RESUMO
DDP-resistant MDA-MB-231 cells (MDA-MB-231/DDP) cells had higher expression of L1CAM than their parental cells. L1CAM siRNA decreased the IC50 of MDA-MB-231/DDP cells to DDP. L1CAM inhibition down-regulated p-AKT/AKT in MDA-MB-231/DDP cells; meanwhile, it could promote MDA-MB-231/DDP cell apoptosis, inhibit cell EMT, invasion, and migration. Moreover, SC79 (an AKT activator) increased the DDP-resistance of MDA-MB-231/DDP cells, which was reversed by L1CAM inhibition. Furthermore, co-treatment of L1CAM shRNA and cisplatin injection had better anti-tumor effects in vivo than these two single treatments with decreased p-AKT/AKT. Thus, silencing L1CAM reversed the DDP resistance by inhibiting the AKT pathway.
Assuntos
Cisplatino , Resistencia a Medicamentos Antineoplásicos , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas , Apoptose , Linhagem Celular Tumoral , Proliferação de Células , Cisplatino/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
The biophysical properties of therapeutic antibodies influence their manufacturability, efficacy, and safety. To develop an anti-cancer antibody, we previously generated a human monoclonal antibody (Ab417) that specifically binds to L1 cell adhesion molecule with a high affinity, and we validated its anti-tumor activity and mechanism of action in human cholangiocarcinoma xenograft models. In the present study, we aimed to improve the biophysical properties of Ab417. We designed 20 variants of Ab417 with reduced aggregation propensity, less potential post-translational modification (PTM) motifs, and the lowest predicted immunogenicity using computational methods. Next, we constructed these variants to analyze their expression levels and antigen-binding activities. One variant (Ab612)-which contains six substitutions for reduced surface hydrophobicity, removal of PTM, and change to the germline residue-exhibited an increased expression level and antigen-binding activity compared to Ab417. In further studies, compared to Ab417, Ab612 showed improved biophysical properties, including reduced aggregation propensity, increased stability, higher purification yield, lower pI, higher affinity, and greater in vivo anti-tumor efficacy. Additionally, we generated a highly productive and stable research cell bank (RCB) and scaled up the production process to 50 L, yielding 6.6 g/L of Ab612. The RCB will be used for preclinical development of Ab612.
Assuntos
Anticorpos Monoclonais/química , Modelos Moleculares , Molécula L1 de Adesão de Célula Nervosa/química , Engenharia de Proteínas , Animais , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/farmacologia , Afinidade de Anticorpos , Células CHO , Fenômenos Químicos , Cricetulus , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Humanos , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Engenharia de Proteínas/métodos , Estabilidade Proteica , TermodinâmicaRESUMO
Targeting the molecular pathways underlying the cardiotoxicity associated with thoracic irradiation and doxorubicin (Dox) could reduce the morbidity and mortality associated with these anticancer treatments. Here, we find that vascular endothelial cells (ECs) with persistent DNA damage induced by irradiation and Dox treatment exhibit a fibrotic phenotype (endothelial-mesenchymal transition, EndMT) correlating with the colocalization of L1CAM and persistent DNA damage foci. We demonstrate that treatment with the anti-L1CAM antibody Ab417 decreases L1CAM overexpression and nuclear translocation and persistent DNA damage foci. We show that in whole-heart-irradiated mice, EC-specific p53 deletion increases vascular fibrosis and the colocalization of L1CAM and DNA damage foci, while Ab417 attenuates these effects. We also demonstrate that Ab417 prevents cardiac dysfunction-related decrease in fractional shortening and prolongs survival after whole-heart irradiation or Dox treatment. We show that cardiomyopathy patient-derived cardiovascular ECs with persistent DNA damage show upregulated L1CAM and EndMT, indicating clinical applicability of Ab417. We conclude that controlling vascular DNA damage by inhibiting nuclear L1CAM translocation might effectively prevent anticancer therapy-associated cardiotoxicity.
