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997 In Vivo Growth Inhibition of Sarcoma 180 by the Medicinal Plant Kielmeyera Rugosa Choisy (Clusiaceae)
Poster Sessions
european journal of cancer 48, suppl. 5 (2012) S25–S288
following results: (I) in the provided system, H-1PV led to partial regression of tumor with prolonged survival of tumor-bearing animals, (II) the method of virus delivery via neck-implanted osmotic pump was found as a most effective method of infection during experimental glioma viral therapy. 997 In Vivo Growth Inhibition of Sarcoma 180 by the Medicinal Plant Kielmeyera Rugosa Choisy (Clusiaceae) D. Bezerra1 , A.C.A. Oliveira1 , A.C.S. Britto1 , R.M. Henriques1 , G.M.B. Cardoso1 , C.S. Anjos2 , A.M. Jesus2 , V.R.S. Moraes2 , P.C.L. Nogueira2 . 1 Federal University of Sergipe, Department of Physiology, ˜ Cristov ´ ao-SE, ˜ Sao Brazil, 2 Federal University of Sergipe, Department of ˜ Cristov ´ ao-SE, ˜ Chemistry, Sao Brazil Background: The medicinal plant Kielmeyera rugosa Choisy (family Clusiaceae, subfamily: Kielmeyeroideae) is popularly known in Brazil as “pausanto” and growing in Sergipe and Bahia, northeastern, Brazil. In a preliminary study, the stem extract of K. rugosa showed cytotoxity against several tumor cell lines in culture. In this study, the in vivo antitumor activity of stem extract of Kielmeyera rugosa (EKR) was demonstrated using experimental model. In addition, systemic toxicological evaluation was also performed. Material and Methods: The antitumor activity of EKR was assessed in mice inoculated with sarcoma 180 cells. Sarcoma 180 cells were injected (2×106 cells/animal/s.c.) in mice left hind limbs. Young healthy male (n = 10) Swiss albino mice (body weight 25−30 g) were used. One day after, the animals were treated intraperitoneally or orally for 7 consecutive days. Negative control was treated with the vehicle (10% DMSO) used for diluting the tested substance. The 5-Fluorouracil was used as the positive control. On day 8, the mice were sacrificed and tumors were excised and weighed. The animal studies committee of the Federal University of Sergipe approved the experimental protocol (number 56/2010). Results: The EKR showed antitumor activity in both administration routes. Tumor growth inhibition rates were 34.9–40.8% and 25.4–51.8% for the EKR treatment by intraperitoneal (50 and 100 mg/kg/day) and oral (100 and 200 mg/kg/day) administration, respectively. The treatment with KRE did not significantly affect the body mass, macroscopy of the organs, or blood leukocyte counts. Conclusions: We can conclude that the EKR possesses significant antitumor activity, when administrated by oral or intraperitoneal routes, and has only low toxicity. 998 Assessment of Antitumor Properties of the Essential Oil From the Leaves of Guatteria Friesiana D.P. Bezerra1 , A.C.S. Britto1 , A.C.A. Oliveira1 , R.M. Henriques1 , G.M.B. Cardoso1 , D.S. Bomfim1 , A.A. Carvalho2 , C. Pessoa2 , M.L.B. Pinheiro3 , E.V. Costa4 . 1 Federal University of Sergipe, Department ´ ˜ Cristov ´ ao-SE, ˜ of Physiology, Sao Brazil, 2 Federal University of Ceara, Department of Physiology and Pharmacology, Fortaleza-CE, Brazil, 3 Federal University of Amazonas, Department of Chemistry, Manaus-AM, Brazil, 4 ˜ Cristov ´ ao-SE, ˜ Federal University of Sergipe, Department of Chemistry, Sao Brazil Background: Guatteria friesiana (W.A. Rodrigues) Erkens & Maas (synonym Guatteriopsis friesiana W.A. Rodrigues, family Annonaceae), popularly known as ‘envireira’, is a medicinal plant found in the Brazilian and Colombian Amazon basin that is used in traditional medicine for various purposes. In this study, the antitumor activity of the essential oil from the leaves of G. friesiana (EOGF) and its main components (a-, b-, and g-eudesmol) were determined using experimental models. Material and Methods: The in vitro cytotoxic activity of EOGF and its components a-, b-, and g-eudesmol were determined by the MTT assay, after 72 hours of incubation, against HL-60 (human leukemia), MDA-MB-435 (human melanoma), SF-295 (human glioblastoma), and HCT-8 (human colon). In vivo antitumor activity of OEGF was assessed in mice inoculated with