- Email: [email protected]
Ginsenoside Rd inhibits adhesion and migration through inactivation of MAPK signaling and induces focal adhesion formation in HepG2 cells
Abstracts
Results: Patents filed by firms from the food and agriculture segments have by far outnumbered those from chemicals and pharmaceuticals. Additionally, when comparing patents according to industry sectors with patents according to subject areas, we could show that there are clear signs of convergence. Discussion: Functional food products are emerging at the borderline of the food as well as the pharmaceutical industry. Their development has led to a new inter-industry segment offering a plethora of opportunities for innovation which indicates the trend of industry convergence at the borderline of foods and drugs. A closer look at probiotics via patent data shows that application areas shift between food, drug and personal care applications. Keywords: Patents, Probiotics, Industry convergence doi:10.1016/j.ejphar.2011.09.230
N−3 PUFAs increase cytotoxicity to cytostatics in vitro: A role for DPA formed by EPA elongation? F.J. Dijka,⁎, M. van Dijka, J.M. Argilésc, A. Lavianod, A. van Helvoorta, K. van Norrena,b et al. a
Danone Research, Netherlands Wageningen University, Netherlands c Universitat de Barcelona, Spain d University La Sapienza, Italy b
Rationale: The cytotoxic effect of cytostatics drugs on cancer cells in vitro has been described to become more efficient after preincubation with n−3 fatty acids. The most described n−3 fatty acids are eicosapentaenoic acid (EPA, 20:5(n−3)) and docosahexaenoic acid (DHA, 22:6(n−3)). Docosapentaenoic acid (DPA, 22:5(n− 3)) is less familiar and can be formed intracellular by the elongation of EPA. In this in vitro study, the effect of the commonly clinically used cytostatics doxorubicin (DOX) and cisplatin (CIS) on metabolic activity was investigated after pre-incubation with EPA, DHA or DPA. Methods: Murine colon C26 adenocarcinoma cells were incubated for 4 days with 50 μM EPA (93%), DHA (99%) or DPA (97%), followed by 24 h incubation with DOX or CIS. Metabolic activity was quantified by WST-1. Gas chromatography was used to measure total cellular fatty acid content of the cells and to confirm purity of the fatty acid stocks. Results: DHA incubation increased cellular DHA content by 25.3%. EPA incubation increased cellular EPA content by 7.2%, but also DPA by 30% and DHA by 0.7%. DPA incubation resulted in 38.2% DPA, 5.7% EPA and 1.1% DHA content of the cancer cells. Furthermore, pre-incubation with EPA, DHA or DPA decreased the metabolic activity of the cells after CIS and DOX incubation (see Table). Decrease in metabolic activity DHA EPA DPA
25 μM CIS 5% (p = 0.219) 23%*(p = 0.013) 38%*(p = 0.013)
e17
Keywords: n−3 PUFAs, Cytostatic drugs, C26 colon adenocarcinoma cells, Cytotoxicity doi:10.1016/j.ejphar.2011.09.231
Nutritional knowledge in breast cancer patients M.N. Patellaa, C. Ghiottob, R. Pertilec, M. Morelloa, D. Fedelea, A. Jirillob,⁎ a
Dietetics and Clinical Nutrition Service, Italy Istituto Oncologico Veneto IRCCS, Italy c University of Verona, Italy E-mail address: [email protected] (A. Jirillo) b
Non-balanced diet and obesity are considered significant risk factors for many cancers. In Breast Cancer (BC) patients heavy association between overweight and disease risk has been noted. Authors suggest that cancer survivors could benefit from interventions of nutritional education. Therefore we verified in detail nutritional knowledge (NK) of 296 adult BC outpatients consecutively attending our Centre from 2004 to 2008 and we evaluated links between nutritional knowledge and demographic, anthropometric and metabolic variables. Patients completed a questionnaire developed in the UK [Parmenter K, Wardle J.]