Assuntos
Anticorpos Neutralizantes/farmacologia , Cardiomiopatias/prevenção & controle , Cardiotoxicidade/prevenção & controle , Doxorrubicina/toxicidade , Raios gama/efeitos adversos , Molécula L1 de Adesão de Célula Nervosa/genética , Animais , Antibióticos Antineoplásicos/toxicidade , Cardiomiopatias/etiologia , Cardiomiopatias/genética , Cardiomiopatias/metabolismo , Cardiotoxicidade/etiologia , Cardiotoxicidade/genética , Cardiotoxicidade/metabolismo , Estudos de Casos e Controles , Técnicas de Cocultura , Dano ao DNA , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Células Endoteliais/efeitos da radiação , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/efeitos da radiação , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Miócitos Cardíacos/efeitos da radiação , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/deficiência , Proteína Supressora de Tumor p53/genéticaRESUMO
OBJECTIVE: Neural cell adhesion molecule L1CAM (L1) is involved in neuroprotection. To investigate a possible neuroprotective effect of L1 during ischemia, we determined whether blocking L1 with an antagonistic antibody would worsen the outcome of focal cerebral ischemia-reperfusion and increase blood-brain barrier (BBB) disruption. METHODS: Transient middle cerebral artery occlusion (MCAO) was performed in anesthetized rats. Five µg of antagonistic mouse IgG monoclonal L1 antibody 324 or non-immune control mouse IgG was applied on the ischemic-reperfused cortex during one hour of MCAO and two hours of reperfusion. At two hours of reperfusion, BBB permeability, size of infarct using tetrazolium staining, number of TUNEL-labeled apoptotic cells, and immunohistochemistry for expression of PTEN and p53 were studied. RESULTS: The antagonistic L1 antibody 324 increased the percentage of cortical infarct area (+36%), but did not affect BBB permeability in the ischemic-reperfused cortex. The antagonistic L1 antibody increased number of apoptotic neurons and p53 expression, but decreased PTEN expression. CONCLUSION: Functional antagonism of L1 increases infarct size by increasing numbers of apoptotic neurons without affecting BBB permeability during the early stage of cerebral ischemia-reperfusion. Our data suggest that L1 affects primarily the brain parenchyma rather than BBB during early stages of cerebral ischemia-reperfusion and that endogenous brain L1 may be neuroprotective.
Assuntos
Barreira Hematoencefálica/fisiopatologia , Isquemia Encefálica/fisiopatologia , Molécula L1 de Adesão de Célula Nervosa/fisiologia , Traumatismo por Reperfusão/fisiopatologia , Animais , Barreira Hematoencefálica/metabolismo , Masculino , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Neuroproteção , Ratos Endogâmicos F344RESUMO
PURPOSE: Cholangiocarcinoma is a malignancy of bile duct with a poor prognosis. Conventional chemotherapy and radiotherapy are generally ineffective, and surgical resection is the only curative treatment for cholangiocarcinoma. L1-cell adhesion molecule (L1CAM) has been known as a novel prognostic marker and therapeutic target for cholangiocarcinoma. This study aimed to evaluate the feasibility of immuno-PET imaging-based radioimmunotherapy using radiolabeled anti-L1CAM antibody in cholangiocarcinoma xenograft model. EXPERIMENTAL DESIGN: We prepared a theranostic convergence bioradiopharmaceutical using chimeric anti-L1CAM antibody (cA10-A3) conjugated with 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) chelator and labeled with 64Cu or 177Lu and evaluated the immuno-PET or SPECT/CT imaging and biodistribution with 64Cu-/177Lu-cA10-A3 in various cholangiocarcinoma xenograft models. Therapeutic efficacy and response monitoring were performed by 177Lu-cA10-A3 and 18F-FDG-PET, respectively, and immunohistochemistry was done by TUNEL and Ki-67. RESULTS: Radiolabeled cA10-A3 antibodies specifically recognized L1CAM in vitro, clearly visualized cholangiocarcinoma tumors in immuno-PET and SPECT/CT imaging, and differentiated the L1CAM expression level in cholangiocarcinoma xenograft models. 177Lu-cA10-A3 (12.95 MBq/100 µg) showed statistically significant reduction in tumor volumes (P < 0.05) and decreased glucose metabolism (P < 0.01). IHC analysis revealed 177Lu-cA10-A3 treatment increased TUNEL-positive and decreased Ki-67-positive cells, compared with saline, cA10-A3, or 177Lu-isotype. CONCLUSIONS: Anti-L1CAM immuno-PET imaging using 64Cu-cA10-A3 could be translated into the clinic for characterizing the pharmacokinetics and selecting appropriate patients for radioimmunotherapy. Radioimmunotherapy using 177Lu-cA10-A3 may provide survival benefit in L1CAM-expressing cholangiocarcinoma tumor. Theranostic convergence bioradiopharmaceutical strategy would be applied as imaging biomarker-based personalized medicine in L1CAM-expressing patients with cholangiocarcinoma.
