Mathias Tesson

The Role of Copper in Disulfiram-Induced Toxicity and Radiosensitization of Cancer Cells

Disulfiram has been used for several decades in the treatment of alcoholism. It now shows promise as an anticancer drug and radiosensitizer. Proposed mechanisms of action include the induction of oxidative stress and inhibition of proteasome activity. Our purpose was to determine the potential of disulfiram to enhance the antitumor efficacy of external-beam γ-irradiation and 131I-metaiodobenzylguanidine (131I-MIBG), a radiopharmaceutical used for the therapy of neuroendocrine tumors. Methods: The role of copper in disulfiram-induced toxicity was investigated by clonogenic assay after treatment of human SK-N-BE(2c) neuroblastoma and UVW/noradrenaline transporter (NAT) glioma cells. The synergistic interaction between disulfiram and radiotherapy was evaluated by combination-index analysis. Tumor growth delay was determined in vitro using multicellular tumor spheroids and in vivo using human tumor xenografts in athymic mice. Results: Escalating the disulfiram dosage caused a biphasic reduction in the surviving fraction of clonogens. Clonogenic cell kill after treatment with disulfiram concentrations less than 4 μM was copper-dependent, whereas cytotoxicity at concentrations greater than 10 μM was caused by oxidative stress. The cytotoxic effect of disulfiram was maximal when administered with equimolar copper. Likewise, disulfiram radiosensitization of tumor cells was copper-dependent. Furthermore, disulfiram treatment enhanced the toxicity of 131I-MIBG to spheroids and xenografts expressing the noradrenaline transporter. Conclusion: The results demonstrate that the cytotoxicity of disulfiram was copper-dependent, the molar excess of disulfiram relative to copper resulted in attenuation of disulfiram-mediated cytotoxicity, copper was required for the radiosensitizing activity of disulfiram, and copper-complexed disulfiram enhanced the efficacy not only of external-beam radiation but also of targeted radionuclide therapy in the form of 131I-MIBG. Therefore, disulfiram may have anticancer potential in combination with radiotherapy.

Inhibition of Poly(ADP-Ribose) Polymerase Enhances the Toxicity of 131I-Metaiodobenzylguanidine/Topotecan Combination Therapy to Cells and Xenografts That Express the Noradrenaline Transporter

Targeted radiotherapy using 131I-metaiodobenzylguanidine (131I-MIBG) has produced remissions in some neuroblastoma patients. We previously reported that combining 131I-MIBG with the topoisomerase I inhibitor topotecan induced long-term DNA damage and supraadditive toxicity to noradrenaline transporter (NAT)–expressing cells and xenografts. This combination treatment is undergoing clinical evaluation. This present study investigated the potential of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP-1) inhibition, in vitro and in vivo, to further enhance 131I-MIBG/topotecan efficacy. Methods: Combinations of topotecan and the PARP-1 inhibitor PJ34 were assessed for synergism in vitro by combination-index analysis in SK-N-BE(2c) (neuroblastoma) and UVW/NAT (NAT-transfected glioma) cells. Three treatment schedules were evaluated: topotecan administered 24 h before, 24 h after, or simultaneously with PJ34. Combinations of PJ34 and 131I-MIBG and of PJ34 and 131I-MIBG/topotecan were also assessed using similar scheduling. In vivo efficacy was measured by growth delay of tumor xenografts. We also assessed DNA damage by γH2A.X assay, cell cycle progression by fluorescence-activated cell sorting analysis, and PARP-1 activity in treated cells. Results: In vitro, only simultaneous administration of topotecan and PJ34 or PJ34 and 131I-MIBG induced supraadditive toxicity in both cell lines. All scheduled combinations of PJ34 and 131I-MIBG/topotecan induced supraadditive toxicity and increased DNA damage in SK-N-BE(2c) cells, but only simultaneous administration induced enhanced efficacy in UVW/NAT cells. The PJ34 and 131I-MIBG/topotecan combination treatment induced G2 arrest in all cell lines, regardless of the schedule of delivery. In vivo, simultaneous administration of PJ34 and 131I-MIBG/topotecan significantly delayed the growth of SK-N-BE(2c) and UVW/NAT xenografts, compared with 131I-MIBG/topotecan therapy. Conclusion: The antitumor efficacy of topotecan, 131I-MIBG, and 131I-MIBG/topotecan combination treatment was increased by PARP-1 inhibition in vitro and in vivo.

