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186: The anti-cancer effects of clioquinol on oral cancer
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EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
184 Roles of digoxin and digitoxin in hepatocellular carcinoma W. Huang1 , Y. Jeng2 , I. Fong3 , H. Hsu4 . 1 Hsin Sheng College of Medical Care and Management, Nursing, Taoyuan, Taiwan, 2 National Taiwan University Hospital and College of Medicine, Pathology, Taipei, Taiwan, 3 College of Medicine National Taiwan University, Pathology, Taipei, Taiwan, 4 Department of Internal Medicine Taipei City Hospital, Cardiology, Taipei, Taiwan Background: Digoxin (DG) and digitoxin (DT), composed of digitalis, were the main drugs for heart disease. DG is widely used in the treatment of various heart conditions, namely atrial fibrillation, atrial flutter and sometimes heart failure. DG and DT were mainly metabolized by kidney and liver. It has been known that DG may be a possible therapy for prostate cancer. DT inhibits the growth of cancer cell lines at concentrations commonly found in cardiac patients. hypoxia-induced factor (HIF) activity is involved in angiogenesis required for cancer tumor growth. It has been known that DG down-regulates VEGF through the inhibition of HIF-1alpha under hypoxic conditions in some human cancer cells lines. The aim of this study is to find the role of DG and DT in hepatocellular carcinoma (HCC) cells by observing the proliferation, apoptosis, and morphology in DG and DT treatment. Material and Methods: Several HCC cancer cell lines were treated with DG or DT in dose- and time-responses. Cells apoptosis was observed by MTT assay, cells containing transforming oncogenes grown in focus-forming assay, and cell migration was proved by Wound healing assay. Western blotting also showed the HIF-1 alpha expression in DG and DT treatment. Results: Our preliminary data in vitro study with MTT and focus-forming assay showed that treatment with DG (0.1−1 uM) in PLC5, HA22T and Hep3B liver cancer cell lines, as well as treatment with DT (0.1−1 uM) in PLC5 and HA22T liver cancer cell lines, significantly inhibited the survival and growth in liver cancer cells in 24 hrs and 48 hrs. The morphology of cells was transformed normal to round shape in DG or DT (0.1−1 uM) compared to normal cells. Cell migration decreased significantly under DG at higher doses (0.05−0.2 uM). Western blotting data showed that HIF-1 alpha over-expressed under the treatment of DG at dose of 1 uM after 24 hrs. Conclusions: Our study investigate that DG and DT might promote the cell apoptosis, inhibit migration, and change the cell morphology by the HIF-related conditions in HCC. This finding may be a basis for clinical HCC therapy. No conflict of interest. 185 Regulation of Panax notoginseng extract on metastasis in human colon cancer cells C.C. Wu1 , Y.H. Kuo2 , C.Y. Lee3 , C.C. Tsai4 , S.L. Hsieh2 . 1 Chang Jung Christian University, Nutrition and Health Sciences, Tainan City, Taiwan, 2 National Kaohsiung Marine University, Department of Seafood Science, Kaohsiung City, Taiwan, 3 E-DA Hospital, Department of Chinese Medicine, Kaohsiung City, Taiwan, 4 I-Shou University, College of Medicine The School of Chinese Medicine for Post Baccalaureate, Kaohsiung City, Taiwan Background: Panax notoginseng is a traditional Chinese medicine for cardiovascular disease, but it is limited on anti-metastasis study. Materials and Methods: To investigate the regulation of Panax notoginseng alcohol extract (PNAE) on metastasismigration and adhesion of colon cancer. The human colon cancer cells (HCT-116) cultured was as an experimental model, and wound healing assay, adhesion reaction and regulate molecular expression were analyzed in this study. Results: According to the results, the inhibition percentage of migration on HCT-116 cells were significantly increased after 0.05, 0.1 or 0.5 mg/mL PNAE treatment for 24 and 48 hrs (P < 0.05). From gelatin zymography analysis result show that 0.1 and 0.5 mg/mL PNAE groups were significantly decreased the activity of MMP-2 after 24 hrs incubation (P < 0.05). In adhesion reaction assay results shown 0.1 and 0.5 mg/mL PNAE groups were significantly decreased HCT-116 cells adhesion to endothelial cells (EA. hy926 cells). E-selectin protein levels of EA. hy926 cells were significantly decreased after 0.1 or 0.5 mg/mL PNAE treatment. Conclusion: These results demonstrate the anti-metastatic properties of PNAE. Furthermore, the mechanism is through the inhibition of cell migration and adhesion. No conflict of interest. 