Assuntos
Neoplasias dos Ductos Biliares/radioterapia , Colangiocarcinoma/radioterapia , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Radioimunoterapia/métodos , Compostos Radiofarmacêuticos/administração & dosagem , Animais , Neoplasias dos Ductos Biliares/diagnóstico por imagem , Neoplasias dos Ductos Biliares/imunologia , Neoplasias dos Ductos Biliares/patologia , Ductos Biliares/diagnóstico por imagem , Ductos Biliares/patologia , Linhagem Celular Tumoral , Colangiocarcinoma/diagnóstico por imagem , Colangiocarcinoma/imunologia , Colangiocarcinoma/patologia , Feminino , Compostos Heterocíclicos com 1 Anel/administração & dosagem , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos com 1 Anel/farmacocinética , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/química , Imunoconjugados/farmacocinética , Camundongos , Molécula L1 de Adesão de Célula Nervosa/imunologia , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Nanomedicina Teranóstica/métodos , Distribuição Tecidual , Tomografia Computadorizada de Emissão de Fóton Único , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Protein kinase inhibitors (PKIs) are currently tested in clinical studies (phase I-III) as an alternative strategy against (recurrent) ovarian cancer. Besides their anti-tumour efficacy, several PKIs have also shown radiosensitizing effects when combined with external beam radiation. Based on these results we asked if the addition of PKIs offers a therapeutic opportunity to improve radioimmunotherapy (RIT) against ovarian cancer. Five PKIs (alisertib, MK1775, MK2206, saracatinib, temsirolimus) were chosen for cytotoxicity screenings based on their current clinical trials in the treatment of ovarian cancer and their influence on cell cycle regulation and DNA damage repair pathways. We combined selected PKIs with 177Lu-labelled anti-L1CAM monoclonal antibody chCE7 for our investigations. METHODS: PKIs cytotoxicity was determined via cell colony-forming assays. Biomarker of DNA double-strand breaks (DSBs, γH2A.X) was analysed by western blot and fluorescence microscopy. Flow cytometric measurements were performed to evaluate levels of apoptosis based on mono- or combination treatments. The best combination was used for in vivo combination therapy studies in nude mice with SKOV3ip and IGROV1 human ovarian cancer xenografts. Bonferroni correction was used to determine statistical significance for multiple comparisons. RESULTS: The highest cytotoxicity against both cell lines was observed for MK1775 and alisertib. Combinations including 177Lu-labelled mAb chCE7 and MK1775 decreased 177Lu-DOTA-chCE7 IC60-values 14-fold, compared to 6-fold, when the radioimmunoconjugate was combined with alisertib. The most effective PKI MK1775 was further evaluated and demonstrated synergistic effects in combination with 177Lu-DOTA-chCE7 against IGROV1 cells. Significantly higher amounts of DSBs were detected in IGROV1 cells after combination (91%) compared to either treatment alone (MK1775: 52%; radioimmunoconjugate: 72%; p < 0.0125). Early-apoptosis was significantly enhanced in IGROV1 cells correlating with induced DSBs (177Lu-DOTA-chCE7: 8%, MK1775: 28%, 177Lu-DOTA-chCE7 + MK1775: 40%, p < 0.0125). Immunohistochemistry analysis of γH2A.X expression levels after therapy in SKOV3ip xenografts revealed a high sensitivity of the tumour cells to MK1775 and a high radioresistance. A prominent effect of tumour growth inhibition of the RIT and of the combination therapy was observed in vivo in a late stage IGROV1 xenograft model. CONCLUSIONS: Our results warrant further evaluation of combination of MK1775 and radioimmunotherapy.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Imunoconjugados/farmacologia , Lutécio , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Radioisótopos , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Pirimidinonas , Radioimunoterapia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Cholangiocarcinoma has a poor prognosis and is refractory to conventional chemotherapy and radiation therapy. Improving survival of patients with advanced cholangiocarcinoma urgently requires the development of new effective targeted therapies in combination with chemotherapy. We previously developed a human monoclonal antibody (mAb) Ab417 that binds to both the human and mouse L1 cell adhesion molecule (L1CAM) with high affinities. In the present study, we observed that Ab417 exhibited tumor targeting ability in biodistribution studies and dose-dependent tumor growth inhibition in an intrahepatic cholangiocarcinoma (Choi-CK) xenograft mouse model. Regarding the mechanism of action, Ab417 was internalized into the tumor cells and thereby down-regulated membrane L1CAM, and inhibited tumor growth by reducing tumor cell proliferation in vivo. Gemcitabine inhibited the tumor growth in a dose-dependent manner in the Choi-CK xenograft model. However, cisplatin inhibited the tumor growth moderately and not in a dose-dependent way, suggesting that the tumors may have developed resistance to apoptosis induced by cisplatin. Combined treatment with Ab417 and gemcitabine or cisplatin exerted enhanced tumor growth inhibition compared to treatment with antibody or drug alone. The results suggest that Ab417 in combination with chemotherapy may have potential as a new therapeutic regimen for cholangiocarcinoma. Our study is the first to show an enhanced therapeutic effect of a therapeutic antibody targeting L1CAM in combination with chemotherapy in cholangiocarcinoma models.
