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Soluble receptor for advanced glycation endproducts (sRAGE) effectively reduces tumor growth as a single agent and in combination with doxorubicin in a spontaneous mammary tumor model
ASSOCIATION FOR ACADEMIC SURGERY—ABSTRACTS serve as a future target to prevent tumor cell invasion in pancreatic adenocarcinoma.
37. Soluble Receptor for Advanced Glycation Endproducts (sRAGE) Effectively Reduces Tumor Growth as a Single Agent and in Combination with Doxorubicin in a Spontaneous Mammary Tumor Model. K. P. Joseph, M.D., M.P.H., L. Liu, M.D., Ph.D., W. Qu, M.D., F. R. Schnabel, M.D., A. Schmidt, M.D. Columbia-Presbyterian Medical Center, New York, NY. Introduction: The receptor for advanced glycation endproducts (RAGE), a member of the immunoglobulin family interacts with distinct ligands that have been implicated in various pathophysiologic states such as diabetes, atherosclerosis, inflammation as well as tumor growth. We have previously demonstrated that blockade of RAGE in tumors raised in mice from rat C6 glioma cells or murine Lewis lung carcinoma effectively reduced tumor growth, invasion and metastases. Blockade of RAGE has also previously been shown to halt the progression of breast tumors in a murine model that spontaneously produces breast tumors. We hypothesize that the addition of RAGE antagonists to standard chemotherapeutic agents for breast cancer may enhance the effectiveness of monotherapy. Methods: Female MMTV transgenic mice (5-8 weeks) were divided into four groups: (1) Control group-no treatment, (2) doxorubicin (DOX) 2 mg/kg IP weekly x 4 weeks (3) sRAGE 20 g IP daily x 4 weeks and (4) DOX 2 mg/kg IP weekly x 4 weeks and sRAGE 20 g IP daily x 4 weeks. The mice were sacrificed and tumors harvested on treatment day 35 and were weighed and measured. Results: There was no difference in tumor weight between the control mice (n ⫽ 9) and DOX-treated mice (n ⫽ 4) (0.192 g v. 0.193). The administration of sRAGE (n ⫽ 7) decreased tumor weight to 0.12 g and the combination of sRAGE and DOX (n ⫽ 8) decreased tumor weight by a third (0.192 g v. 0.061 g, p ⫽ 0.11). While the mean tumor volume in the control mice was 316.7 mm 3, sRAGE decreased tumor volume to 89.65 mm 3 and the combination of sRAGE and DOX decreased tumor volume by 6.5 fold (316.7 mm 3 vs. 48.08 mm 3, p ⫽ 0.006). The mean tumor volume in the DOX treated group was 500.9 mm 3. Conclusions: RAGE antagonists suppress local tumor growth in transgenic mice with spontaneously occurring cancer in an early intervention model. The addition of sRAGE, a low molecular weight RAGE antagonist, to doxorubicin enhanced the effectiveness of this standard chemotherapy agent. 38. Genetic Heterogeneity of Colorectal Cancer Liver Metastases. J. C. Sung, M.D., J.D., D. Boulware, M.S., S. Eschrich, Ph.D., F. Gonzalez, B.S., T. J. Yeatman, M.D. H. Lee Moffitt Cancer Center. Introduction: Although liver metastases are a leading cause of colorectal cancer, the molecular genetic basis of the advanced disease stages remains poorly understood. Whether the metastatic lesions are genetically homogeneous or heterogeneous may determine the response to therapy. We investigated whether synchronouslyoccurring, multi-focal colon cancer liver metastases were of multiclonal origin by using genome-scale microarray analysis. Methods: Five distinct colon cancer liver metastases were biopsied from a single patient harboring 15 or more lesions. All specimens were microdissected to ensure ⬎90% tumor cells and total RNA was
