RESUMO
EGFR is over-expressed in several solid tumors including breast, prostate, pancreas, and lung cancers and is correlated to the metastatic potential of the tumor. Anti-EGFR receptor-binding peptidomimetics (AERP) were examined to assess the small molecule's potential use as tumor-specific imaging agents. The aim of this work was to design and characterize the binding specificity of the radiolabeled peptidomimetics to EGFR over-expressing cell lysate and to A431 xenograft tumors. Our newly designed peptidomimetic, AERP, was conjugated to DTPA and labeled with (99m)Tc. The in vivo tumor accumulation of [(99m)Tc] DTPA-AERP-2 was 1.6±0.1%ID/g and tumor to muscle ratio was 5.5. Our studies suggest that this novel peptidomimetic, AERP-2, warrants further development as an EGFR specific tumor-imaging agent.
Assuntos
Inibidores Enzimáticos/química , Receptores ErbB/antagonistas & inibidores , Neoplasias/diagnóstico por imagem , Compostos Radiofarmacêuticos/química , Animais , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Receptores ErbB/metabolismo , Humanos , Camundongos , Camundongos Nus , Ácido Pentético/química , Peptidomiméticos , Cintilografia , Compostos Radiofarmacêuticos/síntese química , Tecnécio/química , Distribuição Tecidual , Transplante HeterólogoRESUMO
Angiogenesis is crucial for solid tumor growth. By secreting angiogenic factors, tumor cells induce angiogenesis. However, targeting these angiogenic factors for cancer therapy is not always successful, suggesting that other factors may be involved in tumor angiogenesis. This work shows that 25 protein spots were differentially expressed by two-dimensional gel electrophoretic analysis when HepG2 cells induced endothelial cell differentiation to tube in vitro, and most of them were upregulated. Twenty-one proteins were identified with MALDI-TOF-MS, and the other four were identified by LTQ-MS/MS. Keratins were identified as one class of these upregulated proteins. Further study indicated that the expression of keratin 17 in cultured endothelial cells is likely microenvironment regulated, because its expression can be induced by HepG2 cells and bFGF as well as serum in culture media. Increased expression of keratins in endothelial cells, such as keratin 17, may contribute to the angiogenesis induced by HepG2 cells.
Assuntos
Queratina-17/isolamento & purificação , Queratina-17/fisiologia , Neoplasias/irrigação sanguínea , Neovascularização Patológica/metabolismo , Células Cultivadas , Técnicas de Cocultura , Células Endoteliais/metabolismo , Humanos , Queratina-17/genética , Queratina-17/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neovascularização Patológica/genética , Proteômica/métodosRESUMO
OBJECTIVE: To investigate the effect of keratin 17 (K-17) on the migration, proliferation and tube formation of human umbilical vein endothelial cell (HUVEC), and to realize the role of K-17 in angiogenesis. METHODS: After HUVEC were cultured in DMEM medium supplemented with 10%FBS overnight, K-17-siRNA-mixture (experimental group) and control-siRNA-mixture (negative control group) were added into HUVEC, respectively, by Lipofectamine 2000 transfection assay, and the final concentration of the siRNA was 50 nmol/L. Lipofectamine 2000 alone was used as the control. After the cells were cultured for 36 hours, the cell proliferation ability was detected by cell counting. After 30-hour culture, the cell's abilities of migration and differentiation to tube were detected by 24-well Millicell units and the collagen gel assay, respectively. In addition, non-siRNA-treated HUVEC were cultured for 24 hours in DMEM medium supplemented with 10%FBS (group A), 2%FBS (group B) and 2%FBS+10 ng/mL bFGF (group C), respectively, and then the expression of K-17 in HUVEC was detected by RT-PCR and Western blot. RESULTS: After the treatment with K-17-siRNA for 36 hours, HUVEC exhibited no significant difference in the proliferation, compared with both control and negative control groups (P > 0.05). After transfected with K-17-siRNA for 30 hours, the number of HUVEC in the experimental group which migrated from the upper chamber to the lower chamber of Millicell wells within 24 hours (3719.0 +/- 319.0) was smaller than both control (7 437.5 +/- 212.0) and negative control (7 356.3 +/- 795.7) groups, with significant difference (P < 0.01). However, there was no significant difference between the control group and the negative control group (P > 0.05). After HUVEC were transfected with K-17-siRNA for 30 hours, the number of tubes in the experimental group, the negative control group and the control group in 24 hours was (1.1 +/- 0.5), (3.6 +/- 0.5) and (3.2 +/- 0.6) per field, respectively. The experimental group was significantly different from both control and negative control groups (P < 0.01), and there was no significant difference between the negative control group and the control group (P > 0.05). The expression of K-17 protein in HUVEC in groups A, B and C was 0.25 +/- 0.02, 0.08 +/- 0.01 and 0.72 +/- 0.03, respectively. There was significant difference among these three groups (P < 0.01). CONCLUSION: K-17 has no impact on cell proliferation, but may augment endothelial cell migration, which may facilitate angiogenesis.