. The 114 items of the questionnaire cover four areas of nutrition knowledge: 1—awareness of current expert dietary recommendations, 2—understanding of terms such as fibre, saturated and unsaturated fats, cholesterol and knowledge of which foods provide the recommended nutrients, 3—ability to choose the healthiest foods (practical food choice), 4—knowledge of diet– disease relationships. Mean percentage of correct answers was almost 60%. Very few patients (9.6%) knew experts' recommendations to consume more complex carbohydrate foods and only 46.7% of respondents were conscious that the recommended portions of fruit and vegetables are at least five/day. Large confusion was in knowledge of which foods provide saturated or unsaturated fats and few patients were able to identify correctly antioxidant vitamins. Level of education and civil status were variables with the higher impact on global nutritional knowledge. When increasing the level of education, total score increased by 8.18 points (pb 0.00). BC single patients had total score smaller than married (p b 0.01). No significant correlation was between NK scores and other data, as previous slimming diets, body mass index or metabolic diseases. In conclusion, the knowledge about messages of experts and public educational campaigns is still incomplete. Detailed understanding of the deficiencies in NK of BC patients may help to develop more targeted education interventions. Keywords: Breast cancer, Nutritional knowledge doi:10.1016/j.ejphar.2011.09.232
6.25 μM DOX 12%*(p = 0.014) 30%*(p = 0.014) 43%*(p = 0.014)
Conclusions: Like previous publications, this study confirms that incorporation of EPA in cancer cells can further decrease the metabolic activity of cytostatics. However, this study shows firstly that DPA plays an important role in the observed effect on metabolic activity. Since most of the EPA is converted to DPA intracellular, it is most likely that DPA is responsible for the decrease in metabolic activity and consequently the increase in cytotoxicity of the tested cytostatics.
Ginsenoside Rd inhibits adhesion and migration through inactivation of MAPK signaling and induces focal adhesion formation in HepG2 cells J.H. Yoon, Y.J. Choi, L.J. Kang, S.G. Lee⁎ Chonnam National University, Republic of Korea E-mail address: [email protected] (S.G. Lee) Ginsenoside Rd is a protopanaxadiol-type ginsenoside found in ginseng and the active ingredient in several oriental herbal medicines. We investigated the effects of ginsenoside Rd on tumor invasion and metastasis in human hepatocellular carcinoma HepG2 and its possible
e18
Abstracts
mechanism of action. HepG2 cells were treated with ginsenoside Rd at different concentrations. Scratch wound and Boyden chamber assays were used to determine the effects of ginsenoside Rd on the migration and invasiveness of HepG2 cells, respectively. The molecular mechanisms by which ginsenoside Rd inhibits the invasion and migration of HepG2 cells were investigated by RT-PCR, Western blotting, gelatin zymography, and treatment with inhibitors of MAPK signaling. Immunofluorescence analysis was carried out to evaluate the effect of ginsenoside Rd on focal adhesion formation in HepG2 cells. Treatment with ginsenoside Rd dose- and time-dependently inhibited the migration and invasion of HepG2 cells. It was achieved by reducing the expression of MMP-1, MMP-2, and MMP-7, by blocking MAPK signaling by inhibiting the phosphorylation of ERK and p38 MAPK, and by inducing focal adhesion formation and modulating vinculin localization and expression. Treatment of HepG2 cells with ginsenoside Rd significantly inhibited metastasis, most likely by blocking MMP activation and MAPK signaling pathways involved in cancer cell migration. This study may be useful for the development of novel chemotherapeutic agents for the treatment of malignant cancers. [This work was supported by the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Project No. 2010-0020141) to S-G Lee]. Keywords: Ginsenoside Rd, Anti-metastasis, MMP, Vinculin doi:10.1016/j.ejphar.2011.09.233
Quercetin synergistically induces sensitivity to 5-fluorouracil through p53 modulation in colorectal cancer cells C.P.R. Xavier, C.F. Lima, C. Pereira-Wilson⁎ University of Minho, Portugal E-mail address: [email protected] (C. Pereira-Wilson) Colorectal tumors (CRC) with microsatellite instability (MSI) show resistance to chemotherapy with 5-fluorouracil (5-FU), the most widely used pharmacological drug for CRC treatment. The aims of this study were to identify compounds that increase sensitivity of MSI CRC cells to 5-FU and characterize their dependence on the p53 status of the cells. Two MSI human CRC derived cell lines were used: CO115 wildtype for p53 and HCT15 that harbors a p53 mutation. The sensitivity of these cells to 5-FU was evaluated