study aimed to investigate whether a sub-toxic concentration of VT-1 could enhance cisplatin-induced apoptosis and overcome acquired-cisplatin resistance in cultured cancer cell lines. Method: P31 and H1299 cells with corresponding cisplatin-resistant sub-lines (P31res/H1299res) were incubated with VT-1 and/or cisplatin followed by determination of Gb3 expression, cell viability, apoptosis, and signalling pathways. Results: Cells from the resistant sub-lines had elevated Gb3 expression compared with the parental cell lines, and cisplatin further increased Gb3 expression, whereas VT-1 reduced the percentage of Gb3-expressing cells. Combination of cisplatin and sub-toxic concentrations of VT-1 led to a super-additive increase of cytotoxicity and TUNEL staining, especially in the cisplatin-resistant sub-lines. Blockade of Gb3 synthesis by a Gb3 synthesis inhibitor not only led to eradicated TUNEL staining of P31 cells, but also sensitised P31res cells to the induction of apoptosis by cisplatin alone. Cisplatin- and VT-1-induced apoptosis involved the MAPK pathways with increased C-Jun N-terminal kinase and MAPK kinase-3 and -6 phosphorylation. Conclusions: We show the presence of Gb3 in acquired-cisplatin resistance in P31res and H1299res cells. Cisplatin up-regulated Gb3 expression in all cells and thus sensitised the cells to VT-1-induced cytotoxicity. A strong super-additive effect of combined cisplatin and a sub-toxic concentration of VT-1 in cisplatin-resistant malignant pleural mesothelioma cells were observed, indicating a new potential clinical-treatment approach.

Aims: Resistance mechanisms are important limiting factors in the treatment of solid malignancies with cis-diamminedichloroplatinum(II) (cisplatin). To gain further understanding of the effects of acquired cisplatin-resistance, we compared a human malignant pleural mesothelioma cell line (P31) to a sub-line (P31res1.2) with acquired cisplatin-resistance. Methods and Results: The role of Na+,K+,2Cl--cotransport (NKCC1) activity in cisplatin-induced morphological changes and acquired cisplatin-resistance was investigated in a time-resolved manner. Acquisition of cisplatin-resistance resulted in markedly reduced NKCC1 activity, absence of cisplatin-induced early membrane blebbing, and increased basal caspase-3 activity. At equitoxic cisplatin concentrations, P31res1.2 cells had a faster activation of caspase-3 than P31 cells, but the end-stage cytotoxicity and number of cells with DNA fragmentation was similar. Bumetanide inhibition of NKCC1 activity in P31 cells repressed cisplatin-induced early-phase membrane blebbing but did not increase P31 cell resistance to cisplatin. Conclusions: Together, these results suggest that active NKCC1 was necessary for cisplatin-induced early membrane blebbing of P31 cells, but not for cisplatin-resistance. Thus, acquisition of cisplatin-resistance can affect mechanisms that have profound effects on cisplatin-induced morphological changes but are not necessary for the subsequent progression to apoptosis.

Depletion of intracellular potassium ions (K+) is necessary for cells to shrink, activate caspases and induce DNA fragmentation, events which are features of apoptosis. Here we describe a 96-well plate method using the cell permeable form of K+ binding benzofuran isophtalate (PBFI-AM) to measure intracellular K+ content in relation to untreated control. Cultured human pulmonary mesothelioma cells (P31) and small-cell lung cancer cells (U1690) were treated with K+ flux modulators in order to deprive the cells of intracellular K+. The combination of K+ influx inhibition with 10 micromol/L bumetanide plus 10 micromol/L ouabain and K+ efflux stimulation with 3 mg/L amphotericin B or 5 micromol/L nigericin efficiently reduced the intracellular K+ content after 3 h. Manipulation of K+ fluxes with subsequent intracellular K+ depletion induced apoptosis of lung cancer cells, as detected by caspase-3 activity after 3 h K+ depletion followed by 24 h proliferation and TUNEL positive staining after 48 h proliferation. We concluded that the PBFI-AM assay was a useful tool to determine intracellular K+ content in relation to untreated control, and that intracellular K+ depletion of lung cancer cells by clinically used drugs of relevant concentrations induced apoptosis. These findings may lead to novel therapeutic strategies in the treatment of lung cancer.