186 The anti-cancer effects of clioquinol on oral cancer M.W. Lee1 , P.C. Lin2 , W.C. Tsai2 . 1 Chung Shan Medical University, School of Medical Laboratory and Biotechnology, Taichung City, Taiwan, 2 Kaohsiung Medical University, Department of Medical Laboratory Science and Biotechnology, Kaohsiung City, Taiwan Background: Clioquinol (iodochlorhydroxyquin) is an antifungal drug and antiprotozoal drug. It is neurotoxic in large doses. It is a member of a family of drugs called hydroxyquinolines which inhibit certain enzymes related to DNA replication. The drugs have been found to have activity against both viral and protozoal infections. In a recent study, researchers found that the zinc-binding compound, clioquinol, was able to kill cancer cells. The addition of zinc, which would be expected to reverse the activity/property of a sequestering agent,
was found to potentiate the cytotoxic effects of clioquinol, and direct evidence that clioquinol was acting as a zinc ionophore was obtained. More recently, CQ is a newly discovered anticancer agent. In this study, we focus on the anti-cancer effects of CQ on human oral squamous cell carcinoma (OSCC). Material and Methods: OC-2 oral cell lines established from primary tumors from adult male OSCC patients from Taiwan. HSC-3 derived from human tongue carcinoma with lymph node metastasis was from the Taiwan Collection of Research Bioresources. Cell viability was determined via MTT assay. Cell apoptosis, caspase-3 activation, mitochondria membrane potential change and ROS production were analyzed by flow cytometer and analyzed by WinMDI analysis software. Protein expression was detected by western blot. Results: We found that CQ with copper could enhance the cytotoxicity of CQ in two kinds of oral cancer cells, OC-2 and HSC-3 cells. Further, CQ with copper would induce cell apoptosis, decrease mitochondria membrane potential and increase ROS production in OSCC cells. Moreover, we also found the effect of CQ with copper caused aberrant expression of apoptosis-related protein in intrinsic cell apoptosis pathway. Conclusions: CQ with copper could enhance the cytotoxicity of CQ in oral cancer cells compared to CQ alone. CQ with copper could also induce ROS production, mitochondria disruption, and trigger cell apoptosis via intrinsic (mitochondrial) cell apoptosis pathway in OSCC cells. Our results supported that CQ would act as a potential selective anti-cancer drug due to the accumulation of copper in tumor part but not normal tissue, and induce OSCC cells apoptosis by intrinsic apoptosis pathway. No conflict of interest. 187 The expression of the human Sprouty protein-1 (hSpry1) inversely correlates with proliferation, migration and invasion of the SKOV-3 and 1A9 human ovarian cancer cells S. Masoumi-Moghaddam1 , A. Amini1 , D.L. Morris1 . 1 St George Clinical School, The University of New South Wales, Surgery, Sydney NSW, Australia Background: Sprouty proteins are regulators of MAPK/ERK pathway. We have already shown that the human ovarian cancer cell lines SKOV-3 and 1A9 express low and high levels of the Sprouty protein 1 (Spry1), respectively. In the present study, the effect of the Spry1 overexpression or silencing on behaviour of these malignant cells was investigated. Materials and Methods: Spry1 was silenced in 1A9 cells using the specific siRNA. In parallel, SKOV-3 cells were transiently transfected with either the Spry1 plasmid or the pcDNA3.1 vector. The effect of the altered expression was then functionally investigated using proliferation, MTT, scratch-wound, migration and invasion assays. To evaluate the effect of the Spry1 transfection on the SKOV-3 cell survival, the stably-transfected clones were also