by TUNEL assay and the effects on apoptosis induction of co-incubation of the flavonoids, quercetin (Q) or luteolin (L), with 5-FU were characterized. The mechanisms of apoptosis induction were assessed by western blot and p53 mediated effects confirmed by small interference RNA (siRNA) in CO115 and in HCT116 wt and p53 knockout cells. Our results demonstrate that CO115 is more sensitive to 5-FU than the p53 mutated HCT15. Additive effects on apoptosis were shown for L (in both cell lines) and Q (in HCT15). In CO115 Q synergistically induced apoptosis with 5-FU. Apoptosis induction was caspase dependent in CO115 cells but not in HCT15 cells. Both flavonoids increased p53 expression in both cell lines, an effect particularly remarkable for Q. The synergistic effect of Q and 5-FU in CO115 involved the activation of the mitochondrial pathway with an increase in the expression of cleaved caspase 9 and 3 and PARP, as well as a decrease in Bcl-2 expression. Importantly, knockdown of p53 by siRNA in CO115 cells and p53 knockout in HCT116 cells totally abrogated apoptosis induction, demonstrating the dependence on p53 modulation of apoptosis induction by Q. This study suggests the potential applicability of these phytochemicals for enhancement 5-FU efficiency in CRC therapy, especially Q in p53 wild-type tumors.
Keywords: Quercetin, 5-Fluorouracil, Colorectal cancer, p53 doi:10.1016/j.ejphar.2011.09.234
Evaluation of cactus pear (Opuntia spp.) extracts as promising bioactive ingredients for colon cancer therapy J. Poejoa,⁎, A.A. Matiasa,b, M.R. Bronzeb,c, C.M.M. Duartea,b, A.T. Serrab a
ITQB/UNL, Portugal IBET, Portugal c iMED-FFUL, Portugal E-mail address: [email protected] (J. Poejo) b
Cancer is one of the most causes of death worldwide. In particular, colorectal cancer is the second most frequent malignant disease in Europe. Treatments for recurrent and metastatic diseases remain a centre of clinical attention. While continuing efforts have been made for discovering new molecular target-based molecules, there is an emerging interest in chemotherapeutic application of natural substances. Epidemiological data suggests that the ingestion of phytochemicals from fruits and vegetables may contribute to reduce the incidence of cancer in humans. The mechanisms by which these compounds inhibit tumourgenesis include inhibition of tumour cell mediated protease activity, attenuation of tumour angiogenesis, induction of cell cycle arrest and promotion of apoptosis. Cactus (Opuntia spp.) fruits and cladodes have been widely used as food and in folk medicine. Nutraceutical benefits of fruits are believed to be related to the presence of ascorbic acid, flavonoids, betaxanthin and betacyanin. Recently, several studies demonstrated that cactus pear juices inhibit the proliferation of human cancer cell lines suggesting that cactus compounds could be considered as promising ingredients for chemoprevention and chemotherapy. Within this context, the main aim of this study was to develop natural ingredients from cactus pear and evaluate their potential use as natural chemotherapeutic agents on colon cancer. Different varieties of cactus pear were screened for their antiproliferative effect on HT29 cells. The phenolic content was determined using HPLC technique in order to understand which compounds are responsible for the anticancer activity. The most promising varieties were selected and further processed using macroporous resin, aiming to develop polyphenol-rich concentrates. The anticancer activity of final products was evaluated by quantifying the effective dose values and analysing cell cycle arrest on HT29. The results obtained were compared with doxorubicin, a conventional drug used in cancer treatment. Additionally, the development of a drug-resistant HT29 cell culture was performed aiming to evaluate the bioproducts' potential in overcoming the main drawback of chemotherapy. Keywords: Opuntia, Natural ingredients, Colon cancer doi:10.1016/j.ejphar.2011.09.235
Aloe vera and honey solution modulates the oxidative stress, calpain activity and survival in tumour-bearing rats R. Tomasin⁎, R.S. Andrade, M.C.C. Gomes-Marcondes State University of Campinas, Brazil E-mail address: [email protected] (R. Tomasin) Oxidative stress is linked to several tumorigenic events and hostwaste, and also to senescence and apoptosis, the major mechanisms in