The aim of this study was to examine the cytotoxicity and mechanism of apoptosis induction of verotoxin-1 (VT-1) in human glioma cell lines. VT-1 is a member of the shiga-toxin family expressed by some serotypes of Escherichia coli and Shigella dysenteriae. Shiga-toxins have been shown to induce apoptosis by binding to its membrane receptor Gb3. The human glioma cell lines SF-767, U-343 MG, and U-251 MG were studied together with BT4C, a rat glioma cell line. Cells were first screened for Gb3 expression by flow cytometry. Fluorescein diacetate was used to determine cell viability after VT-1 and irradiation exposure and apoptosis was studied by TUNEL staining, a mitochondrial membrane potential assay, and caspase activity assays. SF-767 and U-343 MG cells were found to express Gb3 and were also sensitive to VT-1-induced cytotoxicity, whereas nonGb3-expressing U-251 MG and BT4C glioma cells were not. VT-1 depolarized the mitochondrial membrane and activated caspase-9 and -3 of SF-767 and U-343 MG cells. VT-1 exposure for 72 h resulted in approx. 60 and 90% TUNEL-stained cells, respectively. D, L-Threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP) an inhibitor of glucosylceramide synthesis was used to block Gb3 synthesis. Two mumol/L PPMP for 72 h abolished SF-767 and U-343 MG expression of Gb3 and made the cells completely resistant to VT-1 induced apoptosis. Key components of MAP kinase signalling pathways that control BAX and mitochondrial function were investigated. VT-1 induced JNK phosphorylation in both cell lines, suggesting that survival signal pathways were overruled by VT-1-induced JNK activation leading to mitochondrial depolarization, caspase-9 activation and apoptosis. Immunohistochemistry of cryostat section from glioma biopsies demonstrated expression of Gb3 was in the vascular endothelial cells as well as tumor cells, but not in astrocytes. The high specificity and apoptosis inducing properties of verotoxin-1 indicates that the toxin may be a potential anti-neoplastic agent for Gb3-expressing gliomas.

The anticancer drug cisplatin induces cell death by apoptosis. Apoptosis is dependent on cellular loss of potassium ions (K + ). We have recently shown that the antifungal drug amphotericin B (enhancing K + efflux), combined with the Na + , K + , 2Cl - -cotransport blocker bumetanide (decreasing K + influx), augmented cisplatin-induced apoptosis in vitro. We therefore quantified K + fluxes with the K + analogue rubidium ( 86 Rb + ) in cisplatin-induced apoptosis of mesothelioma cells treated with bumetanide and amphotericin B. Bumetanide combined with amphotericin B enhanced cisplatin-induced apoptosis by a pronounced initial reduction of K + influx due (in addition to Na + , K + , 2Cl - -cotransport inhibition) also to Na + , K + , ATPase pump inhibition. As 86 Rb + efflux was initially preserved, combination of the drugs would lead to net K + loss. Combinations of K + flux modulators leading to cellular potassium ion deprivation thus augments cisplatin-induced apoptosis and could therefore possibly be used to enhance the antitumour efficacy of cisplatin treatment.