sarcoma 180 cells. Sarcoma 180 cells were injected (2×106 cells/animal/s.c.) in mice left hind limbs. Young healthy male (n = 10) Swiss albino mice (body weight 25−30 g) were used. One day after, the animals were treated intraperitoneally or orally for 7 consecutive days. Negative control was treated with the vehicle (10% DMSO) used for diluting the tested substance. The 5-Fluorouracil was used as the positive control. On day 8, the mice were sacrificed and tumors were excised and weighed. The animal studies committee of the Federal University of Sergipe approved the experimental protocol (number 60/2010). Results: EOGF and its components a-, b-, and g-eudesmol displayed cytotoxicity against tumor cell lines, showing IC50 values in the range of 1.7 to 9.4 mg/mL in the HCT-8 and HL-60 cell lines for EOGF, 5.7 to 19.4 mg/mL in the HL-60 and MDA-MB-435 cell lines for a-eudesmol, 24.1 to >25 mg/mL in the SF-295 and MDA-MB-435 cell lines for b-eudesmol, and 7.1 to 20.6 mg/mL in the SF-295 and MDA-MB-435 cell lines for g-eudesmol, respectively. In vivo tumor growth inhibition rates were 43.4–54.2% and 6.6–42.8% for the EOGF treatment by intraperitoneal (50 and 100 mg/kg/day) and oral (100
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and 200 mg/kg/day) administration, respectively. The treatment with EOGF did not significantly affect the body mass, macroscopy of the organs, or blood leukocyte counts. Conclusions: Based on these results, we can conclude that EOGF possesses significant antitumor activity and has only low systemic toxicity. These effects could be assigned to its components a-, b-, and g-eudesmol. 999 Correlation Between Differences in the Increase in MAPK (ERK1/2) Activity Due to Driver Mutations and Prognosis in Non-small-cell Lung Cancer I. Tsujino1 , Y. Nakanishi2 , T. Shimizu1 , Y. Obana2 , S. Ohni2 , N. Takahashi1 , N. Nemoto2 , S. Hashimoto1 . 1 Nihon University School of Medicine, Department of Respiratory Medicine, Tokyo, Japan, 2 Nihon University School of Medicine, Department of Pathology, Tokyo, Japan Background: Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor that, on ligand binding, triggers the RAS-RAFmitogen-activated protein kinase (MAPK) signaling pathway, which is mainly associated with cell proliferation. In non-small-cell lung carcinoma (NSCLC), known mutations, which are called driver mutations, in EGFR, KRAS, BRAF, and other oncogenes cause continuous activation of tyrosine kinase. In this study, the aim is to clarify the correlation between driver mutations and the activity of MAPK (ERK1/2) in advanced NSCLCs. Material and Methods: Paraffin-embedded lung biopsy samples were obtained from 110 non-squamous NSCLC patients (study period, 2009–2010). EGFR mutations were analyzed using the PCR clamp method. KRAS codons 12 and 13 and BRAF V600E mutations were assessed by mutation-based PCR performed using a quenching probe (MBP-QP), with the i-densy system (ARKRAY Inc.). ALK rearrangement was analyzed by immunohistochemistry (ALK detection kit; Nichirei Biosciences Inc.). Phosphorylation of MAPK (ERK1/2) was assessed by immunohistochemical analysis with the antiphospho-p44/42 MAPK (ERK1/2, Thr202/Thr204) antibody (Cell Signaling Technology). Clinical and prognostic assessments were performed using the Kaplan–Meier method. Results: Phospshorylated p44/42 MAPK was detected in 84 (76.4%) of the 110 patients. In the 79 patients with phospho-p44/42 expression, 14 (17.7%) had EGFR mutations, 8 (10.1%) had a KRAS mutation, and 2 (2.5%) had a BRAF mutation. The phospho-p44/42 expression level, which was assessed on the basis of the immunohistochemical score, was found to be lower in the patients with EGFR mutations and higher in the patients with a KRAS/BRAF mutation. The patients’ prognosis tended to worsen with increase in the phospho-p44/42 expression level. These results showed that the activity of the MAPK signaling pathway increased to a greater extent because of KRAS/BRAF mutations than because of EGFR mutations and may have caused poor prognosis. No correlation was found between ALK rearrangement and the phospho-p44/42 expression level. Conclusions: The expression level of phosphorylated MAPK was higher in the NSCLC patients with a KRAS/BRAF mutation than with EGFR mutations, and this higher activity level was associated with poor prognosis. 