selected. Results and discussion: The Spry1 silencing led to a significant increase in growth and proliferation of 1A9 cells at 48 h and 72 h time points as shown by growth (48 h, p: 0.0365; 72 h, p: 0.0228) and MTT assays (48 h, p: 0.0011; 72 h, p: 0.0024) as well as a higher percentage closure of the scratch at the 40 h endpoint (p: 0.0259). In the migration assay, the number of the migrated cells in the silenced group was significantly higher than control examined at hours 14 (p: 0.0125) and 20 (p: 0.0090). Similarly, our invasion assay showed an increased number of the invaded cells in test group at hours 14 (p-value: 0.0298) and 20 (p-value: 0.0373). In SKOV-3 cells, the proliferation of the transfected cells was significantly lower than control on day 3 post-transfection as evaluated by growth (p-value: 0.0003) and MTT (p-value: 0.0042) assays. In the migration assay, the number of the migrated cells in the transfection group was significantly lower examined at hours 6 (p-value: 0.0090) and 12 (p-value: 0.0002). Invasion assay similarly showed a decreased number of the invading cells in the Spry1 group assayed at hours 6 (p-value: 0.0159) and 12 (p-value: 0.0005). Also, a significantly-decreased percentage of the scratch closure was observed at hours 20 and 24 (p-values of 0.0232 and 0.0046, respectively). Finally, the Spry1 stably-transfected clones were almost undetectable after two weeks post selection. Given its pivotal role in modulation of MAPK/ERK, Sprouty regulates key cellular events, including cell proliferation, differentiation and apoptosis. In agreement with our earlier study indicating differential expression of Spry1 in a range of ovarian cancer cells with different invasiveness, our results argue that the Spry1 deregulation confers functional changes in human ovarian cancer cells. Conclusion: Here, we report for the first time that the expression of Spry1 inversely correlates with proliferation, migration, invasion and survival of the human ovarian cancer cells. The clinicopathological relevance of the Sprouty deregulation in ovarian cancer is currently under investigation. No conflict of interest.
EACR-23 Poster Sessions / European Journal of Cancer 50, Suppl. 5 (2014) S23–S242
184 Roles of digoxin and digitoxin in hepatocellular carcinoma W. Huang1 , Y. Jeng2 , I. Fong3 , H. Hsu4 . 1 Hsin Sheng College of Medical Care and Management, Nursing, Taoyuan, Taiwan, 2 National Taiwan University Hospital and College of Medicine, Pathology, Taipei, Taiwan, 3 College of Medicine National Taiwan University, Pathology, Taipei, Taiwan, 4 Department of Internal Medicine Taipei City Hospital, Cardiology, Taipei, Taiwan Background: Digoxin (DG) and digitoxin (DT), composed of digitalis, were the main drugs for heart disease. DG is widely used in the treatment of various heart conditions, namely atrial fibrillation, atrial flutter and sometimes heart failure. DG and DT were mainly metabolized by kidney and liver. It has been known that DG may be a possible therapy for prostate cancer. DT inhibits the growth of cancer cell lines at concentrations commonly found in cardiac patients. hypoxia-induced factor (HIF) activity is involved in angiogenesis required for cancer tumor growth. It has been known that DG down-regulates VEGF through the inhibition of HIF-1alpha under hypoxic conditions in some human cancer cells lines. The aim of this study is to find the role of DG and DT in hepatocellular carcinoma (HCC) cells by observing the proliferation, apoptosis, and morphology in DG and DT treatment. Material and Methods: Several HCC cancer cell lines were treated with DG or DT in dose- and time-responses. Cells apoptosis was observed by MTT assay, cells containing transforming oncogenes grown in focus-forming assay, and cell migration was proved by Wound healing assay. Western blotting also showed the HIF-1 alpha expression in DG and DT treatment. Results: Our preliminary data in vitro study with MTT and focus-forming assay showed that treatment with DG (0.1−1 uM) in PLC5, HA22T and Hep3B liver cancer cell lines, as well as treatment with DT (0.1−1 uM) in PLC5 and HA22T liver cancer cell lines, significantly