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extracted and prepared for hybridization. Each specimen was analyzed using the Affymetrix U133 GeneChip TM containing approximately 22,000 gene elements. The gene expression profiles of the five specimens were then compared using two independent methods: unsupervised hierarchical clustering to determine the relatedness of the samples and the chi-square test of independence to assess whether the distribution of ranks was different across the samples. Results: No histological difference was observed among the microdissected samples. Each specimen appeared to have equivalent numbers of tumor cells versus stromal cells. However, unsupervised hierarchical clustering of all 22,000 genes by five samples yielded five distinct clusters. Moreover, 2 analysis identified 5 statistically significant (p ⬍ 0.001) groups. Conclusions: Genetic profiling of multiple liver metastases using genome scale profiling suggests that colon cancer metastases are muti-clonal in origin. These results have negative implications for the effective application of single agent systemic therapy. 39. Endocytosis of VEGFR-2 on Endothelial Progenitor Cells by Dopamine Attenuates Tumour Growth and Vasculogenesis. B. D. Barry, M.D., E. Condon, M.D., J. H. Wang, Ph.D., H. P. Redmond, M.D. Cork University Hospital, Cork, Ireland. Introduction: Endothelial progenitor cells (EPC’s) andvasculogenesis are intricately involved in the initial neovascularisation of tumours. EPC’s are characterised by the expression of VEGFR-2 and AC133. Vasculogenesis offers a novel possibility for the targeting of anti-cancer therapies. Methods: Ex-vivo expanded EPC’s were cultured from peripheral blood samples from healthy male volunteers. To endocytose VEFGR-2 on EPC’s, cells were treated with incremental doses of dopamine and VEGFR-2 expression was analysed using anti-VEGFR-2 mAb on flow cytometry. Dil-labelled ex-vivo expanded EPC’s were transplanted into tumour bearing (3LL tumour cell line) MF1-nude mice (n⫽12). The mice received a daily i.p. injection of PBS or Dopamine (50 mg/kg). Tumour volume was assessed on alternate days. Vasculogenesis and angiogenesis were assessed using fluorescent microscopy and CD31 staining. To determine if Dopamine has a direct effect on 3LL cells, cells were cultured with incremental doses of dopamine for 48 h and cell apoptosis (PIstaining), proliferation (BrdU-Assay) and Cytotoxicity (LDH-Assay) were analysed. Statistical analysis was performed on SigmaStat using ANOVA. Results: Ex-vivo expanded EPC cultures were 80 – 85% pure. Dopamine caused an endocytosis of VEGFR-2 on EPC’s with the maximal effect seen at 1M (p ⬍ 0.05). Dopamine had no effect on 3LL cell apoptosis, proliferation or cytotoxicity in all doses performed. Dopamine, by endocytosing VEGFR-2 on EPC’s, attenuated tumour growth by decreasing both tumour volume (p ⬍ 0.05; 241.8 ⫹/- 106.8 v’s 59.7 ⫹/- 20.5 at day 10) and tumour weight (p ⬍ 0.05). Absolute and relative levels of vasculogenesis were decreased in the dopamine group as compared to controls (p ⬍ 0.05). Conclusion: Induction of VEGFR-2 endocytosis on EPC’s decreases tumour growth and vasculogenesis. This data suggests that VEGFR-2 is a target for attenuating tumour vasculogenesis. 40. Doxycycline and Indomethacin Synergistically Downregulate Beta-catenin Signaling and Inhibit Colon Cancer Cell Growth. N. K. Veeramachaneni, M.D., L. Lin, M.D., J. A. Pippin, B.A., E. R. Winslow, M.D., J. A. Drebin, M.D., Ph.D. Washington University. Introduction: Aberrant beta-catenin signaling, due to loss of the APC tumor suppressor gene, or mutation of beta-catenin, is found in the majority of colon cancers. We have recently demonstrated that doxycycline (DOX) decreases beta-catenin expression and transcriptional activity in colon cancer cells. NSAIDS also downregulate betacatenin by an undefined mechanism. We demonstrate synergistic inhibition of beta-catenin signaling and colon cancer cell growth by DOX and indomethacin (INDO). Methods: APC-mutant SW480 hu-