Assuntos
Anticorpos Monoclonais/farmacologia , Neoplasias dos Ductos Biliares/metabolismo , Colangiocarcinoma/metabolismo , Cisplatino/farmacologia , Desoxicitidina/análogos & derivados , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Animais , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/patologia , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Humanos , Camundongos , Terapia de Alvo Molecular , Distribuição Tecidual , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Childhood neuroblastoma is one of the most malignant types of cancers leading to a high mortality rate. These cancerous cells can be highly metastatic and malignant giving rise to disease recurrence and poor prognosis. The proto-oncogene myelocytomatosis neuroblastoma (MycN) is known to be amplified in this type of cancer, thus, promoting high malignancy and resistance. The L1 cell adhesion molecule (L1-CAM) cleavage has been found upregulated in many types of malignant cancers. In the present study, we explored the interplay between L1-CAM, MycN and PTEN as well as the role played by PDGFR and VEGFR on tumorigenicity in neuroblastoma cells. We investigated the effect of L1-CAM knock-down (KD) and PDGFR/VEGFR inhibition with sunitinib malate (Sutent®) treatment on subsequent tumorsphere formation and cellular proliferation and migration in the MycN-amplified IMR-32 neuroblastoma cells. We further examined the effect of combined L1-CAM KD with Sutent treatment or radiotherapy on these cellular functions in our cells. Tumorsphere formation is one of the indicators of aggressiveness in malignant cancers, which was significantly inhibited in IMR-32 cells after L1-CAM KD or Sutent treatment, however, no synergistic effect was observed with dual treatments, rather L1-CAM KD alone showed a greater inhibition on tumorsphere formation compared to Sutent treatment alone. In addition, cellular proliferation and migration were significantly inhibited after L1-CAM KD in the IMR-32 cells with no synergistic effect observed on the rate of cell proliferation when combined with Sutent treatment. Again, L1-CAM KD alone exhibited greater inhibitory effect than Sutent treatment on cell proliferation. L1-CAM KD led to the simultaneous downregulation of MycN, but the upregulation of PTEN protein expression. Notably, radiotherapy (2 Gy) of the IMR-32 cells led to significant upregulation of both L1-CAM and MycN, which was abrogated with L1-CAM KD in our cells. In addition, L1-CAM KD radiosensitized the cells as exhibited by the synergistic effect on the reduction in cell proliferation compared to radiotherapy alone. Taken together, our data show the importance of L1-CAM interplay with MycN and PTEN on the MycN amplified neuroblastoma cell radioresistance, proliferation and motility.
Assuntos
Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Proteína Proto-Oncogênica N-Myc/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuroblastoma/radioterapia , Tumores Neuroectodérmicos Primitivos Periféricos/radioterapia , PTEN Fosfo-Hidrolase/metabolismo , Tolerância a Radiação , Western Blotting , Movimento Celular , Proliferação de Células , Raios gama , Humanos , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Tumores Neuroectodérmicos Primitivos Periféricos/metabolismo , Tumores Neuroectodérmicos Primitivos Periféricos/patologia , Proto-Oncogene Mas , Radioterapia , Células Tumorais CultivadasRESUMO
L1CAM is a cell adhesion molecule of the immunoglobulin superfamily which was originally discovered as a major player in the development of the nervous system. L1CAM was demonstrated to have prognostic value in different cancers and to be a promising target for anti-cancer therapy. Here we overview the present data on L1CAM structure and function, regulation of its expression, role in cancer and therapeutic potential.
Assuntos
Transformação Celular Neoplásica/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Molécula L1 de Adesão de Célula Nervosa/genética , Neurônios/metabolismo , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos/uso terapêutico , Adesão Celular/efeitos dos fármacos , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Transição Epitelial-Mesenquimal/genética , Fibronectinas/química , Fibronectinas/genética , Fibronectinas/metabolismo , Humanos , Domínios de Imunoglobulina/genética , Neoplasias/metabolismo , Neoplasias/patologia , Neoplasias/terapia , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Molécula L1 de Adesão de Célula Nervosa/química , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neurônios/patologia , Prognóstico , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
L1 cell adhesion molecule (L1CAM) is the prototype member of the L1-family of closely related neural adhesion molecules. L1CAM is differentially expressed in the normal nervous system as well as pathological tissues and displays a wide range of biological activities. In human malignancies, L1CAM plays a vital role in tumor growth, invasion and metastasis. Recently, increasing evidence has suggested that L1CAM exerts a variety of functions at different steps of tumor progression through a series of signaling pathways. In addition, L1CAM has been identified as a promising target for cancer therapy by using synthetic and natural inhibitors. In this review, we provide an up-to-date overview of the role of L1CAM involved in cancers and the rationale for L1CAM as a novel molecular target for cancer therapy.
