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Action of hexachlorobenzene on MDA-MB-231 human breast cancer cell invasion and tumour growth
S80
Abstracts / Toxicology Letters 205S (2011) S60–S179
P1063 Action of hexachlorobenzene on MDA-MB-231 human breast cancer cell invasion and tumour growth C.A. Pontillo 1 , P. Rojas 2 , F. Chiappini 1 , C. Ventura 3 , C.M. Cocca 3,∗ , C. Lanari 2 , D.L. Kleiman de Pisarev 1 , A.S. Randi 1 1
Laboratory of Biological Effects of Environmental Pollutants, Human Biochemistry Department, School of Medicine., University of Buenos Aires, Buenos Aires, Argentina, 2 Laboratory of Hormonal Carcinogenesis, Experimental Medicine and Biology Institute (IBYME-CONICET), Buenos Aires, Argentina, 3 Laboratory of Radioisotopes. Physicomathematic Department, School of Pharmacy And Biochemistry, University of Buenos Aires, Buenos Aires, Argentina
Hexachlorobenzene (HCB) is a widespread environmental pollutant and a dioxin-like compound. We demonstrated that HCB stimulates cell migration of MDA-MB-231 breast cancer cells via cSrc/HER1/STAT5b and HER1/ERK1/2 signaling pathways. Increased activity of metalloprotease-9 (MMP-9) is involved in angiogenesis, invasion, and metastasis. We examined HCB action on cell invasion and MMP-9 activity in vitro, and tumor growth and metastasis in vivo. Cells were exposed to HCB (0, 0.005, 0.05, 0.5 and 5 M) for 24 h and then, cell invasion (matrigel coated transwells) and MMP9 activity (gelatine zymography) were assayed. To analyse tumour growth and spontaneous metastasis, MDA-MB-231 cells (6 × 106 ) were injected s.c. into both flanks of female athymic nude mice. After 10 days, HCB (0.3, 3, 30 mg/kg) or vehicle was administered i.p. into nude mice and tumours were measured. Animals were sacrificed at 30 days after treatment and lung, liver and thoracic lymph node tissues were processed for histological analysis. HCB in vitro significantly increased cell invasion (117%, p < 0.05) at 5 M and MMP-9 activity at 0.05, 0.5 and 5 M (15%, 42% and 40%, p < 0.001). In the mouse xenograft assay, HCB (0.3 and 3 mg/kg) increased tumour volume (3244% and 3093%, p < 0.001) and tumour weight (1230%, p < 0.05). Lung, liver and lymph node metastases were registered in few control and treated mice, however the increase induced by HCB was not statistically significant. Our data indicate that HCB promotes MDA-MB-231 breast cancer growth in vivo and invasion and MMP-9 activity in vitro. More experiments are necessary to prove the prometastatic effect of HCB in xenografts. doi:10.1016/j.toxlet.2011.05.297
P1064 Effects of chlorpyrifos on growth of estrogen-dependent human breast cancer cell line ˜ 1 , V. Gaido 1 , D. Martinel Lamas 1 , A.S. C. Ventura 1 , M.A. Núnez Randi 2 , A. Venturino 3 , E.S. Rivera 1 , C.M. Cocca 1,∗ 1 Laboratory of Radioisotopes. Physicomathematic Department, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina, 2 Laboratory of Biological Effects of Environmental Pollutants, Human Biochemistry Department, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina, 3 Chemistry Deparment, School of Engineering, LIBIQUIMA, CONICET, Neuquen, Argentina
It has been reported that environmental chemicals are involved in breast cancer risk. Chlorpyrifos (CPF) is an organophosphorus insecticide, commonly applied for pest control in fruit producing regions of our country. It was also described as endocrine disruptor affecting estrogen responses. Our objective was to evaluate the
effect of CPF on estrogen dependent MCF-7 breast cancer cells. CPF was tested in vitro at 5 × 10−2 ; 0.5; 5 and 50 M. Citotoxicity was studied by MTT assay and proliferation by clonogenic assay and bromodeoxyuridine (BrdU) incorporation. Cell cycle progression was studied by flow cytometry. Western blots were performed for estrogen receptor alpha (ER␣) phosphorylation and Proliferating Cell Nuclear Antigen (PCNA) and p27 expression. Reactive oxygen species (ROS) were determined using DCF fluorescent staining and flow cytometry. Results obtained show that CPF at 50 M induced a non-significant decrease in cell viability and a diminished clonogenic proliferation (41%; p < 0.01). This effect was associated with an increase in ROS content (32%; p < 0.001) and a cell increment in phase sub-G0 (23%; p < 0.001). Conversely, CPF at 5 × 10−2 M induced an increase in BrdU incorporation (38%; p < 0.05) and it was related to a decrease in p27 expression. PCNA (30%; p < 0.05) and ER␣ phosphorylation (36%; p < 0.01) levels were increased. We conclude that high doses of xenobiotics inhibit cell proliferation and our results were not unexpected at those concentrations. However, low levels of CPF significantly induced proliferation in MCF-7 cells and it seems to be related to estrogen dependence of the cells. doi:10.1016/j.toxlet.2011.05.298