37. Soluble Receptor for Advanced Glycation Endproducts (sRAGE) Effectively Reduces Tumor Growth as a Single Agent and in Combination with Doxorubicin in a Spontaneous Mammary Tumor Model. K. P. Joseph, M.D., M.P.H., L. Liu, M.D., Ph.D., W. Qu, M.D., F. R. Schnabel, M.D., A. Schmidt, M.D. Columbia-Presbyterian Medical Center, New York, NY. Introduction: The receptor for advanced glycation endproducts (RAGE), a member of the immunoglobulin family interacts with distinct ligands that have been implicated in various pathophysiologic states such as diabetes, atherosclerosis, inflammation as well as tumor growth. We have previously demonstrated that blockade of RAGE in tumors raised in mice from rat C6 glioma cells or murine Lewis lung carcinoma effectively reduced tumor growth, invasion and metastases. Blockade of RAGE has also previously been shown to halt the progression of breast tumors in a murine model that spontaneously produces breast tumors. We hypothesize that the addition of RAGE antagonists to standard chemotherapeutic agents for breast cancer may enhance the effectiveness of monotherapy. Methods: Female MMTV transgenic mice (5-8 weeks) were divided into four groups: (1) Control group-no treatment, (2) doxorubicin (DOX) 2 mg/kg IP weekly x 4 weeks (3) sRAGE 20 g IP daily x 4 weeks and (4) DOX 2 mg/kg IP weekly x 4 weeks and sRAGE 20 g IP daily x 4 weeks. The mice were sacrificed and tumors harvested on treatment day 35 and were weighed and measured. Results: There was no difference in tumor weight between the control mice (n ⫽ 9) and DOX-treated mice (n ⫽ 4) (0.192 g v. 0.193). The administration of sRAGE (n ⫽ 7) decreased tumor weight to 0.12 g and the combination of sRAGE and DOX (n ⫽ 8) decreased tumor weight by a third (0.192 g v. 0.061 g, p ⫽ 0.11). While the mean tumor volume in the control mice was 316.7 mm 3, sRAGE decreased tumor volume to 89.65 mm 3 and the combination of sRAGE and DOX decreased tumor volume by 6.5 fold (316.7 mm 3 vs. 48.08 mm 3, p ⫽ 0.006). The mean tumor volume in the DOX treated group was 500.9 mm 3. Conclusions: RAGE antagonists suppress local tumor growth in transgenic mice with spontaneously occurring cancer in an early intervention model. The addition of sRAGE, a low molecular weight RAGE antagonist, to doxorubicin enhanced the effectiveness of this standard chemotherapy agent. 38. Genetic Heterogeneity of Colorectal Cancer Liver Metastases. J. C. Sung, M.D., J.D., D. Boulware, M.S., S. Eschrich, Ph.D., F. Gonzalez, B.S., T. J. Yeatman, M.D. H. Lee Moffitt Cancer Center. Introduction: Although liver metastases are a leading cause of colorectal cancer, the molecular genetic basis of the advanced disease stages remains poorly understood. Whether the metastatic lesions are genetically homogeneous or heterogeneous may determine the response to therapy. We investigated whether synchronouslyoccurring, multi-focal colon cancer liver metastases were of multiclonal origin by using genome-scale microarray analysis. Methods: Five distinct colon cancer liver metastases were biopsied from a single patient harboring 15 or more lesions. All specimens were microdissected to ensure ⬎90% tumor cells and total RNA was
251