The combined endocrine receptor in breast cancer, a novel approach to traditional hormone receptor interpretation and a better discriminator of outcome than ER and PR alone

Background:The functional role of progesterone receptor (PR) signalling was previously unclear and PR testing in breast cancer is controversial. Recent defining work has highlighted the functional crosstalk that exists between the oestrogen receptor (ER) and PR. The purpose of this retrospective cohort study was to compare the prognostic value of the combined ER and PR score with either ER or PR alone.Methods:Tumour Allred ER and PR scores were reclassified as negative, low and high. The combined endocrine receptor (CER) was calculated as the average of the reclassified ER and PR scores, resulting in three groups: CER negative, impaired and high. Cox proportional hazards models were used to estimate disease-free survival (DFS) and breast cancer-specific survival (BCSS).Results:The CER was a more powerful predictor of 5-year DFS and BCSS than either ER or PR alone. In multivariate analysis that included ER, PR and CER, only CER remained an independent prognostic variable for 5-year DFS (hazard ratio (HR) 0.393; CI: 0.283–0.548, P=0.00001) and BCSS (HR 0.553; CI: 0.423–0.722; P=2.506 × 10−8). In ER-positive (ER+) patients impaired CER was an independent marker of poor outcome for 5-year DFS (HR 2.469; CI: 1.049–5.810; P=0.038) and BCSS (HR 1.946; CI: 1.054–3.596; P=0.033) in multivariate analysis that included grade, lymph node, tumour size, HER2 status and PR status. The results were validated in a separate cohort of patients.Conclusions:Combined endocrine receptor is a more powerful discriminator of patient outcome than either ER or PR alone. Economical and simple, it can identify risk in ER+ early breast cancer and potentially be used for adjuvant cytotoxic chemotherapy decision-making.

Radiosensitization of noradrenaline transporter-expressing tumour cells by proteasome inhibitors and the role of reactive oxygen species

BackgroundThe radiopharmaceutical 131I-metaiodobenzylguanidine (131I-MIBG) is used for the targeted radiotherapy of noradrenaline transporter (NAT)-expressing neuroblastoma. Enhancement of 131I-MIBGs efficacy is achieved by combination with the topoisomerase I inhibitor topotecan - currently being evaluated clinically. Proteasome activity affords resistance of tumour cells to radiation and topoisomerase inhibitors. Therefore, the proteasome inhibitor bortezomib was evaluated with respect to its cytotoxic potency as a single agent and in combination with 131I-MIBG and topotecan. Since elevated levels of reactive oxygen species (ROS) are induced by bortezomib, the role of ROS in tumour cell kill was determined following treatment with bortezomib or the alternative proteasome inhibitor, MG132.MethodsClonogenic assay and growth of tumour xenografts were used to investigate the effects of proteasome inhibitors alone or in combination with radiation treatment. Synergistic interactions in vitro were evaluated by combination index analysis. The dependency of proteasome inhibitor-induced clonogenic kill on ROS generation was assessed using antioxidants.ResultsBortezomib, in the dose range 1 to 30 nM, decreased clonogenic survival of both SK-N-BE(2c) and UVW/NAT cells, and this was prevented by antioxidants. It also acted as a sensitizer in vitro when administered with X-radiation, with 131I-MIBG, or with 131I-MIBG and topotecan. Moreover, bortezomib enhanced the delay of the growth of human tumour xenografts in athymic mice when administered in combination with 131I-MIBG and topotecan. MG132 and bortezomib had similar radiosensitizing potency, but only bortezomib-induced cytotoxicity was ROS-dependent.ConclusionsProteasome inhibition shows promise for the treatment of neuroblastoma in combination with 131I-MIBG and topotecan. Since the cytotoxicity of MG132, unlike that of bortezomib, was not ROS-dependent, the latter proteasome inhibitor may have a favourable toxicity profile in normal tissues.

Preliminary evaluation of prostate-targeted radiotherapy using 131I-MIP-1095 in combination with radiosensitising chemotherapeutic drugs

Despite recent advances in the treatment of metastatic prostate cancer, survival rates are low and treatment options are limited to chemotherapy and hormonal therapy. 131I‐MIP‐1095 is a recently developed prostate‐specific membrane antigen (PSMA)‐targeting, small molecular weight radiopharmaceutical which has anti‐tumour activity as a single agent. Our purpose was to determine in vitro the potential benefit to be gained by combining 131I‐MIP‐1095 with cytotoxic drug treatments.

An evaluation in vitro of the efficacy of nutlin-3 and topotecan in combination with 177 Lu-DOTATATE for the treatment of neuroblastoma

Targeted radiotherapy of metastatic neuroblastoma using the somatostatin receptor (SSTR)-targeted octreotide analogue DOTATATE radiolabelled with lutetium-177 (177Lu-DOTATATE) is a promising strategy. This study evaluates whether its effectiveness may be enhanced by combination with radiosensitising drugs. The growth rate of multicellular tumour spheroids, derived from the neuroblastoma cell lines SK-N-BE(2c), CHLA-15 and CHLA-20, was evaluated following treatment with 177Lu-DOTATATE, nutlin-3 and topotecan alone or in combination. Immunoblotting, immunostaining and flow cytometric analyses were used to determine activation of p53 signalling and cell death. Exposure to 177Lu-DOTATATE resulted in a significant growth delay in CHLA-15 and CHLA-20 spheroids, but not in SK-N-BE(2c) spheroids. Nutlin-3 enhanced the spheroid growth delay induced by topotecan in CHLA-15 and CHLA-20 spheroids, but not in SK-N-BE(2c) spheroids. Importantly, the combination of nutlin-3 with topotecan enhanced the spheroid growth delay induced by X-irradiation or by exposure to 177Lu-DOTATATE. The efficacy of the combination treatments was p53-dependent. These results indicate that targeted radiotherapy of high risk neuroblastoma with 177Lu-DOTATATE may be improved by combination with the radiosensitising drugs nutlin-3 and topotecan.