Results: Patents filed by firms from the food and agriculture segments have by far outnumbered those from chemicals and pharmaceuticals. Additionally, when comparing patents according to industry sectors with patents according to subject areas, we could show that there are clear signs of convergence. Discussion: Functional food products are emerging at the borderline of the food as well as the pharmaceutical industry. Their development has led to a new inter-industry segment offering a plethora of opportunities for innovation which indicates the trend of industry convergence at the borderline of foods and drugs. A closer look at probiotics via patent data shows that application areas shift between food, drug and personal care applications. Keywords: Patents, Probiotics, Industry convergence doi:10.1016/j.ejphar.2011.09.230
N−3 PUFAs increase cytotoxicity to cytostatics in vitro: A role for DPA formed by EPA elongation? F.J. Dijka,⁎, M. van Dijka, J.M. Argilésc, A. Lavianod, A. van Helvoorta, K. van Norrena,b et al. a
Danone Research, Netherlands Wageningen University, Netherlands c Universitat de Barcelona, Spain d University La Sapienza, Italy b
Rationale: The cytotoxic effect of cytostatics drugs on cancer cells in vitro has been described to become more efficient after preincubation with n−3 fatty acids. The most described n−3 fatty acids are eicosapentaenoic acid (EPA, 20:5(n−3)) and docosahexaenoic acid (DHA, 22:6(n−3)). Docosapentaenoic acid (DPA, 22:5(n− 3)) is less familiar and can be formed intracellular by the elongation of EPA. In this in vitro study, the effect of the commonly clinically used cytostatics doxorubicin (DOX) and cisplatin (CIS) on metabolic activity was investigated after pre-incubation with EPA, DHA or DPA. Methods: Murine colon C26 adenocarcinoma cells were incubated for 4 days with 50 μM EPA (93%), DHA (99%) or DPA (97%), followed by 24 h incubation with DOX or CIS. Metabolic activity was quantified by WST-1. Gas chromatography was used to measure total cellular fatty acid content of the cells and to confirm purity of the fatty acid stocks. Results: DHA incubation increased cellular DHA content by 25.3%. EPA incubation increased cellular EPA content by 7.2%, but also DPA by 30% and DHA by 0.7%. DPA incubation resulted in 38.2% DPA, 5.7% EPA and 1.1% DHA content of the cancer cells. Furthermore, pre-incubation with EPA, DHA or DPA decreased the metabolic activity of the cells after CIS and DOX incubation (see Table). Decrease in metabolic activity DHA EPA DPA
25 μM CIS 5% (p = 0.219) 23%*(p = 0.013) 38%*(p = 0.013)
e17
Keywords: n−3 PUFAs, Cytostatic drugs, C26 colon adenocarcinoma cells, Cytotoxicity doi:10.1016/j.ejphar.2011.09.231
Nutritional knowledge in breast cancer patients M.N. Patellaa, C. Ghiottob, R. Pertilec, M. Morelloa, D. Fedelea, A. Jirillob,⁎ a
Dietetics and Clinical Nutrition Service, Italy Istituto Oncologico Veneto IRCCS, Italy c University of Verona, Italy E-mail address: [email protected] (A. Jirillo) b
Non-balanced diet and obesity are considered significant risk factors for many cancers. In Breast Cancer (BC) patients heavy association between overweight and disease risk has been noted. Authors suggest that cancer survivors could benefit from interventions of nutritional education. Therefore we verified in detail nutritional knowledge (NK) of 296 adult BC outpatients consecutively attending our Centre from 2004 to 2008 and we evaluated links between nutritional knowledge and demographic, anthropometric and metabolic variables. Patients completed a questionnaire developed in the UK [Parmenter K, Wardle J.]