We used 86 Rb + (K + analogue) to study potassium influx during the interaction of highly specific 5-HT 3 -receptor antagonists, ondansetron and granisetron, with the effects of the anticancer drug, estramustine phosphate, on P31 mesothelioma cells. Estramustine phosphate (80 mg/l, 142 μmol/l) for 120 min. reduced 86 Rb + influx by 18.7%. The reduction was inhibited by ondansetron (0.1 μmol/l), but augmented by granisetron (0.1 μmol/l). Serotonin (1.0 μmol/l) antagonized ondansetron inhibition and restored granisetron-augmented reduction of estramustine phosphate-induced 86 Rb + influx to the level of the drug itself. Estramustine phosphate inhibited cellular Na + , K + , 2Cl - -cotransport activity whereas Na + , K + , ATPase activity was unaffected. Ondansetron blockade of estramustine phosphate-induced reduction of 86 Rb + influx was due to increased Na + , K + , ATPase and Na + , K + , 2Cl - -cotransport whereas augmentation of estramustine phosphate-induced reduction of 86 Rb + influx by granisetron, or combination of 5-HT 3 receptor antagonists with serotonin was due mainly to inhibition of cellular Na + , K + , ATPase activity. Thus, ondansetron possesses a distinct ability to reverse K + influx of tumour cells exposed to estramustine phosphate whereas granisetron does not, due to different effect on cellular Na + , K + , ATPase and Na + , K + , 2Cl - -cotransport activity. Highly 5-HT 3 receptor-specific antiemetic agents may have different effects on ion transport of tumour cells during treatment with cytotoxic drugs.

Acquired resistance to chemotherapy is a major problem during cancer treatment. One mechanism for drug resistance is overexpression of the MDR1 (multidrug resistance) gene encoding for the transmembrane efflux pump, P-glycoprotein (P-gp). The calcium channel blocker verapamil has been shown to reverse cellular drug resistance by inhibiting P-gp drug efflux. This study evaluated whether the new antihypertensive drug carvedilol influenced doxorubicin (Dox) cytotoxicity and P-gp activity in a P-gp-expressing cell line compared to a non-expressing subline. Verapamil (10 μmol/L), and even more markedly, carvedilol (10 μmol/L) increased cellular uptake of P-gp-transported calcein of a P-gp-expressing breast cancer cell line (Hs578T-Dox). In the subline (Hs578T) not expressing P-gp, no effects of carvedilol or verapamil on calcein uptake were seen. Carvedilol and verapamil (10 μmol/L) reduced the ld 50 (dose which results in the death of half the number of cells) of the Hs578T-Dox subline from 200 mg/L to approx. 10 mg/L Dox, whereas the ld 50 of the Hs578T subline was only marginally affected. Carvedilol (10 μmol/L) reduced P-gp activity approximately twice as effectively as verapamil at an equimolar concentration. Carvedilol did not affect pyrogallol cytotoxicity and pyrogallol was without effect on calcein accumulation of the Hs578T-Dox cell line, indicating the lack of antioxidative properties affecting P-gp activity and associated toxicity of the drug. The results suggest that carvedilol has the clinical potential to reverse tumour MDR involving the efflux protein P-gp.

A technique of microscopy with computerised detection of early morphological changes during continuous perifusion was used to monitor the geometry changes of cultured glioma cells (MG-251) when exposed to 40 mg/L estramustine phosphate (EMP) alone or in combination with granisetron (0.1 mumol/L), ondansetron (0.1 mumol/L), or serotonin (1 mumol/L). When the cells were exposed to EMP, cell volume measured as projected cell area (PCA) rapidly increased. Serotonin and ondansetron, but not granisetron, prevented the acute EMP response (PCA). Serotonin, but none of the 5-HT3 receptor antagonists, protected against the cytotoxicity of EMP to the glioma cells as measured by a fluorometric microculture assay. Our results demonstrate hitherto unknown differences between selective 5-HT3 receptor antagonist on the cellular response to EMP and shows the necessity to study the receptor antagonists from viewpoint of interference with the antitumour drug effects on malignant cells. The perifusion technique could be used to study the effects of serotoninergic agonists and antagonists on cell volume regulation of cells exposed to anticancer drugs.