1000 Investigating the Efficacy of the Irreversible Pan-ERBB Inhibitor PF-00299804 Using Paediatric Embryonal Tumor Models C.L. Burchill1 , H.L. Hii1 , P.B. Dallas1 , C. Cole2 , R. Endersby1 , N.G. Gottardo3 . 1 Telethon Institute for Child Health Research and Centre for Child Health Research, Brain Tumour Research Program Division of Children’s Leukaemia and Cancer Research, Perth, Australia, 2 The University of Western Australia, School of Paediatrics and Child Health, Perth, Australia, 3 Princess Margaret Hospital for Children, Department of Paediatric Oncology and Haematology, Perth, Australia Background: Medulloblastoma (MB) and pineoblastoma (PB) are malignant paediatric brain tumours. Despite improved treatment regimens many children with these tumours remain incurable. Moreover, survivors are often left with devastating long-term side effects, highlighting the need for innovative therapies. Previous studies have revealed that over-expression of transmembrane receptors ERBB2 and ERBB4 is associated with poor clinical outcomes in children with MB. PF-00299804 is an irreversible pan-ERBB inhibitor, with anti-tumour activity in lung cancers that harbour deregulated ERBB receptors. We hypothesise that increased ERBB expression is associated with decreased sensitivity of MB cells to chemotherapy due to upregulation of survival pathways and that PF-00299804 will improve the efficacy of conventional anti-cancer drugs. Material and Methods: To evaluate the efficacy of PF-00299804, as a single agent and in combination with conventional chemotherapeutics, we have developed a panel of cell lines representing PB and various paediatric MB subtypes which were examined in vitro and in vivo using orthotopic xenograft. In addition, this study utilised a transgenic spontaneous mouse model of MB, the ND2-SmoA1 mouse, which represents the Sonic Hedgehog subgroup of human MB. Immunoblot and immunohistochemical analysis for ERBB1−4 was used to determine ERBB pathway deregulation in our models. In vitro cell
european journal of cancer 48, suppl. 5 (2012) S25–S288
following results: (I) in the provided system, H-1PV led to partial regression of tumor with prolonged survival of tumor-bearing animals, (II) the method of virus delivery via neck-implanted osmotic pump was found as a most effective method of infection during experimental glioma viral therapy. 997 In Vivo Growth Inhibition of Sarcoma 180 by the Medicinal Plant Kielmeyera Rugosa Choisy (Clusiaceae) D. Bezerra1 , A.C.A. Oliveira1 , A.C.S. Britto1 , R.M. Henriques1 , G.M.B. Cardoso1 , C.S. Anjos2 , A.M. Jesus2 , V.R.S. Moraes2 , P.C.L. Nogueira2 . 1 Federal University of Sergipe, Department of Physiology, ˜ Cristov ´ ao-SE, ˜ Sao Brazil, 2 Federal University of Sergipe, Department of ˜ Cristov ´ ao-SE, ˜ Chemistry, Sao Brazil Background: The medicinal plant Kielmeyera rugosa Choisy (family Clusiaceae, subfamily: Kielmeyeroideae) is popularly known in Brazil as “pausanto” and growing in Sergipe and Bahia, northeastern, Brazil. In a preliminary study, the stem extract of K. rugosa showed cytotoxity against several tumor cell lines in culture. In this study, the in vivo antitumor activity of stem extract of Kielmeyera rugosa (EKR) was demonstrated using experimental model. In addition, systemic toxicological evaluation was also performed. Material and Methods: The antitumor activity of EKR was assessed in mice inoculated with sarcoma 180 cells. Sarcoma 180 cells were injected (2×106 cells/animal/s.c.) in mice left hind limbs. Young healthy male (n = 10) Swiss albino mice (body weight 25−30 g) were used. One day after, the animals were treated intraperitoneally or orally for 7 consecutive days. Negative control was treated with the vehicle (10% DMSO) used for diluting the tested substance. The 5-Fluorouracil was used as the positive control. On day 8, the mice were sacrificed and tumors were excised and weighed. The animal studies committee of the Federal University of Sergipe approved the experimental protocol (number 56/2010). Results: The EKR showed antitumor activity in both administration routes. Tumor growth inhibition rates were 34.9–40.8% and 25.4–51.8% for the EKR treatment by intraperitoneal (50 and 100 mg/kg/day) and oral (100 and 200 mg/kg/day) administration, respectively. The treatment with KRE did not significantly affect the body mass, macroscopy of the organs, or blood leukocyte counts. Conclusions: We can conclude that the EKR possesses significant antitumor activity, when administrated by oral or intraperitoneal routes, and has only low toxicity. 