Assuntos
Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Progressão da Doença , Humanos , Invasividade Neoplásica , Metástase Neoplásica , Neoplasias/patologia , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Transdução de Sinais/fisiologiaRESUMO
L1 cell adhesion molecule (L1CAM) is aberrantly expressed in malignant tumors and plays important roles in tumor progression. Thus, L1CAM could serve as a therapeutic target and anti-L1CAM antibodies may have potential as anticancer agents. However, L1CAM is expressed in neural cells and the druggability of anti-L1AM antibody must be validated at the earliest stages of preclinical study. Here, we generated a human monoclonal antibody that is cross-reactive with mouse L1CAM and evaluated its pharmacokinetic properties and anti-tumor efficacy in rodent models. First, we selected an antibody (Ab4) that binds human and mouse L1CAM from the human naïve Fab library using phage display, then increased its affinity 45-fold through mutation of 3 residues in the complementarity-determining regions (CDRs) to generate Ab4M. Next, the affinity of Ab4M was increased 1.8-fold by yeast display of single-chain variable fragment containing randomly mutated light chain CDR3 to generate Ab417. The affinities (KD) of Ab417 for human and mouse L1CAM were 0.24 nM and 79.16 pM, respectively. Ab417 specifically bound the Ig5 domain of L1CAM and did not exhibit off-target activity, but bound to the peripheral nerves embedded in normal human tissues as expected in immunohistochemical analysis. In a pharmacokinetics study, the mean half-life of Ab417 was 114.49 h when a single dose (10 mg/kg) was intravenously injected into SD rats. Ab417 significantly inhibited tumor growth in a human cholangiocarcinoma xenograft nude mouse model and did not induce any adverse effect in in vivo studies. Thus, Ab417 may have potential as an anticancer agent.
Assuntos
Anticorpos Antineoplásicos , Especificidade de Anticorpos/imunologia , Neoplasias Experimentais/tratamento farmacológico , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Anticorpos de Cadeia Única , Animais , Anticorpos Antineoplásicos/química , Anticorpos Antineoplásicos/genética , Anticorpos Antineoplásicos/imunologia , Anticorpos Antineoplásicos/farmacologia , Células CHO , Cricetinae , Cricetulus , Reações Cruzadas/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias Experimentais/imunologia , Neoplasias Experimentais/patologia , Molécula L1 de Adesão de Célula Nervosa/imunologia , Células PC12 , Ratos , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/genética , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
L1 cell adhesion molecule (L1CAM) is overexpressed in many human cancers, confers bad prognosis and augments cell motility, invasion and metastasis. Results from xenograft mouse models suggested that L1CAM antibodies might be promising tools for cancer therapy. Here, we generated human L1CAM-transgenic mice to study therapeutic efficacy and putative side effects in a model system. We established three transgenic lines (M2, M3 and F4) expressing the human L1CAM transgene in brain, kidney and colon with decreasing intensity (M2, M3 > F4). The expression pattern was similar to that of L1CAM in humans. No interference of the transgene with the expression of endogenous L1CAM was observed. Immunohistochemical analysis revealed correct expression of the transgene in mouse cortex and collective duct of the kidney. Injection of (125)I-labeled L1CAM antibodies resulted in specific enrichment in the kidney but not in the brain. The injection of the therapeutic anti-human L1CAM mAb L1-9.3/2a into transgenic mice even at high doses did not cause behavioral changes or other side effects. Similar results were obtained using a mouse specific L1CAM mAb in normal mice. Tumor therapy experiments were performed using syngeneic mouse tumor cells (RET melanoma and Panc02 pancreatic adenocarcinoma) transduced with human L1CAM. MAb L1-9.3/2a efficiently and specifically attenuated local tumor growth in both model systems without apparent side effects. The therapeutic effect was dependent on immune effector mechanisms. Analysis of Panc02-huL1CAM tumors after therapy showed elevated levels of EGF and evidence of immune-induced epithelial-mesenchymal transition. The results suggest that our transgenic mice are valuable tools to study L1CAM-based antibody therapy.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Citotoxicidade Celular Dependente de Anticorpos/imunologia , Modelos Animais de Doenças , Transição Epitelial-Mesenquimal , Melanoma/terapia , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Neoplasias Pancreáticas/terapia , Adenocarcinoma/genética , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Adenocarcinoma/terapia , Animais , Western Blotting , Movimento Celular , Proliferação de Células , Feminino , Humanos , Técnicas Imunoenzimáticas , Radioisótopos do Iodo/uso terapêutico , Melanoma/genética , Melanoma/imunologia , Melanoma/patologia , Camundongos , Camundongos Transgênicos , Molécula L1 de Adesão de Célula Nervosa/imunologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , RNA Mensageiro/genética , Radioimunoterapia , Ratos , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