extracted and prepared for hybridization. Each specimen was analyzed using the Affymetrix U133 GeneChip TM containing approximately 22,000 gene elements. The gene expression profiles of the five specimens were then compared using two independent methods: unsupervised hierarchical clustering to determine the relatedness of the samples and the chi-square test of independence to assess whether the distribution of ranks was different across the samples. Results: No histological difference was observed among the microdissected samples. Each specimen appeared to have equivalent numbers of tumor cells versus stromal cells. However, unsupervised hierarchical clustering of all 22,000 genes by five samples yielded five distinct clusters. Moreover, 2 analysis identified 5 statistically significant (p ⬍ 0.001) groups. Conclusions: Genetic profiling of multiple liver metastases using genome scale profiling suggests that colon cancer metastases are muti-clonal in origin. These results have negative implications for the effective application of single agent systemic therapy. 39. Endocytosis of VEGFR-2 on Endothelial Progenitor Cells by Dopamine Attenuates Tumour Growth and Vasculogenesis. B. D. Barry, M.D., E. Condon, M.D., J. H. Wang, Ph.D., H. P. Redmond, M.D. Cork University Hospital, Cork, Ireland. Introduction: Endothelial progenitor cells (EPC’s) andvasculogenesis are intricately involved in the initial neovascularisation of tumours. EPC’s are characterised by the expression of VEGFR-2 and AC133. Vasculogenesis offers a novel possibility for the targeting of anti-cancer therapies. Methods: Ex-vivo expanded EPC’s were cultured from peripheral blood samples from healthy male volunteers. To endocytose VEFGR-2 on EPC’s, cells were treated with incremental doses of dopamine and VEGFR-2 expression was analysed using anti-VEGFR-2 mAb on flow cytometry. Dil-labelled ex-vivo expanded EPC’s were transplanted into tumour bearing (3LL tumour cell line) MF1-nude mice (n⫽12). The mice received a daily i.p. injection of PBS or Dopamine (50 mg/kg). Tumour volume was assessed on alternate days. Vasculogenesis and angiogenesis were assessed using fluorescent microscopy and CD31 staining. To determine if Dopamine has a direct effect on 3LL cells, cells were cultured with incremental doses of dopamine for 48 h and cell apoptosis (PIstaining), proliferation (BrdU-Assay) and Cytotoxicity (LDH-Assay) were analysed. Statistical analysis was performed on SigmaStat using ANOVA. Results: Ex-vivo expanded EPC cultures were 80 – 85% pure. Dopamine caused an endocytosis of VEGFR-2 on EPC’s with the maximal effect seen at 1M (p ⬍ 0.05). Dopamine had no effect on 3LL cell apoptosis, proliferation or cytotoxicity in all doses performed. Dopamine, by endocytosing VEGFR-2 on EPC’s, attenuated tumour growth by decreasing both tumour volume (p ⬍ 0.05; 241.8 ⫹/- 106.8 v’s 59.7 ⫹/- 20.5 at day 10) and tumour weight (p ⬍ 0.05). Absolute and relative levels of vasculogenesis were decreased in the dopamine group as compared to controls (p ⬍ 0.05). Conclusion: Induction of VEGFR-2 endocytosis on EPC’s decreases tumour growth and vasculogenesis. This data suggests that VEGFR-2 is a target for attenuating tumour vasculogenesis. 40. Doxycycline and Indomethacin Synergistically Downregulate Beta-catenin Signaling and Inhibit Colon Cancer Cell Growth. N. K. Veeramachaneni, M.D., L. Lin, M.D., J. A. Pippin, B.A., E. R. Winslow, M.D., J. A. Drebin, M.D., Ph.D. Washington University. Introduction: Aberrant beta-catenin signaling, due to loss of the APC tumor suppressor gene, or mutation of beta-catenin, is found in the majority of colon cancers. We have recently demonstrated that doxycycline (DOX) decreases beta-catenin expression and transcriptional activity in colon cancer cells. NSAIDS also downregulate betacatenin by an undefined mechanism. We demonstrate synergistic inhibition of beta-catenin signaling and colon cancer cell growth by DOX and indomethacin (INDO). Methods: APC-mutant SW480 hu-