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586: Metallothionein up-regulation in response to cadmium exposure in a normal human urothelial cell system
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242 extracts were carried out and used to analyze the activity and expression of diverse XMEs and that of their regulatory transcription factors, by Real Time-PCR, Co-immuno-precipitation, Western Blot and activity assays. The formation of the mammary gland DMBA-DNA adducts was detected by ImmnoHistochemistry. Results and Discussion: An increase in the hepatic activity of the Phase I enzymes (CYP1A1 and CYP1B1) 12 hours after the DMBA induction was observed, reaching the maximum levels at this time point. The steady-state levels were achieved five days after induction. The Phase I mRNA expression in the liver and the DMBA-DNA adducts formation in the mammary gland showed similar patterns to that of the enzyme activity. The Phase II enzymes (GSTP1 and NQO1) response to the DMBA induction was slightly delayed, reaching the highest activity levels around 5 days after induction, not matching with the mRNA expression levels. The HCO diet mainly modulated the Phase I enzymes triggering a faster response and a higher adducts formation in the early hours after DMBA induction compared with the LF diet. In contrast, the HEVOO diet displayed an effect similar to the LF or, in some cases, intermediate between the LF and the HCO diets. Conclusion: Our results drive us to hypothesise that the differential XMEs modulation by the dietary lipids may be important in the cancer susceptibility window in the early hours after the PAH exposure. These results are in accordance with the previously demonstrated stimulating action of the HCO diet and the potentially chemopreventive action of the HEVOO diet on experimental mammary carcinogenesis. No conflict of interest. 585 The synthetic Tryptanthrin analogue suppresses STATs signalling and induces caspase dependent apoptosis via ERK up regulation in human leukemia HL-60 cells A. Pathania1 , S. Kumar1 , S. Guru1 , F. Malik1 , S. Bhushan1 , A. Kumar1 . 1 Indian Institute of Integrative Medicine, Cancer Pharmacology, Jammu, India Background: The aim of the study is to explore the anticancer mechanism of natural product Tryptanthrin analogue, Tryptanthrin bromo (TBr) on human acute myleiod leukemia cell line HL-60. Material and Methods: The antiproliferative effect of TBr in leukemia cell lines HL-60, K-562 and MOLT-4 was determined by 3-(4,5-dimethylthiazol2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Mitochondrial membrane potential loss and cell cycle analysis of TBr treated HL-60 cells were analysed by flow cytometry. Effect of TBr on STATs, Erk and apoptotic signalling cascade in HL-60 cells were analysed by using western blotting and fluorescence microscopy. Immunoprecipitation studies were performed to explore the ubiquitin dependent STAT-3 degradation by TBr. Results: Tryptanthrin is a natural product, which has been reported to have several medicinal properties. In this study, we tried to investigate the detailed molecular mechanism of its bromo analogue (TBr), a potent cytotoxic agent, in the induction of cancer cell death. It was found that TBr primarily targets STAT3 and ERK signalling during the induction of apoptosis in several human leukemia cell lines. In HL-60 cells, TBr treatment caused early down regulation of p-STAT3 with concomitant up regulation of p-ERK leading to the activation of extrinsic and intrinsic pathways of apoptosis. The mechanism of TBr mediated inhibition of p-STAT3 was found to be due to the activation of ubiquitin dependent degradation of Tyrosine705 and Serine727 p-STAT3. As IL-6 is the main driver of STAT3 pathway, the effect of TBr on cell death was subdued when treated in the combination with IL-6 in HL60 cells. Interestingly, it was also observed that TBr mediated activation of p-ERK also played a role in the induction of cell death as was evidenced by using PD98059 mediated inhibition of ERK pathway. It was further shown that apoptotic protein Bax silencing in HL-60 cells, resist TBr mediated ERK dependent apoptosis, signifying that apoptotic effect of ERK occurred through bax. In summary, Our data suggested that TBr induced cytotoxicity required two independent events, which involved activation of Bax through ERK pathway and down regulation of STAT signalling by inhibition of ubiquitin dependent proteasomal degradation in HL-60 cells. This study has attempted to explore in depth the mechanism of cytotoxicity induced by TBr in human leukemia HL-60 cells. Conclusion: We first time report the STATs inhibitory activity of Tryptanthrin analogue TBr and the role of ERK and Bax in TBr mediated apoptosis in human leukemia HL-60 cells. TBr is potent analogue of Trypthanrin and perhaps serves as a potent chemotherapeutic agent for myleiod and lymphoid leukemia. No conflict of interest. 586 Metallothionein up-regulation in response to cadmium exposure in a normal human urothelial cell system R. McNeill1 , J. Southgate1 . 1 University of York, Jack Birch Unit of Molecular Carcinogenesis, York, United Kingdom Introduction: The development of bladder cancer is being increasingly associated with occupational exposure to heavy metals such as cadmium