A perifusion technique for microscopy with computerized detection of early changes in cell morphology during continuous perifusion was used to show that the geometry of cultured glioma cells (MG-251) changes rapidly when they are exposed to estramustine phosphate (EMP). When the cells were exposed to 20 or 40 mg l(-1) EMP, cell volume projected cell area (PCA) rapidly increased. When the Na+,K+-ATPase blocker ouabain (100 micromol l(-1)) was added to the EMP (40 mg l(-1)) perifusion, the acute EMP response was eradicated. When the PCA curve for ouabain alone was subtracted from the curve of combined ouabain and EMP perifusion, the resulting curve showed that ouabain completely blocked the EMP-induced increase in PCA. When the Na+, K+, Cl- co-transport inhibitors bumetanide (10 micromol l(-1)), or furosemide (100 micromol l(-1)), were added to EMP (40 mg l(-1)), the acute increase in PCA seen for EMP alone was also completely blocked. This study shows that inhibitors of ion transmembrane transport can modify EMP-induced cell volume increases. This may be of particular importance since the blockers have been found to interfere also with the cytotoxic function of EMP during cell culture. Thus, it is possible that cell volume changes could serve as a rapid technique for predicting the cytotoxic activity of antineoplastic drugs.

Non–small cell lung carcinoma patients are frequently treated with cisplatin (CDDP), most often yielding temporary clinical responses. Here, we show that PARP1 is highly expressed and constitutively hyperactivated in a majority of human CDDP-resistant cancer cells of distinct histologic origin. Cells manifesting elevated intracellular levels of poly(ADP-ribosyl)ated proteins (PARhigh) responded to pharmacologic PARP inhibitors as well as to PARP1-targeting siRNAs by initiating a DNA damage response that translated into cell death following the activation of the intrinsic pathway of apoptosis. Moreover, PARP1-overexpressing tumor cells and xenografts displayed elevated levels of PAR, which predicted the response to PARP inhibitors in vitro and in vivo more accurately than PARP1 expression itself. Thus, a majority of CDDP-resistant cancer cells appear to develop a dependency to PARP1, becoming susceptible to PARP inhibitor–induced apoptosis. Cancer Res; 73(7); 2271–80. ©2013 AACR.

Resistance mechanisms are important limiting factors in the treatment of solid malignancies with cis-diamminedichloroplatinum(II) (cisplatin). To gain further understanding of the effects of acquired cisplatin-resistance, we compared a human malignant pleural mesothelioma cell line (p31) to a sub-line (p31res1.2) with acquired cisplatin-resistance.The role of Na(+),K(+),2Cl(-)-cotransport (NKCC1) activity in cisplatin-induced morphological changes and acquired cisplatin-resistance was investigated in a time-resolved manner. Acquisition of cisplatin-resistance resulted in markedly reduced NKCC1 activity, absence of cisplatin-induced early membrane blebbing, and increased basal caspase-3 activity. At equitoxic cisplatin concentrations, P31res1.2 cells had a faster activation of caspase-3 than P31 cells, but the end-stage cytotoxicity and number of cells with DNA fragmentation was similar. Bumetanide inhibition of NKCC1 activity in P31 cells repressed cisplatin-induced early-phase membrane blebbing but did not increase P31 cell resistance to cisplatin.Together, these results suggest that active NKCC1 was necessary for cisplatin-induced early membrane blebbing of P31 cells, but not for cisplatin-resistance. Thus, acquisition of cisplatin-resistance can affect mechanisms that have profound effects on cisplatin-induced morphological changes but are not necessary for the subsequent progression to apoptosis.

Cisplatin treatment efficacy of malignant pleural mesothelioma (MPM) is aggravated by resistance and adverse effects. In P31 MPM cells, cisplatin induces morphological changes and apoptosis. To determine if very early (10 minutes) morphological responses corresponded to apoptosis-induction, cisplatin effects on P31 morphology were examined with phase-contrast microscopy (PCM), scanning electron microscopy (SEM), and flow cytometry (fluorescence-activated cell sorting [FACS]), and compared to apoptosis-induction over time. Increased membrane protrusions were identified with PCM and SEM, but these were not consistent with the induction of apoptosis. The authors concluded that very early morphological changes can be determined with PCM in MPM, but they did not convincingly correspond to apoptosis induction.