. The 114 items of the questionnaire cover four areas of nutrition knowledge: 1—awareness of current expert dietary recommendations, 2—understanding of terms such as fibre, saturated and unsaturated fats, cholesterol and knowledge of which foods provide the recommended nutrients, 3—ability to choose the healthiest foods (practical food choice), 4—knowledge of diet– disease relationships. Mean percentage of correct answers was almost 60%. Very few patients (9.6%) knew experts' recommendations to consume more complex carbohydrate foods and only 46.7% of respondents were conscious that the recommended portions of fruit and vegetables are at least five/day. Large confusion was in knowledge of which foods provide saturated or unsaturated fats and few patients were able to identify correctly antioxidant vitamins. Level of education and civil status were variables with the higher impact on global nutritional knowledge. When increasing the level of education, total score increased by 8.18 points (pb 0.00). BC single patients had total score smaller than married (p b 0.01). No significant correlation was between NK scores and other data, as previous slimming diets, body mass index or metabolic diseases. In conclusion, the knowledge about messages of experts and public educational campaigns is still incomplete. Detailed understanding of the deficiencies in NK of BC patients may help to develop more targeted education interventions. Keywords: Breast cancer, Nutritional knowledge doi:10.1016/j.ejphar.2011.09.232
6.25 μM DOX 12%*(p = 0.014) 30%*(p = 0.014) 43%*(p = 0.014)
Conclusions: Like previous publications, this study confirms that incorporation of EPA in cancer cells can further decrease the metabolic activity of cytostatics. However, this study shows firstly that DPA plays an important role in the observed effect on metabolic activity. Since most of the EPA is converted to DPA intracellular, it is most likely that DPA is responsible for the decrease in metabolic activity and consequently the increase in cytotoxicity of the tested cytostatics.
Ginsenoside Rd inhibits adhesion and migration through inactivation of MAPK signaling and induces focal adhesion formation in HepG2 cells J.H. Yoon, Y.J. Choi, L.J. Kang, S.G. Lee⁎ Chonnam National University, Republic of Korea E-mail address: [email protected] (S.G. Lee) Ginsenoside Rd is a protopanaxadiol-type ginsenoside found in ginseng and the active ingredient in several oriental herbal medicines. We investigated the effects of ginsenoside Rd on tumor invasion and metastasis in human hepatocellular carcinoma HepG2 and its possible
e18
Abstracts
mechanism of action. HepG2 cells were treated with ginsenoside Rd at different concentrations. Scratch wound and Boyden chamber assays were used to determine the effects of ginsenoside Rd on the migration and invasiveness of HepG2 cells, respectively. The molecular mechanisms by which ginsenoside Rd inhibits the invasion and migration of HepG2 cells were investigated by RT-PCR, Western blotting, gelatin zymography, and treatment with inhibitors of MAPK signaling. Immunofluorescence analysis was carried out to evaluate the effect of ginsenoside Rd on focal adhesion formation in HepG2 cells. Treatment with ginsenoside Rd dose- and time-dependently inhibited the migration and invasion of HepG2 cells. It was achieved by reducing the expression of MMP-1, MMP-2, and MMP-7, by blocking MAPK signaling by inhibiting the phosphorylation of ERK and p38 MAPK, and by inducing focal adhesion formation and modulating vinculin localization and expression. Treatment of HepG2 cells with ginsenoside Rd significantly inhibited metastasis, most likely by blocking MMP activation and MAPK signaling pathways involved in cancer cell migration. This study may be useful for the development of novel chemotherapeutic agents for the treatment of malignant cancers. [This work was supported by the Priority Research Centers Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Project No. 2010-0020141) to S-G Lee]. Keywords: Ginsenoside Rd, Anti-metastasis, MMP, Vinculin doi:10.1016/j.ejphar.2011.09.233
Quercetin synergistically induces sensitivity to 5-fluorouracil through p53 modulation in colorectal cancer cells C.P.R. Xavier, C.F. Lima, C. Pereira-Wilson⁎ University of Minho, Portugal E-mail address: [email protected] (C. Pereira-Wilson) Colorectal tumors (CRC) with microsatellite instability (MSI) show resistance to chemotherapy with 5-fluorouracil (5-FU), the most widely used pharmacological drug for CRC treatment. The aims of this study were to identify compounds that increase sensitivity of MSI CRC cells to 5-FU and characterize their dependence on the p53 status of the cells. Two MSI human CRC derived cell lines were used: CO115 wildtype for p53 and HCT15 that harbors a p53 mutation. The sensitivity of these cells to 5-FU was evaluated by TUNEL assay and the effects on apoptosis induction of co-incubation