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in the steel, haulage and fishing industries. Heavy metals are only weakly mutagenic and have poor DNA binding affinity, meaning their mechanism of carcinogenesis remains unclear. The metallothioneins (MT) are a multi-gene family of proteins which bind to and sequester heavy metals as part of a stress response to exposure, thereby reducing cytotoxicity. Little is known about the specificity of metallothionein induction, or the consequences of elevated metallothionein expression on normal cell phenotype and initiation of carcinogenesis. Most previous work has focussed on the expression of MT in immortalised cell systems, and so the aim of this work was to examine the metallothionein response in normal human urothelial (NHU) cells exposed to cadmium in vitro. Materials and Methods: Normal human urothelium was obtained with informed consent from patients undergoing urological surgery. NHU cells were isolated and established in culture using previously optimised techniques. The proliferating cells were exposed for different periods of time to a human-relevant concentration of cadmium chloride (10 mM; CdCl2 ), and the metallothionein response was assessed using RT-PCR and western blotting. Results and Discussion: 12 hours of cadmium exposure resulted in a striking induction of 5 metallothionein genes in proliferating NHU cells from the MT-1 protein subfamily (MT-1A, 1E, 1F, 1G and 1H), whereas constitutive expression of MT-1X and 2A was observed. Interestingly, no MT-3 expression was found. This is of note as high MT-3 expression has been reported in bladder cancer specimens and in immortalised urothelial cells malignantly transformed by prolonged cadmium exposure. At 48 hours of exposure MT1A protein expression was found to be 5 times more abundant in exposed cells, verifying the up-regulation observed at the transcript level. The induction of MT transcript expression appeared to be transient, with expression returning to control levels 24 hours after the removal of exposure. However MT protein expression remained elevated compared to control cells 24 hours after removal. Conclusion: This is the first work to study the relationship between cadmium exposure and metallothionein response in a normal urothelial cell system. The results indicate that cadmium exposure results in the induction of a number of MT genes in NHU cells, with some findings contradicting past studies that used immortalised urothelial cell lines. The consequences of this elevated expression on the normal cell have not yet been investigated, but may provide a potential mechanism for the onset of carcinogenesis. No conflict of interest. 587 Are molecular findings of papillary thyroid microcarcinoma associated with its morphology? C. Cacchi1 , E. Maldi2 , R. Boldorini2 , C.J. Haas3 , H. Gabbert1 , S. Allegrini2 . 1 ¨ Universitatsklinikum Dusseldorf, ¨ Pathologie, Dusseldorf, ¨ Germany, 2 University of Piemonte Orientale, Pathologie, Novara, Italy, 3 Klinikum Augsburg, Pathologie, Augsburg, Germany Introduction: Papillary thyroid microcarcinoma (PMC), defined (WHO 2004) as papillary carcinoma 1 cm or less in diameter and found incidentally, represent about 40% of all the thyroid papillary carcinomas having overall excellent prognosis. Their biology and molecular features remain largely unclear. Characteristics associated with higher recurrence include tumor multifocality and lymph node metastasis. Recently it has been proposed a correlation between BRAF V600E mutation and an aggressive behavior (extrathyroideal extension, lymph node metastasis and multifocality) of the tumor. Aim of the present research is to study the relationship between the histology of PMCs and molecular characteristics such BRAF and RAS mutations. Material and Methods: The files of the Pathology Unit of the University Hospital of Novara (Italy) and Klinikum Augsburg (Germany) were searched for all cases of PMCs from 2000 to 2013. A total of 97 PMCs were found, and all of them were reviewed analyzing following aspects: architecture (classical, follicular other type), tumor fibrosis, superficial localisation, multifocality/intraglandular neopalstc spread, psammomabodies, tumor capsule. For 90 cases, after tumor microdissection, the BRAF V600E and N-RAS and H-RAS mutations were assessed by DNA sequencing; only the wild type BRAF cases have been screened for N-RAS and the for H-RAS mutation statuts. Results and Discussion: 37 cases of 90 showed a BRAF V600E mutation, 11 N-RAS (Q61) mutation and 42 are wild type (wt) for both. No H-RAS mutation has been found. The morphology differs among wt PMCs, BRAF mutated PMC and mutated PMCs (NRAS and BRAF). The tumoral fibrosis frequence was 42% in wt PMCs, 70% in BRAF mut PMCs and 62% the mutated PMCs group. They also differ each other respectively for: superficial localization 81%, 75% and 69%, psammomabodies 28%, 18% and 7% and for the presence of tumorcapsule: 32%, 42% and 71%. The follicular type of mutated PMCs had 50% of BRAF and 50% of N-RAs mutations both group with a very similar histology. On the other hand ca 94% of PMCs with classical morphology were BRAF V600E mutated. Conclusion: PMCs with and without BRAF (thought to be per se an unfavorable prognostic factor) and N-RAS mutations appear to be morphologic