inhibited the survival and growth in liver cancer cells in 24 hrs and 48 hrs. The morphology of cells was transformed normal to round shape in DG or DT (0.1−1 uM) compared to normal cells. Cell migration decreased significantly under DG at higher doses (0.05−0.2 uM). Western blotting data showed that HIF-1 alpha over-expressed under the treatment of DG at dose of 1 uM after 24 hrs. Conclusions: Our study investigate that DG and DT might promote the cell apoptosis, inhibit migration, and change the cell morphology by the HIF-related conditions in HCC. This finding may be a basis for clinical HCC therapy. No conflict of interest. 185 Regulation of Panax notoginseng extract on metastasis in human colon cancer cells C.C. Wu1 , Y.H. Kuo2 , C.Y. Lee3 , C.C. Tsai4 , S.L. Hsieh2 . 1 Chang Jung Christian University, Nutrition and Health Sciences, Tainan City, Taiwan, 2 National Kaohsiung Marine University, Department of Seafood Science, Kaohsiung City, Taiwan, 3 E-DA Hospital, Department of Chinese Medicine, Kaohsiung City, Taiwan, 4 I-Shou University, College of Medicine The School of Chinese Medicine for Post Baccalaureate, Kaohsiung City, Taiwan Background: Panax notoginseng is a traditional Chinese medicine for cardiovascular disease, but it is limited on anti-metastasis study. Materials and Methods: To investigate the regulation of Panax notoginseng alcohol extract (PNAE) on metastasismigration and adhesion of colon cancer. The human colon cancer cells (HCT-116) cultured was as an experimental model, and wound healing assay, adhesion reaction and regulate molecular expression were analyzed in this study. Results: According to the results, the inhibition percentage of migration on HCT-116 cells were significantly increased after 0.05, 0.1 or 0.5 mg/mL PNAE treatment for 24 and 48 hrs (P < 0.05). From gelatin zymography analysis result show that 0.1 and 0.5 mg/mL PNAE groups were significantly decreased the activity of MMP-2 after 24 hrs incubation (P < 0.05). In adhesion reaction assay results shown 0.1 and 0.5 mg/mL PNAE groups were significantly decreased HCT-116 cells adhesion to endothelial cells (EA. hy926 cells). E-selectin protein levels of EA. hy926 cells were significantly decreased after 0.1 or 0.5 mg/mL PNAE treatment. Conclusion: These results demonstrate the anti-metastatic properties of PNAE. Furthermore, the mechanism is through the inhibition of cell migration and adhesion. No conflict of interest. 186 The anti-cancer effects of clioquinol on oral cancer M.W. Lee1 , P.C. Lin2 , W.C. Tsai2 . 1 Chung Shan Medical University, School of Medical Laboratory and Biotechnology, Taichung City, Taiwan, 2 Kaohsiung Medical University, Department of Medical Laboratory Science and Biotechnology, Kaohsiung City, Taiwan Background: Clioquinol (iodochlorhydroxyquin) is an antifungal drug and antiprotozoal drug. It is neurotoxic in large doses. It is a member of a family of drugs called hydroxyquinolines which inhibit certain enzymes related to DNA replication. The drugs have been found to have activity against both viral and protozoal infections. In a recent study, researchers found that the zinc-binding compound, clioquinol, was able to kill cancer cells. The addition of zinc, which would be expected to reverse the activity/property of a sequestering agent,
was found to potentiate the cytotoxic effects of clioquinol, and direct evidence that clioquinol was acting as a zinc ionophore was obtained. More recently, CQ is a newly discovered anticancer agent. In this study, we focus on the anti-cancer effects of CQ on human oral squamous cell carcinoma (OSCC). Material and Methods: OC-2 oral cell lines established from primary tumors from adult male OSCC patients from Taiwan. HSC-3 derived from human tongue carcinoma with lymph node metastasis was from the Taiwan Collection of Research Bioresources. Cell viability was determined via MTT assay. Cell apoptosis, caspase-3 activation, mitochondria membrane potential change and ROS production were analyzed by flow cytometer and analyzed by WinMDI analysis software. Protein expression was detected by western blot. Results: We found that CQ with copper