alpha-Toxin (alpha-hemolysin) of Staphylococcus aureus is a pore-forming bacterial toxin which after caveolin-1-dependent assembly induces apoptosis in eukaryotic cells. We investigated if a sub-toxic concentration of staphylococcal alpha-toxin could enhance cisplatin-induced apoptosis and overcome acquired cisplatin-resistance in cultured malignant pleural mesothelioma (MPM) cells. MPM cells (P31wt) and a cisplatin-resistant sub-line (P31res) was incubated with alpha-toxin and/or cisplatin followed by determination of cell viability, apoptosis, and signaling pathways. P31res cells were more sensitive to alpha-toxin than P31 wt cells due to induction of apoptosis. A low-toxic concentration of alpha-toxin re-sensitized cisplatin P31res cytotoxicity by apoptosis-induced through the mitochondrial pathway without detectable activation of common up-stream apoptosis signaling proteins. The toxin/drug combination should be tested for cisplatin-resistant mesothelioma treatment.

Amplified epidermal growth factor receptor (EGFR) signaling is supposed to contribute to clinical radiation resistance of glioblastoma multiforme (GBM). Therefore, inhibition of EGFR signaling pathways by the selective EGFR tyrosine kinase inhibitor, gefitinib (ZD1839, Iressa), may increase the therapeutic effects of radiotherapy. The effects of different schedules for administration of gefitinib on sensitivity to irradiation of the human glioma cell lines (251MG and SF-767), a rat glioma cell line (BT4C), and an immortalized rat brain endothelial cell line (RBE4) is reported. Differences in effects of the combined treatment on cell toxicity were determined by a fluorometric cytotoxicity assay, and nuclear DNA fragmentation was used for quantification of apoptosis. Pre-administration with gefitinib for 30 min prior to irradiation followed by continuous incubation with gefitinib significantly increased the cytotoxicity of SF-767, BT4C, and RBE4 cells. However, the human glioma cell line 251MG was protected against radiation-induced damage by this treatment schedule, at lower concentrations of gefitinib. Pre-administration with gefitinib for 24 h prior to irradiation without following incubation with gefitinib increased the cytotoxicity of SF-767 and BT4C cells. Post-irradiation treatment with gefitinib significantly increased the cytotoxicity in all cell lines except for 251MG. We demonstrated heterogeneity in the cytotoxic effects of gefitinib between cell lines. Response to gefitinib might be due to other mechanisms than through the EGF receptor as some of the cell lines showed sensitivity to gefitinib despite no or low expression of EGFR. This study also demonstrates the importance of timing of gefitinib administration when this agent is combined with irradiation.

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.

Depletion of intracellular potassium ions (K+) is necessary for cells to shrink, induce DNA fragmentation and activate caspases, events which are features of apoptosis.We used 86Rb+ as a K+ analogue to evaluate the possibility of pharmacologically depleting human pulmonary mesothelioma (P31) and small cell lung cancer (U1690) cells of K+, for future use in studies of apoptosis induction.The Na+, K+, 2CI(-)-cotransport inhibitor bumetanide transiently inhibited 86Rb+ influx, but when combined with the Na+, K+, ATPase pump inhibitor ouabain there was a marked and lasting (up to 6 h) 86Rb+ influx inhibition. Cellular K+ efflux was augmented by amphotericin B, digitonin and nigericin. Amphotericin B was an effective 86Rb+ efflux stimulator with low cytotoxicity, whereas digitonin caused cell detachment and nigericin increased LDH release in the U1690 cell line, indicating considerable toxicity of the drugs.It is possible to efficiently reduce intracellular K+ by persistent K+ influx inhibition and simultaneous K+ efflux stimulation with clinically available drugs.

The anticancer drug cisplatin induces cell death by apoptosis. Apoptosis is dependent on cellular loss of potassium ions (K+). We have recently shown that the antifungal drug amphotericin B (enhancing K+ efflux), combined with the Na+, K+, 2Cl(-)-cotransport blocker bumetanide (decreasing K+ influx), augmented cisplatin-induced apoptosis in vitro. We therefore quantified K+ fluxes with the K+ analogue rubidium (86Rb+) in cisplatin-induced apoptosis of mesothelioma cells treated with bumetanide and amphotericin B. Bumetanide combined with amphotericin B enhanced cisplatin-induced apoptosis by a pronounced initial reduction of K+ influx due (in addition to Na+, K+, 2Cl(-)-cotransport inhibition) also to Na+, K+, ATPase pump inhibition. As 86Rb+ efflux was initially preserved, combination of the drugs would lead to net K+ loss. Combinations of K+ flux modulators leading to cellular potassium ion deprivation thus augments cisplatin-induced apoptosis and could therefore possibly be used to enhance the antitumour efficacy of cisplatin treatment.