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in the steel, haulage and fishing industries. Heavy metals are only weakly mutagenic and have poor DNA binding affinity, meaning their mechanism of carcinogenesis remains unclear. The metallothioneins (MT) are a multi-gene family of proteins which bind to and sequester heavy metals as part of a stress response to exposure, thereby reducing cytotoxicity. Little is known about the specificity of metallothionein induction, or the consequences of elevated metallothionein expression on normal cell phenotype and initiation of carcinogenesis. Most previous work has focussed on the expression of MT in immortalised cell systems, and so the aim of this work was to examine the metallothionein response in normal human urothelial (NHU) cells exposed to cadmium in vitro. Materials and Methods: Normal human urothelium was obtained with informed consent from patients undergoing urological surgery. NHU cells were isolated and established in culture using previously optimised techniques. The proliferating cells were exposed for different periods of time to a human-relevant concentration of cadmium chloride (10 mM; CdCl2 ), and the metallothionein response was assessed using RT-PCR and western blotting. Results and Discussion: 12 hours of cadmium exposure resulted in a striking induction of 5 metallothionein genes in proliferating NHU cells from the MT-1 protein subfamily (MT-1A, 1E, 1F, 1G and 1H), whereas constitutive expression of MT-1X and 2A was observed. Interestingly, no MT-3 expression was found. This is of note as high MT-3 expression has been reported in bladder cancer specimens and in immortalised urothelial cells malignantly transformed by prolonged cadmium exposure. At 48 hours of exposure MT1A protein expression was found to be 5 times more abundant in exposed cells, verifying the up-regulation observed at the transcript level. The induction of MT transcript expression appeared to be transient, with expression returning to control levels 24 hours after the removal of exposure. However MT protein expression remained elevated compared to control cells 24 hours after removal. Conclusion: This is the first work to study the relationship between cadmium exposure and metallothionein response in a normal urothelial cell system. The results indicate that cadmium exposure results in the induction of a number of MT genes in NHU cells, with some findings contradicting past studies that used immortalised urothelial cell lines. The consequences of this elevated expression on the normal cell have not yet been investigated, but may provide a potential mechanism for the onset of carcinogenesis. No conflict of interest. 587 Are molecular findings of papillary thyroid microcarcinoma associated with its morphology? C. Cacchi1 , E. Maldi2 , R. Boldorini2 , C.J. Haas3 , H. Gabbert1 , S. Allegrini2 . 1 ¨ Universitatsklinikum Dusseldorf, ¨ Pathologie, Dusseldorf, ¨ Germany, 2 University of Piemonte Orientale, Pathologie, Novara, Italy, 3 Klinikum Augsburg, Pathologie, Augsburg, Germany Introduction: Papillary thyroid microcarcinoma (PMC), defined (WHO 2004) as papillary carcinoma 1 cm or less in diameter and found incidentally, represent about 40% of all the thyroid papillary carcinomas having overall excellent prognosis. Their biology and molecular features remain largely unclear. Characteristics associated with higher recurrence include tumor multifocality and lymph node metastasis. Recently it has been proposed a correlation between BRAF V600E mutation and an aggressive behavior (extrathyroideal extension, lymph node metastasis and multifocality) of the tumor. Aim of the present research is to study the relationship between the histology of PMCs and molecular characteristics such BRAF and RAS mutations. Material and Methods: The files of the Pathology Unit of the University Hospital of Novara (Italy) and Klinikum Augsburg (Germany) were searched for all cases of PMCs from 2000 to 2013. A total of 97 PMCs were found, and all of them were reviewed analyzing following aspects: architecture (classical, follicular other type), tumor fibrosis, superficial localisation, multifocality/intraglandular neopalstc spread, psammomabodies, tumor capsule. For 90 cases, after tumor microdissection, the BRAF V600E and N-RAS and H-RAS mutations were assessed by DNA sequencing; only the wild type BRAF cases have been screened for N-RAS and the for H-RAS mutation statuts. Results and Discussion: 37 cases of 90 showed a BRAF V600E mutation, 11 N-RAS (Q61) mutation and 42 are wild type (wt) for both. No H-RAS mutation has been found. The morphology differs among wt PMCs, BRAF mutated PMC and mutated PMCs (NRAS and BRAF). The tumoral fibrosis frequence was 42% in wt PMCs, 70% in BRAF mut PMCs and 62% the mutated PMCs group. They also differ each other respectively for: superficial localization 81%, 75% and 69%, psammomabodies 28%, 18% and 7% and for the presence of tumorcapsule: 32%, 42% and 71%. The follicular type of mutated PMCs had 50% of BRAF and 50% of N-RAs mutations both group with a very similar histology. On the other hand ca 94% of PMCs with classical morphology were BRAF V600E mutated. Conclusion: PMCs with and without BRAF (thought to be per se an unfavorable prognostic factor) and N-RAS mutations appear to be morphologic