could enhance the cytotoxicity of CQ in two kinds of oral cancer cells, OC-2 and HSC-3 cells. Further, CQ with copper would induce cell apoptosis, decrease mitochondria membrane potential and increase ROS production in OSCC cells. Moreover, we also found the effect of CQ with copper caused aberrant expression of apoptosis-related protein in intrinsic cell apoptosis pathway. Conclusions: CQ with copper could enhance the cytotoxicity of CQ in oral cancer cells compared to CQ alone. CQ with copper could also induce ROS production, mitochondria disruption, and trigger cell apoptosis via intrinsic (mitochondrial) cell apoptosis pathway in OSCC cells. Our results supported that CQ would act as a potential selective anti-cancer drug due to the accumulation of copper in tumor part but not normal tissue, and induce OSCC cells apoptosis by intrinsic apoptosis pathway. No conflict of interest. 187 The expression of the human Sprouty protein-1 (hSpry1) inversely correlates with proliferation, migration and invasion of the SKOV-3 and 1A9 human ovarian cancer cells S. Masoumi-Moghaddam1 , A. Amini1 , D.L. Morris1 . 1 St George Clinical School, The University of New South Wales, Surgery, Sydney NSW, Australia Background: Sprouty proteins are regulators of MAPK/ERK pathway. We have already shown that the human ovarian cancer cell lines SKOV-3 and 1A9 express low and high levels of the Sprouty protein 1 (Spry1), respectively. In the present study, the effect of the Spry1 overexpression or silencing on behaviour of these malignant cells was investigated. Materials and Methods: Spry1 was silenced in 1A9 cells using the specific siRNA. In parallel, SKOV-3 cells were transiently transfected with either the Spry1 plasmid or the pcDNA3.1 vector. The effect of the altered expression was then functionally investigated using proliferation, MTT, scratch-wound, migration and invasion assays. To evaluate the effect of the Spry1 transfection on the SKOV-3 cell survival, the stably-transfected clones were also selected. Results and discussion: The Spry1 silencing led to a significant increase in growth and proliferation of 1A9 cells at 48 h and 72 h time points as shown by growth (48 h, p: 0.0365; 72 h, p: 0.0228) and MTT assays (48 h, p: 0.0011; 72 h, p: 0.0024) as well as a higher percentage closure of the scratch at the 40 h endpoint (p: 0.0259). In the migration assay, the number of the migrated cells in the silenced group was significantly higher than control examined at hours 14 (p: 0.0125) and 20 (p: 0.0090). Similarly, our invasion assay showed an increased number of the invaded cells in test group at hours 14 (p-value: 0.0298) and 20 (p-value: 0.0373). In SKOV-3 cells, the proliferation of the transfected cells was significantly lower than control on day 3 post-transfection as evaluated by growth (p-value: 0.0003) and MTT (p-value: 0.0042) assays. In the migration assay, the number of the migrated cells in the transfection group was significantly lower examined at hours 6 (p-value: 0.0090) and 12 (p-value: 0.0002). Invasion assay similarly showed a decreased number of the invading cells in the Spry1 group assayed at hours 6 (p-value: 0.0159) and 12 (p-value: 0.0005). Also, a significantly-decreased percentage of the scratch closure was observed at hours 20 and 24 (p-values of 0.0232 and 0.0046, respectively). Finally, the Spry1 stably-transfected clones were almost undetectable after two weeks post selection. Given its pivotal role in modulation of MAPK/ERK, Sprouty regulates key cellular events, including cell proliferation, differentiation and apoptosis. In agreement with our earlier study indicating differential expression of Spry1 in a range of ovarian cancer cells with different invasiveness, our results argue that the Spry1 deregulation confers functional changes in human ovarian cancer cells. Conclusion: Here, we report for the first time that the expression of Spry1 inversely correlates with proliferation, migration, invasion and survival of the human ovarian cancer cells. The clinicopathological relevance of the Sprouty deregulation in ovarian cancer is currently under investigation. No conflict of interest.