Cell cycle specific radiosensitisation by the disulfiram and copper complex

The disulfiram and copper complex (DSF:Cu) has emerged as a potent radiosensitising anti-cancer agent. The ability of copper to stabilise DSF in a planar conformation and to inhibit DNA replication enzymes stimulated our investigation of the effect of DSF:Cu on cell cycle regulation. Flow cytometry and immunoblotting were used to assess the effect of DSF:Cu on cell cycle progression of the neuroblastoma cell line SK-N-BE(2c) and the glioma cell line UVW. Treatment with 0.1 and 0.3 μM DSF:Cu inhibited DNA synthesis in SK-N-BE(2c) and UVW cells, respectively. The increased potency of ionising radiation treatment induced by DSF:Cu and/or gemcitabine was determined by clonogenic assay. Treatment with 0.3 μM DSF:Cu resulted in greater radiation kill, exemplified by dose enhancement factor values of 2.64 and 2.84 in SK-N-BE(2c) and UVW cells, respectively. Although DSF:Cu failed to sensitise S phase cells to irradiation, we observed that DSF:Cu radiosensitisation was potentiated by the S phase-specific cytotoxic drug gemcitabine. The efficacy of the combination treatment consisting of DSF:Cu, gemcitabine and ionising radiation was schedule-dependent. Together, these results describe cell cycle specific radiosensitisation by DSF:Cu. The well-established toxicity profiles of DSF and gemcitabine should facilitate their evaluation as a combination treatment in patients undergoing radiotherapy.

The use of targeted radionuclide therapy for the treatment of malignant melanoma in vivo

The Syrian hamster embryo (SHE) assay (pH6.7) is being touted as a ‘‘3R’s’’ alternative in animal laboratory studies. In the SHE assay, traditionally, colonies are counted and scored by eye to determine the transforming potential of test chemicals. Application of infrared (IR) spectroscopy opens up the possibility of comparing test chemicals with negative and positive controls in a high-throughput fashion (1) through objective pattern recognition methods. Such methods are under development under the 1) ‘‘openness’’, and 2) multiple-class requirements: 1) computer systems need to be ‘‘open’’ to new data to refine existing classifiers; 2) furthermore, the existence of multiple classes (i.e., chemical treatment conditions) calls for composite architectures containing many simple classifiers instead a single complicated one. In this study we present two classification strategies contemplating these two principles. The proposed strategies are compared to well established ‘‘closed’’, single-model classifiers. The dataset used in the study was derived from a SHE assay where eight treatment conditions were present [vehicle control (DMSO), D-M, B[a]P, 3-MCA, Anthracene, o-T, 2,4-dT, and MNNG] (2). From the assay, IR spectra (n¼14,000) were obtained using attenuated total reflection Fourier-transform IR spectroscopy. Gradual feeding of the proposed models with training data is shown to gradually improve the classification of test data. Segregation of data along chemical mode of action was observed. Overall, the results strengthen arguments towards using the SHE assay in toxicological assessments and point to IR spectroscopy as a possible alternative to visual scoring.DNA is not chemically inert but faces constant challenges to its stability. One of these is the fusion of adjacent pyrimidine bases by ultra violet (UV) radiation to create cyclobutane pyrimidine dimers (CPDs). Numerous methods of DNA repair have evolved within cells, of which nucleotide excision repair (NER) is responsible for the removal of CPDs and other bulky adducts. To investigate this and other repair pathways various techniques have been developed to detect DNA damage at low resolutions in whole genomes or high resolutions over small sections of a genome. We have developed a novel microarray based method for the genome wide high resolution analysis of DNA damage in yeast which combines the advantages of these, allowing detailed measurement of repair across entire genomes. A program has been written to predict the expected CPD formation based on sequence; this has shown that the genome wide damage detection method is accurate. Additionally, ChIPchip has been used to determine the binding positions of proteins involved in NER and analyse histone modifications after damage induction. Combining these datasets allows protein binding and acetylation levels to be correlated with repair rates. These datasets require bioinformatic tools to analyse and extract results. I have developed a suite of novel tools to process, normalise, display and interrogate these datasets including a new normalisation method which allows accurate comparisons to be made between different factors, revealing changes in acetylation profiles following UV and between different mutant strains, a peak detection method to distinguish protein binding peaks from a background of nonbound regions, revealing many novel binding sites for proteins such as Abf1 and Rad16, and graphical displays to determine patterns that occur at multiple positions throughout genomes, revealing patterns of varying repair rates at regions such as centromeres and telomeres.