BACKGROUND: Alcohol causes fetal alcohol spectrum disorders in part by disrupting the function of the neural cell adhesion molecule L1. Alcohol inhibits L1-mediated cell-cell adhesion in diverse cell types and inhibits L1-mediated neurite outgrowth in cerebellar granule neurons (CGNs). A recent report indicates that ethanol (EtOH) induces the translocation of L1 into CGN lipid rafts and that disruption of lipid rafts prevents EtOH inhibition of L1-mediated neurite outgrowth. The same butanol-pentanol cutoff was noted for alcohol-induced translocation of L1 into lipid rafts that was reported previously for alcohol inhibition of L1 adhesion, suggesting that EtOH might inhibit L1 adhesion by shifting L1 into lipid rafts. METHODS: The NIH/3T3 cell line, 2A2-L1s , is a well-characterized EtOH-sensitive clonal cell line that stably expresses human L1. Cells were treated with 25 mM EtOH, 5 µM filipin, or both. Lipid rafts were enriched in membrane fractions by preparation of detergent-resistant membrane (DRMs) fractions. Caveolin-1 was used as a marker of lipid rafts, and L1 and Src were quantified by Western blotting in lipid-raft-enriched membrane fractions and by immunohistochemistry. RESULTS: EtOH (25 mM) increased the percentage of L1, but not Src, in 2A2-L1s membrane fractions enriched in lipid rafts. Filipin, an agent known to disrupt lipid rafts, decreased the percentage of caveolin and L1 in DRMs from 2A2-L1s cells. Filipin also blocked EtOH-induced translocation of L1 into lipid rafts from 2A2-L1s cells but did not significantly affect L1 adhesion or EtOH inhibition of L1 adhesion. CONCLUSIONS: These findings indicate that EtOH does not inhibit L1 adhesion in NIH/3T3 cells by inducing the translocation of L1 into lipid rafts.
Assuntos
Adesão Celular/efeitos dos fármacos , Etanol/farmacologia , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Animais , Adesão Celular/fisiologia , Linhagem Celular , Humanos , Camundongos , Células NIH 3T3RESUMO
BACKGROUND: Transforming Growth Factor beta (TGF-ß) acts as a tumor suppressor early in carcinogenesis but turns into tumor promoter in later disease stages. In fact, TGF-ß is a known inducer of integrin expression by tumor cells which contributes to cancer metastatic spread and TGF-ß inhibition has been shown to attenuate metastasis in mouse models. However, carcinoma cells often become refractory to TGF-ß-mediated growth inhibition. Therefore identifying patients that may benefit from anti-TGF-ß therapy requires careful selection. METHODS: We performed in vitro analysis of the effects of exposure to TGF-ß in NSCLC cell chemotaxis and adhesion to lymphatic endothelial cells. We also studied in an orthotopic model of NSCLC the incidence of metastases to the lymph nodes after inhibition of TGF-ß signaling, ß3 integrin expression or both. RESULTS: We offer evidences of increased ß3-integrin dependent NSCLC adhesion to lymphatic endothelium after TGF-ß exposure. In vivo experiments show that targeting of TGF-ß and ß3 integrin significantly reduces the incidence of lymph node metastasis. Even more, blockade of ß3 integrin expression in tumors that did not respond to TGF-ß inhibition severely impaired the ability of the tumor to metastasize towards the lymph nodes. CONCLUSION: These findings suggest that lung cancer tumors refractory to TGF-ß monotherapy can be effectively treated using dual therapy that combines the inhibition of tumor cell adhesion to lymphatic vessels with stromal TGF-ß inhibition.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/terapia , Regulação Neoplásica da Expressão Gênica , Integrina beta3/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/terapia , Fator de Crescimento Transformador beta1/genética , Animais , Anticorpos Monoclonais/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quimiotaxia/efeitos dos fármacos , Técnicas de Cocultura , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Humanos , Integrina beta3/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Metástase Linfática , Camundongos , Terapia de Alvo Molecular , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Molécula L1 de Adesão de Célula Nervosa/genética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/antagonistas & inibidores , Fator de Crescimento Transformador beta1/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
L1CAM promotes cell motility, invasion and metastasis formation in various human cancers and can be considered as a driver of tumor progression. Knowledge about genetic processes leading to the presence of L1CAM in cancers is of considerable importance. Experimentally, L1CAM expression can be achieved by various means. Over-expression of the transcription factor SLUG or treatment of cells with TGF-ß1 can induce or augment L1CAM levels in cancer cells. Likewise, hypomethylation of the L1CAM promoter on the X chromosome correlates with L1CAM expression. However, presently no mechanisms that might control transcriptional activity are known. Here we have identified miR-34a as a suppressor of L1CAM. We observed that L1CAM positive endometrial carcinoma (EC) cell lines HEC1B and SPAC1L lost L1CAM protein and mRNA by treatment with demethylating agents or knock-down of the DNA-methyltransferase-1 (DNMT1). Concomitantly, several miRNAs were up-regulated. Using miRNA profiling, luciferase reporter assays and mutagenesis, we identified miR-34a as a putative binder to the L1CAM-3'UTR. Over-expression of miR-34a in HEC1B cells blocked L1CAM expression and inhibited cell migration. In ECC1 cells (wildtype p53) the activation of p53 caused miR-34a up-regulation and loss of L1CAM expression that was miR-34a dependent. We observed an inverse correlation between L1CAM and miR-34a levels in EC cell lines. In primary tumor sections areas expressing high amounts of L1CAM had less miR-34a expression than those with low L1CAM levels. Our data suggest that miR-34a can regulate L1CAM expression by targeting L1CAM mRNA for degradation. These findings shed new light on the complex regulation of L1CAM in human tumors.