Assuntos
Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Queratina-17/farmacologia , RNA Interferente Pequeno/farmacologia , Células Cultivadas , Células Endoteliais , Humanos , Queratina-17/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção , Veias Umbilicais/citologiaRESUMO
UNLABELLED: The level of alpha(V)beta(3) integrins on endothelial cells is elevated in angiogenesis. The high binding specificity to alpha(V)beta(3) integrins of peptides containing Arg-Gly-Asp (RGD) residues suggests that the radiolabeled RGD peptides may be useful as tumor specific imaging agents. In this research, cyclised peptides containing Arg-Gly-Asp (RGD) and Arg-Gly-Glu (RGE, as control) residues were conjugated with HYNIC and labeled with (99m)Tc. OBJECTIVE: The goal was to evaluate the influence of co-ligand, either tricine or ethylenediamine-N,N'-diacetic acid (EDDA) on protein and integrin binding and on cellular uptake in culture. METHODS: The n-octanol/water partition coefficient, binding to bovine serum albumin (BSA) and human umbilical vein endothelial (HUVE) cells, and cell lysate distributions of the radiolabeled peptides were evaluated. RESULTS: The co-ligands had a significant effect on the labeling efficiency of the HYNIC conjugates and on certain properties of the (99m)Tc complexes. The labeling efficiency with tricine was 10 fold higher and BSA binding was over 8 fold greater compared to EDDA. Both RGD labels showed higher (6 to 28 fold) binding to HUVE cells than that of the RGE labels, indicating binding specificity. After cell-lysis, only a small percentage of the total RGD label that accumulated in the cells was found bound to cellular proteins (9% of RGD/tricine and 5% of RGD/EDDA), implying that over 90% of the radiolabeled peptides were internalized for both radiolabeled RGDs. The number of the RGD molecules bound to proteins was estimated to be approximately three per cell, suggesting that only a small number of alpha(V)beta(3) integrin proteins are expressed on the cells. CONCLUSIONS: Apart from the differences in radiolabeling, the only important effect of substituting EDDA for tricine as co-ligand on the HYNIC-peptides was the lower degree of serum protein binding. In spite of the lower serum protein binding potential, in vivo tumor accumulation of the RGD/EDDA may not be improved compared to RGD/tricine since quantitation of the cell binding results suggests that the number of alpha(V)beta(3) integrin proteins per cell might be limited.
Assuntos
Proteínas Sanguíneas/metabolismo , Ácido Edético/análogos & derivados , Endotélio Vascular/metabolismo , Glicina/análogos & derivados , Hidrazinas/farmacocinética , Integrinas/metabolismo , Ácidos Nicotínicos/farmacocinética , Oligopeptídeos/farmacocinética , Tecnécio/farmacocinética , Células Cultivadas , Ácido Edético/farmacocinética , Endotélio Vascular/diagnóstico por imagem , Glicina/farmacocinética , Humanos , Ligantes , Taxa de Depuração Metabólica , Ligação Proteica , Cintilografia , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Veias Umbilicais/diagnóstico por imagem , Veias Umbilicais/metabolismoRESUMO
Three amino acids residues, Arg-Gly-Asp (RGD), in vitronectin and fibronectin show affinity for alpha(V)beta(3) integrins expressed in vascular endothelial cells. That tumor growth can upregulate the expression of these integrins on tumor cells for invasion and metastasis and in tissue neovasculature suggests the potential of developing radiolabeled RGD peptides as antagonists of alpha(V)beta(3) integrins for broad spectrum tumor specific imaging. The polypeptide RGD-4C, which contains four cysteine residues for cyclization, has shown preferential localization on integrins at sites of tumor angiogenesis. Both RGD-4C and RGE (Arg-Gly-Glu)-4C (as control) were purchased and conjugated with 6-hydrazinopyridine-3-carboxylic acid (HYNIC) for 99mTc radiolabeling. After purification of the conjugated peptides by a C18 Sep-Pak cartridge with 20% methanol, both peptides were radiolabeled using tricine. For cell binding studies, both 99mTc peptides were further purified by SE HPLC. High specific radioactivity of labeled cyclized RGD/E (cyclized RGD/E will be simplified as RGD/E through out the text) of about 20 Ci/micromol was achieved. Both 99mTc complexes were stable in the labeling solution for over 24 h at room temperature. In the human umbilical vein endothelial (HUVE) cell studies, the binding at 1 h of radiolabeled RGD/E was determined at 4 degrees C and at concentrations in the picomolar to nanomolar range. Under these conditions, cell accumulation of 99mTc in the case of RGD was as much as 16 times greater than the control RGE. As a check on specificity, 7 nM of native cyclized RGD blocked 50% of the binding of 99mTc-labeled RGD to cells. The binding percentage of 99mTc-labeled RGD to purified alpha(V)beta(3) integrin protein, as determined by SE HPLC, increased with the concentration of the integrin while 99mTc-labeled RGE showed no binding. The association constant for 99mTc-RGD was modest at 7 x 10(6) M(-)(1). In both human renal adenocarcinoma (ACHN) and human colon cancer cell line (LS174T) nude mouse tumor models, the accumulation of 99mTc-labeled RGD/E exhibited no statistical difference. In conclusion, possibly because of limited numbers of alpha(V)beta(3) integrin receptors per tumor cell and low binding affinity, radiolabeled RGD peptides may have limitations as tumor imaging agents.