During cancer treatment, the use of antiemetics are often needed due to induction of nausea and vomiting by antineoplastic drugs. Some antiemetics have however been afflicted with cytotoxic effects during chemotherapy. The influence of the commonly used antiemetic metoclopramide on the cytotoxicity of epirubicin and cisplatin was tested on fibroblasts (V79) and lung cancer cells (P31) in vitro. The clonogenic survival of fibroblast and lung cancer cells were reduced when the cells were exposed to epirubicin or cisplatin. Metoclopramide (0.5 or 5 mg/l) enhanced epirubicin-induced toxicity to both fibroblast and lung cancer cells, but inhibited the cytotoxicity of cisplatin. The demonstrated effect of metoclopramide on cells in vitro and the fact that metoclopramide is used as a routine antiemetic during cancer treatment, may indicate that a possible clinical interaction with the antineoplastic action of cancer treatment drugs should be given attention.

The influence of radiotherapy on malignant glioma multidrug resistance to chemotherapy was evaluated because patients with glioma often are treated with a combination of radiotherapy and chemotherapy. Multidrug resistance gene (MDR1, mdr1a, and mdr1b) transcripts were found in human and rat glioma cell lines. P-Glycoprotein (Pgp) was immunohistochemically detected in glioma cell lines and in the rat brain vascular endothelial cell line (RBE4). A multidrug resistance pump efflux activity assay demonstrated increased calcein efflux of RBE4 endothelial cells, but not glioma cells, 2 h after irradiation and still increased 14 d after irradiation. The increased efflux was equally inhibited by verapamil with or without irradiation. In the rat intracranial glioma model (BT4C), Pgp was demonstrated in capillary endothelial cells of the tumor tissue and surrounding normal brain, but not in tumor cells. The expression of gene transcripts or Pgp was not affected by irradiation. The results indicate that long-lasting verapamil-resistant drug efflux mechanisms are activated in brain endothelial cells after irradiation. The results might explain the poor efficacy of chemotherapy following radiotherapy and contribute to consideration of new treatment strategies in the management of malignant glioma.

Purpose. Highly 5-HT3-receptor-specific antiemetic agents may have different effects on ion transport of tumour cells during treatment with cytotoxic drugs. Cell volume regulation, which is dependent on potassium ion (K+) flux, is involved the control of cell growth, proliferation, and apoptosis. K+-flux response mechanisms to the antiemetics ondansetron and granisetron were therefore correlated to malignant glioma cell (Mg251) volume response to estramustine phosphate (EMP) in vitro. Methods. We quantified the influx and efflux of potassium ions (using the K+ analogue 86Rb+) as well as cell volume changes (with image analysis) of glioma cells incubated with the 5-HT3-receptor antagonists ondansetron and granisetron (0.1 µmol/l) combined with 40 mg/l EMP. Results. The EMP-induced cell volume increase was fully inhibited by ondansetron but not affected by granisetron. Ondansetron retained high cellular K+-efflux and reduced Na+, K+, 2Cl--cotransport activity, whereas granisetron (0.1 µmol/l) reduced K+-efflux and retained an augmented Na+, K+, 2Cl--cotransport activity in the presence of 40 mg/l EMP. Conclusions. Ondansetron affects K+ transport with ensuing effects on cell volume of tumour cells treated with EMP, whereas granisetron does not. Since ondansetron and other 5-HT3-receptor antagonists are used routinely to prevent nausea during anticancer treatment, an increased awareness of possible interactions with the antitumour efficacy of anticancer drugs seems warranted.