Assuntos
Neoplasias do Endométrio/genética , Neoplasias do Endométrio/metabolismo , MicroRNAs/genética , Molécula L1 de Adesão de Célula Nervosa/genética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Regiões 3' não Traduzidas , Azacitidina/farmacologia , Sequência de Bases , Processos de Crescimento Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Humanos , Imidazóis/farmacologia , MicroRNAs/biossíntese , MicroRNAs/metabolismo , Dados de Sequência Molecular , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Molécula L1 de Adesão de Célula Nervosa/biossíntese , Piperazinas/farmacologia , Fatores de Transcrição/genética , Ativação Transcricional , TransfecçãoRESUMO
BACKGROUND: L1 cell adhesion molecule (L1CAM), which belongs to the immunoglobulin superfamily, has recently been observed in a variety of human malignancies. However, its clinical implication in gastric cancer remains unclear. The aim of this study was to explore the role of L1CAM in gastric cancer and to analyze its correlation with tumor progression and prognosis. METHODS: L1CAM expression was measured in human gastric cancer cell lines and knockdown was conducted using siRNA. Cell proliferation, invasion and migration ability was assessed in vitro. The downstream pathway of L1CAM was explored by western blot analysis. L1CAM expression was measured in 112 pairs of human gastric cancer and adjacent noncancerous tissues using real-time quantitative RT-PCR, and the correlation with clinicopathological features and prognosis was analyzed. RESULTS: L1CAM downregulation by siRNA significantly decreased cell proliferation, migration, and invasion in gastric cancer cell lines. Phosphorylated ERK levels began to decline more rapidly in L1CAM knockdown cells compared with parental cells. L1CAM overexpression was significantly correlated with local tumor cell growth (P = 0.041), distant metastasis (P = 0.047), and tumor stage (P = 0.031). The overall survival in patients with high L1CAM expression was significantly shorter than that of patients with low L1CAM expression (P = 0.02). CONCLUSIONS: L1CAM overexpression may be a critical prognostic factor in patients with gastric cancer, and was strongly associated with tumor proliferation, migration, and invasion through the ERK pathway. L1CAM might be an attractive therapeutic molecular target for the treatment of gastric cancer patients.
Assuntos
Adenocarcinoma/secundário , Movimento Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neoplasias Peritoneais/secundário , Neoplasias Gástricas/patologia , Adenocarcinoma/metabolismo , Adenocarcinoma/mortalidade , Apoptose , Western Blotting , Adesão Celular , Proliferação de Células , MAP Quinases Reguladas por Sinal Extracelular/genética , Feminino , Humanos , Técnicas Imunoenzimáticas , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Estadiamento de Neoplasias , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Molécula L1 de Adesão de Célula Nervosa/genética , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/mortalidade , Prognóstico , RNA Mensageiro/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/mortalidade , Taxa de Sobrevida , Células Tumorais Cultivadas , CicatrizaçãoRESUMO
Retinal ganglion cells transmit the visual signal from the retina to the brain. We have previously shown that the activator protein 2 (AP-2)δ (TFAP2D) transcription factor is expressed in one third of ganglion cells in developing retina suggesting a specialized role for these AP-2δ-expressing cells. Here, we address the role of AP-2δ in retina by in ovo electroporation of RCAS/AP-2δ retroviral constructs into the eyes of chick embryos at day 2 of gestation. Ectopic expression of AP-2δ does not affect lineage differentiation in the developing retina. However, immunostaining of retinal tissue with markers associated with axonal growth such as growth-associated protein 43 and polysialic acid-neural cell adhesion molecule (PSA-NCAM) demonstrates axonal misrouting and abnormal axonal bundling. Treatment of AP-2δ-misexpressing retinal cell cultures with endoneuraminidase, an enzyme that removes PSA from NCAM, decreases AP-2δ-induced axonal bundling. Our data suggest a role for AP-2δ in polysialylation of NCAM, with ectopic expression of AP-2δ resulting in premature bundling of emerging axons and misrouting of axons. We propose that expression of AP-2δ in a subset of ganglion cells contributes to the fine-tuning of axonal growth in the developing retina.