of the flavonoids, quercetin (Q) or luteolin (L), with 5-FU were characterized. The mechanisms of apoptosis induction were assessed by western blot and p53 mediated effects confirmed by small interference RNA (siRNA) in CO115 and in HCT116 wt and p53 knockout cells. Our results demonstrate that CO115 is more sensitive to 5-FU than the p53 mutated HCT15. Additive effects on apoptosis were shown for L (in both cell lines) and Q (in HCT15). In CO115 Q synergistically induced apoptosis with 5-FU. Apoptosis induction was caspase dependent in CO115 cells but not in HCT15 cells. Both flavonoids increased p53 expression in both cell lines, an effect particularly remarkable for Q. The synergistic effect of Q and 5-FU in CO115 involved the activation of the mitochondrial pathway with an increase in the expression of cleaved caspase 9 and 3 and PARP, as well as a decrease in Bcl-2 expression. Importantly, knockdown of p53 by siRNA in CO115 cells and p53 knockout in HCT116 cells totally abrogated apoptosis induction, demonstrating the dependence on p53 modulation of apoptosis induction by Q. This study suggests the potential applicability of these phytochemicals for enhancement 5-FU efficiency in CRC therapy, especially Q in p53 wild-type tumors.
Keywords: Quercetin, 5-Fluorouracil, Colorectal cancer, p53 doi:10.1016/j.ejphar.2011.09.234
Evaluation of cactus pear (Opuntia spp.) extracts as promising bioactive ingredients for colon cancer therapy J. Poejoa,⁎, A.A. Matiasa,b, M.R. Bronzeb,c, C.M.M. Duartea,b, A.T. Serrab a
ITQB/UNL, Portugal IBET, Portugal c iMED-FFUL, Portugal E-mail address: [email protected] (J. Poejo) b
Cancer is one of the most causes of death worldwide. In particular, colorectal cancer is the second most frequent malignant disease in Europe. Treatments for recurrent and metastatic diseases remain a centre of clinical attention. While continuing efforts have been made for discovering new molecular target-based molecules, there is an emerging interest in chemotherapeutic application of natural substances. Epidemiological data suggests that the ingestion of phytochemicals from fruits and vegetables may contribute to reduce the incidence of cancer in humans. The mechanisms by which these compounds inhibit tumourgenesis include inhibition of tumour cell mediated protease activity, attenuation of tumour angiogenesis, induction of cell cycle arrest and promotion of apoptosis. Cactus (Opuntia spp.) fruits and cladodes have been widely used as food and in folk medicine. Nutraceutical benefits of fruits are believed to be related to the presence of ascorbic acid, flavonoids, betaxanthin and betacyanin. Recently, several studies demonstrated that cactus pear juices inhibit the proliferation of human cancer cell lines suggesting that cactus compounds could be considered as promising ingredients for chemoprevention and chemotherapy. Within this context, the main aim of this study was to develop natural ingredients from cactus pear and evaluate their potential use as natural chemotherapeutic agents on colon cancer. Different varieties of cactus pear were screened for their antiproliferative effect on HT29 cells. The phenolic content was determined using HPLC technique in order to understand which compounds are responsible for the anticancer activity. The most promising varieties were selected and further processed using macroporous resin, aiming to develop polyphenol-rich concentrates. The anticancer activity of final products was evaluated by quantifying the effective dose values and analysing cell cycle arrest on HT29. The results obtained were compared with doxorubicin, a conventional drug used in cancer treatment. Additionally, the development of a drug-resistant HT29 cell culture was performed aiming to evaluate the bioproducts' potential in overcoming the main drawback of chemotherapy. Keywords: Opuntia, Natural ingredients, Colon cancer doi:10.1016/j.ejphar.2011.09.235
Aloe vera and honey solution modulates the oxidative stress, calpain activity and survival in tumour-bearing rats R. Tomasin⁎, R.S. Andrade, M.C.C. Gomes-Marcondes State University of Campinas, Brazil E-mail address: [email protected] (R. Tomasin) Oxidative stress is linked to several tumorigenic events and hostwaste, and also to senescence and apoptosis, the major mechanisms in