Background: 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. Methods: 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. Results: 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 6 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. Conclusions: 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. Cytometry 40:198 ‐208, 2000.

At cancer treatment, the use of antiemetics are often needed due to induction of nausea and vomiting. Some antiemetics have been shown to interact with the direct cytotoxic effects. The newly developed antiemetics have, as far as we know, not been studied in this respect. In the present study, the effects of the 5-HT3 receptor antagonists ondansetron and granisetron were evaluated on the cytotoxicity, induced by irradiation, bleomycin, epirubicin, estramustine, and cisplatin using fibroblasts (V79) and lung cancer cells (P31) in vitro. Ondansetron or granisetron (10(-5) mol/l) had no effect on the survival of irradiated cells. Granisetron (10(-5) mol/l) significantly potentiated cytotoxicity of 2.5 mg/l epirubicin on fibroblasts whereas the effect of granisetron (10(-7) mol/l) on the cytotoxic effect of 25 mg/l bleomycin, and estramustine (80 mg/l) seemed additive to lung cancer cells. Ondansetron was non-interactive with the cytotoxicity induced by any of the anti-cancer drugs. Although the encountered observation with an enhancing effect of granisetron on the epirubicin-induced cytotoxicity is seen in a specific experimental situation in vitro, the fact that 5-HT3 receptor antagonists are routinely used during cancer treatment indicate that attention should be given to a possible interaction with the antineoplastic action of cancer treatment.

Co-expression of Globotriasosylceramide (Gb3) With MDR1 in Cisplatin-resistant Pleural Mesothelioma and Non-small Cell Lung Cancer Cell May Lead to a New Tumour Resistance Treatment Approach

Background The prerequisite for the potential use of the bacterial toxin verotoxin-1 in the treatment of breast cancer was investigated by first determining the expression of its receptor Gb3 (CD77) in clinical breast cancer tissue specimens. We then examined the cytotoxicity and mechanism of apoptosis induction of Escherichia coli verotoxin-1 (VT-1) in two human breast cancer cell lines. Methods Immunohistochemistry for Gb3 expression was performed on cryostat section from 25 breast cancer specimens. The human breast cancer cell lines T47D and MCF-7 were screened for Gb3 expression by flow cytometry. Fluorescein diacetate and LDH release was used to determine cell viability after VT-1 exposure. Apoptosis was studied by measuring caspase activity and DNA-fragmentation. Signal transduction studies were performed on T47D cells with immunoblotting. Results Gb3 expression was detected in the vascular endothelial cells of all tumours specimens, and in tumour cells in 17 of the specimens. We found no associations between tumour cell Gb3-expression and age, tumour size, TNM-classification, histological type, hormone receptor expression, or survival time. T47D cells strongly expressed Gb3 and were sensitive to the cytotoxicity, caspase activation and DNA fragmentation by VT-1, whereas MCF-7 cells with faint Gb3-expression were insensitive to VT-1. VT-1 (0.01 – 5 μg/L) exposure for 72 h resulted in a small percentage of viable T47D cells whereas the cytotoxicity of cells pre-treated with 2 μmol/L D, L-treo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol (PPMP, an inhibitor of glucosylceramide synthesis) was eliminated (≤ 0.1 μg/L VT-1) or reduced (0.5 – 5 μg/L VT-1). VT-1 did not cause cellular LDH-release or cell cycle arrest. VT-1 induction of caspase-3 (0.1, 1, and 5 μg/L VT-1), -8, and -9 (1 and 5 μg/L VT-1) activity and DNA fragmentation of T47D cells was blocked by PPMP. Key components of MAP kinase signalling pathways that control mitochondrial function were investigated. VT-1 0.1 – 5 μg/L induced phosphorylation of JNK as well as MKK3/6 suggesting that survival signal pathways were overruled by VT-1-induced JNK activation leading to mitochondrial depolarization, caspase-9 activation and apoptosis. Conclusion The high specificity and apoptosis-inducing properties of verotoxin-1 indicates that the toxin potentially may be used for treatment of Gb3-expressing breast cancer.