P1065 Evaluation of p53 Arg72Pro and XRCC1 Arg399Gln polymorphisms and oxidative stress in a Turkish population with colorectal carcinoma A.B. Engin 1 , B. Karahalil 1 , A. Engin 2 , A.E. Karakaya 1,∗ 1
Department of Toxicology, Gazi University, Faculty of Pharmacy, Ankara, Turkey, 2 Department of General Surgery, Gazi University, Faculty of Medicine, Ankara, Turkey
Despite of the increased knowledge on the molecular and genetic pathways leading to colorectal carcinoma, it is still the most common cause of morbidity and mortality, worldwide. During the cellular metabolism as well as extra cellular processes, reactive oxygen species are constantly generated and when accumulated in the cell, they are assumed to contribute to the process of carcinogenesis by oxidative DNA damage. X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1) is one of the prominent base excision repair enzymes of DNA. On the other hand, the functional loss or deletion of the p53 tumor suppression gene contributes to the oncogenic transformation. Thus, eligible 96 colorectal cancer patients and 108 cancer-free individuals constituting the control group included in the study. The association of p53 Arg72Pro, XRCC1 Arg399Gln polymorphisms and the susceptibility to colorectal carcinoma, and the extend of oxidative stress were evaluated. Genotypes were determined by PCR-RFLP using DNA extracted from peripheral blood cells. Albumin which is the major and predominant circulating antioxidant in serum, was measured by auto-analyzer. The albumin concentrations of colorectal cancer patients were significantly decreased compared to the controls (p > 0.05). The carriers of the variant genotype of neither p53 (OR = 0.371, 95% confidence interval; 0.125–1.107) nor XRCC1 (1.418, 0.548–3.673) did not associated with the increased risk of colorectal cancer, in spite of the increased oxidative stress in cancer patients. doi:10.1016/j.toxlet.2011.05.299
Abstracts / Toxicology Letters 205S (2011) S60–S179
P1063 Action of hexachlorobenzene on MDA-MB-231 human breast cancer cell invasion and tumour growth C.A. Pontillo 1 , P. Rojas 2 , F. Chiappini 1 , C. Ventura 3 , C.M. Cocca 3,∗ , C. Lanari 2 , D.L. Kleiman de Pisarev 1 , A.S. Randi 1 1
Laboratory of Biological Effects of Environmental Pollutants, Human Biochemistry Department, School of Medicine., University of Buenos Aires, Buenos Aires, Argentina, 2 Laboratory of Hormonal Carcinogenesis, Experimental Medicine and Biology Institute (IBYME-CONICET), Buenos Aires, Argentina, 3 Laboratory of Radioisotopes. Physicomathematic Department, School of Pharmacy And Biochemistry, University of Buenos Aires, Buenos Aires, Argentina
Hexachlorobenzene (HCB) is a widespread environmental pollutant and a dioxin-like compound. We demonstrated that HCB stimulates cell migration of MDA-MB-231 breast cancer cells via cSrc/HER1/STAT5b and HER1/ERK1/2 signaling pathways. Increased activity of metalloprotease-9 (MMP-9) is involved in angiogenesis, invasion, and metastasis. We examined HCB action on cell invasion and MMP-9 activity in vitro, and tumor growth and metastasis in vivo. Cells were exposed to HCB (0, 0.005, 0.05, 0.5 and 5 M) for 24 h and then, cell invasion (matrigel coated transwells) and MMP9 activity (gelatine zymography) were assayed. To analyse tumour growth and spontaneous metastasis, MDA-MB-231 cells (6 × 106 ) were injected s.c. into both flanks of female athymic nude mice. After 10 days, HCB (0.3, 3, 30 mg/kg) or vehicle was administered i.p. into nude mice and tumours were measured. Animals were sacrificed at 30 days after treatment and lung, liver and thoracic lymph node tissues were processed for histological analysis. HCB in vitro significantly increased cell invasion (117%, p < 0.05) at 5 M and MMP-9 activity at 0.05, 0.5 and 5 M (15%, 42% and 40%, p < 0.001). In the mouse xenograft assay, HCB (0.3 and 3 mg/kg) increased tumour volume (3244% and 3093%, p < 0.001) and tumour weight (1230%, p < 0.05). Lung, liver and lymph node metastases were registered in few control and treated mice, however the increase induced by HCB was not statistically significant. Our data indicate that HCB promotes MDA-MB-231 breast cancer growth in vivo and invasion and MMP-9 activity in vitro. More experiments are necessary to prove the prometastatic effect of HCB in xenografts. doi:10.1016/j.toxlet.2011.05.297