998 Assessment of Antitumor Properties of the Essential Oil From the Leaves of Guatteria Friesiana D.P. Bezerra1 , A.C.S. Britto1 , A.C.A. Oliveira1 , R.M. Henriques1 , G.M.B. Cardoso1 , D.S. Bomfim1 , A.A. Carvalho2 , C. Pessoa2 , M.L.B. Pinheiro3 , E.V. Costa4 . 1 Federal University of Sergipe, Department ´ ˜ Cristov ´ ao-SE, ˜ of Physiology, Sao Brazil, 2 Federal University of Ceara, Department of Physiology and Pharmacology, Fortaleza-CE, Brazil, 3 Federal University of Amazonas, Department of Chemistry, Manaus-AM, Brazil, 4 ˜ Cristov ´ ao-SE, ˜ Federal University of Sergipe, Department of Chemistry, Sao Brazil Background: Guatteria friesiana (W.A. Rodrigues) Erkens & Maas (synonym Guatteriopsis friesiana W.A. Rodrigues, family Annonaceae), popularly known as ‘envireira’, is a medicinal plant found in the Brazilian and Colombian Amazon basin that is used in traditional medicine for various purposes. In this study, the antitumor activity of the essential oil from the leaves of G. friesiana (EOGF) and its main components (a-, b-, and g-eudesmol) were determined using experimental models. Material and Methods: The in vitro cytotoxic activity of EOGF and its components a-, b-, and g-eudesmol were determined by the MTT assay, after 72 hours of incubation, against HL-60 (human leukemia), MDA-MB-435 (human melanoma), SF-295 (human glioblastoma), and HCT-8 (human colon). In vivo antitumor activity of OEGF was assessed in mice inoculated with sarcoma 180 cells. Sarcoma 180 cells were injected (2×106 cells/animal/s.c.) in mice left hind limbs. Young healthy male (n = 10) Swiss albino mice (body weight 25−30 g) were used. One day after, the animals were treated intraperitoneally or orally for 7 consecutive days. Negative control was treated with the vehicle (10% DMSO) used for diluting the tested substance. The 5-Fluorouracil was used as the positive control. On day 8, the mice were sacrificed and tumors were excised and weighed. The animal studies committee of the Federal University of Sergipe approved the experimental protocol (number 60/2010). Results: EOGF and its components a-, b-, and g-eudesmol displayed cytotoxicity against tumor cell lines, showing IC50 values in the range of 1.7 to 9.4 mg/mL in the HCT-8 and HL-60 cell lines for EOGF, 5.7 to 19.4 mg/mL in the HL-60 and MDA-MB-435 cell lines for a-eudesmol, 24.1 to >25 mg/mL in the SF-295 and MDA-MB-435 cell lines for b-eudesmol, and 7.1 to 20.6 mg/mL in the SF-295 and MDA-MB-435 cell lines for g-eudesmol, respectively. In vivo tumor growth inhibition rates were 43.4–54.2% and 6.6–42.8% for the EOGF treatment by intraperitoneal (50 and 100 mg/kg/day) and oral (100
S241
and 200 mg/kg/day) administration, respectively. The treatment with EOGF did not significantly affect the body mass, macroscopy of the organs, or blood leukocyte counts. Conclusions: Based on these results, we can conclude that EOGF possesses significant antitumor activity and has only low systemic toxicity. These effects could be assigned to its components a-, b-, and g-eudesmol. 999 Correlation Between Differences in the Increase in MAPK (ERK1/2) Activity Due to Driver Mutations and Prognosis in Non-small-cell Lung Cancer I. Tsujino1 , Y. Nakanishi2 , T. Shimizu1 , Y. Obana2 , S. Ohni2 , N. Takahashi1 , N. Nemoto2 , S. Hashimoto1 . 