P1064 Effects of chlorpyrifos on growth of estrogen-dependent human breast cancer cell line ˜ 1 , V. Gaido 1 , D. Martinel Lamas 1 , A.S. C. Ventura 1 , M.A. Núnez Randi 2 , A. Venturino 3 , E.S. Rivera 1 , C.M. Cocca 1,∗ 1 Laboratory of Radioisotopes. Physicomathematic Department, School of Pharmacy and Biochemistry, University of Buenos Aires, Buenos Aires, Argentina, 2 Laboratory of Biological Effects of Environmental Pollutants, Human Biochemistry Department, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina, 3 Chemistry Deparment, School of Engineering, LIBIQUIMA, CONICET, Neuquen, Argentina
It has been reported that environmental chemicals are involved in breast cancer risk. Chlorpyrifos (CPF) is an organophosphorus insecticide, commonly applied for pest control in fruit producing regions of our country. It was also described as endocrine disruptor affecting estrogen responses. Our objective was to evaluate the
effect of CPF on estrogen dependent MCF-7 breast cancer cells. CPF was tested in vitro at 5 × 10−2 ; 0.5; 5 and 50 M. Citotoxicity was studied by MTT assay and proliferation by clonogenic assay and bromodeoxyuridine (BrdU) incorporation. Cell cycle progression was studied by flow cytometry. Western blots were performed for estrogen receptor alpha (ER␣) phosphorylation and Proliferating Cell Nuclear Antigen (PCNA) and p27 expression. Reactive oxygen species (ROS) were determined using DCF fluorescent staining and flow cytometry. Results obtained show that CPF at 50 M induced a non-significant decrease in cell viability and a diminished clonogenic proliferation (41%; p < 0.01). This effect was associated with an increase in ROS content (32%; p < 0.001) and a cell increment in phase sub-G0 (23%; p < 0.001). Conversely, CPF at 5 × 10−2 M induced an increase in BrdU incorporation (38%; p < 0.05) and it was related to a decrease in p27 expression. PCNA (30%; p < 0.05) and ER␣ phosphorylation (36%; p < 0.01) levels were increased. We conclude that high doses of xenobiotics inhibit cell proliferation and our results were not unexpected at those concentrations. However, low levels of CPF significantly induced proliferation in MCF-7 cells and it seems to be related to estrogen dependence of the cells. doi:10.1016/j.toxlet.2011.05.298
P1065 Evaluation of p53 Arg72Pro and XRCC1 Arg399Gln polymorphisms and oxidative stress in a Turkish population with colorectal carcinoma A.B. Engin 1 , B. Karahalil 1 , A. Engin 2 , A.E. Karakaya 1,∗ 1
Department of Toxicology, Gazi University, Faculty of Pharmacy, Ankara, Turkey, 2 Department of General Surgery, Gazi University, Faculty of Medicine, Ankara, Turkey
Despite of the increased knowledge on the molecular and genetic pathways leading to colorectal carcinoma, it is still the most common cause of morbidity and mortality, worldwide. During the cellular metabolism as well as extra cellular processes, reactive oxygen species are constantly generated and when accumulated in the cell, they are assumed to contribute to the process of carcinogenesis by oxidative DNA damage. X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1) is one of the prominent base excision repair enzymes of DNA. On the other hand, the functional loss or deletion of the p53 tumor suppression gene contributes to the oncogenic transformation. Thus, eligible 96 colorectal cancer patients and 108 cancer-free individuals constituting the control group included in the study. The association of p53 Arg72Pro, XRCC1 Arg399Gln polymorphisms and the susceptibility to colorectal carcinoma, and the extend of oxidative stress were evaluated. Genotypes were determined by PCR-RFLP using DNA extracted from peripheral blood cells. Albumin which is the major and predominant circulating antioxidant in serum, was measured by auto-analyzer. The albumin concentrations of colorectal cancer patients were significantly decreased compared to the controls (p > 0.05). The carriers of the variant genotype of neither p53 (OR = 0.371, 95% confidence interval; 0.125–1.107) nor XRCC1 (1.418, 0.548–3.673) did not associated with the increased risk of colorectal cancer, in spite of the increased oxidative stress in cancer patients. doi:10.1016/j.toxlet.2011.05.299