1 Nihon University School of Medicine, Department of Respiratory Medicine, Tokyo, Japan, 2 Nihon University School of Medicine, Department of Pathology, Tokyo, Japan Background: Epidermal growth factor receptor (EGFR) is a transmembrane tyrosine kinase receptor that, on ligand binding, triggers the RAS-RAFmitogen-activated protein kinase (MAPK) signaling pathway, which is mainly associated with cell proliferation. In non-small-cell lung carcinoma (NSCLC), known mutations, which are called driver mutations, in EGFR, KRAS, BRAF, and other oncogenes cause continuous activation of tyrosine kinase. In this study, the aim is to clarify the correlation between driver mutations and the activity of MAPK (ERK1/2) in advanced NSCLCs. Material and Methods: Paraffin-embedded lung biopsy samples were obtained from 110 non-squamous NSCLC patients (study period, 2009–2010). EGFR mutations were analyzed using the PCR clamp method. KRAS codons 12 and 13 and BRAF V600E mutations were assessed by mutation-based PCR performed using a quenching probe (MBP-QP), with the i-densy system (ARKRAY Inc.). ALK rearrangement was analyzed by immunohistochemistry (ALK detection kit; Nichirei Biosciences Inc.). Phosphorylation of MAPK (ERK1/2) was assessed by immunohistochemical analysis with the antiphospho-p44/42 MAPK (ERK1/2, Thr202/Thr204) antibody (Cell Signaling Technology). Clinical and prognostic assessments were performed using the Kaplan–Meier method. Results: Phospshorylated p44/42 MAPK was detected in 84 (76.4%) of the 110 patients. In the 79 patients with phospho-p44/42 expression, 14 (17.7%) had EGFR mutations, 8 (10.1%) had a KRAS mutation, and 2 (2.5%) had a BRAF mutation. The phospho-p44/42 expression level, which was assessed on the basis of the immunohistochemical score, was found to be lower in the patients with EGFR mutations and higher in the patients with a KRAS/BRAF mutation. The patients’ prognosis tended to worsen with increase in the phospho-p44/42 expression level. These results showed that the activity of the MAPK signaling pathway increased to a greater extent because of KRAS/BRAF mutations than because of EGFR mutations and may have caused poor prognosis. No correlation was found between ALK rearrangement and the phospho-p44/42 expression level. Conclusions: The expression level of phosphorylated MAPK was higher in the NSCLC patients with a KRAS/BRAF mutation than with EGFR mutations, and this higher activity level was associated with poor prognosis. 1000 Investigating the Efficacy of the Irreversible Pan-ERBB Inhibitor PF-00299804 Using Paediatric Embryonal Tumor Models C.L. Burchill1 , H.L. Hii1 , P.B. Dallas1 , C. Cole2 , R. Endersby1 , N.G. Gottardo3 . 1 Telethon Institute for Child Health Research and Centre for Child Health Research, Brain Tumour Research Program Division of Children’s Leukaemia and Cancer Research, Perth, Australia, 2 The University of Western Australia, School of Paediatrics and Child Health, Perth, Australia, 3 Princess Margaret Hospital for Children, Department of Paediatric Oncology and Haematology, Perth, Australia Background: Medulloblastoma (MB) and pineoblastoma (PB) are malignant paediatric brain tumours. Despite improved treatment regimens many children with these tumours remain incurable. Moreover, survivors are often left with devastating long-term side effects, highlighting the need for innovative therapies. Previous studies have revealed that over-expression of transmembrane receptors ERBB2 and ERBB4 is associated with poor clinical outcomes in children with MB. PF-00299804 is an irreversible pan-ERBB inhibitor, with anti-tumour activity in lung cancers that harbour deregulated ERBB receptors. We hypothesise that increased ERBB expression is associated with decreased sensitivity of MB cells to chemotherapy due to upregulation of survival pathways and that PF-00299804 will improve the efficacy of conventional anti-cancer drugs. Material and Methods: To evaluate the efficacy of PF-00299804, as a single agent and in combination with conventional chemotherapeutics, we have developed a panel of cell lines representing PB and various paediatric MB subtypes which were examined in vitro and in vivo using orthotopic xenograft. In addition, this study utilised a transgenic spontaneous mouse model of MB, the ND2-SmoA1 mouse, which represents the Sonic Hedgehog subgroup of human MB. Immunoblot and immunohistochemical analysis for ERBB1−4 was used to determine ERBB pathway deregulation in our models. In vitro cell