Assuntos
Axônios/fisiologia , Coristoma , Regulação da Expressão Gênica no Desenvolvimento , Molécula L1 de Adesão de Célula Nervosa/biossíntese , Retina/embriologia , Retina/metabolismo , Ácidos Siálicos/biossíntese , Fator de Transcrição AP-2/biossíntese , Animais , Embrião de Galinha , Galinhas , Glicosídeo Hidrolases/farmacologia , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Retina/efeitos dos fármacos , Ácidos Siálicos/antagonistas & inibidores , Fator de Transcrição AP-2/antagonistas & inibidoresRESUMO
BACKGROUND/AIMS: The neural cell adhesion molecule L1CAM is a transmembrane glycoprotein abnormally expressed in tumors and previously associated with cell proliferation, adhesion and invasion, as well as neurite outgrowth in endometriosis. Being an attractive target molecule for antibody-based therapy, the present study assessed the ability of the monoclonal anti-L1 antibody (anti-L1 mAb) to impair the development of endometriotic lesions in vivo and endometriosis-associated nerve fiber growth. METHODS AND RESULTS: Endometriosis was experimentally induced in sexually mature B6C3F1 (n=34) and CD-1 nude (n=21) mice by autologous and heterologous transplantation, respectively, of endometrial fragments into the peritoneal cavity. Transplantation was confirmed four weeks post-surgery by in vivo magnetic resonance imaging and laparotomy, respectively. Mice were then intraperitoneally injected with anti-L1 mAb or an IgG isotype control antibody twice weekly, over a period of four weeks. Upon treatment completion, mice were sacrificed and endometrial implants were excised, measured and fixed. Endometriosis was histologically confirmed and L1CAM was detected by immunohistochemistry. Endometriotic lesion size was significantly reduced in anti-L1-treated B6C3F1 and CD-1 nude mice compared to mice treated with control antibody (P<0.05). Accordingly, a decreased number of PCNA positive epithelial and stromal cells was detected in autologously and heterologously induced endometriotic lesions exposed to anti-L1 mAb treatment. Anti-L1-treated mice also presented a diminished number of intraperitoneal adhesions at implantation sites compared with controls. Furthermore, a double-blind counting of anti-neurofilament L stained nerves revealed significantly reduced nerve density within peritoneal lesions in anti-L1 treated B6C3F1 mice (P=0.0039). CONCLUSIONS: Local anti-L1 mAb treatment suppressed endometriosis growth in B6C3F1 and CD-1 nude mice and exerted a potent anti-neurogenic effect on induced endometriotic lesions in vivo. The findings of this preliminary study in mice provide a strong basis for further testing in in vivo models.
Assuntos
Anticorpos Monoclonais/farmacologia , Endometriose/metabolismo , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Adulto , Animais , Anticorpos Monoclonais/administração & dosagem , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Endometriose/tratamento farmacológico , Endometriose/patologia , Feminino , Humanos , Camundongos , Molécula L1 de Adesão de Célula Nervosa/antagonistas & inibidores , Proteínas de Neurofilamentos/metabolismo , Adulto JovemRESUMO
BACKGROUND: Fetal alcohol spectrum disorder is an immense public health problem. In vitro studies support the hypothesis that L1 cell adhesion molecule (L1) is a target for ethanol (EtOH) developmental neurotoxicity. L1 is critical for the development of the central nervous system. It functions through signal transduction leading to phosphorylation and dephosphorylation of tyrosines on its cytoplasmic domain. The function of L1 is also dependent on trafficking through lipid rafts (LRs). Our hypothesis is that L1 is a target for EtOH neurotoxicity in vivo. Our objective is to demonstrate changes in L1 phosphorylation/dephosphorylation and LR association in vivo. METHODS: Rat pups on postnatal day 6 are administered 4.5, 5.25, and 6 g/kg of EtOH divided into 2 doses 2 hours apart, then killed. Cerebella are rapidly frozen for assay. Blood is analyzed for blood EtOH concentration. L1 tyrosine phosphorylation is determined by immunoprecipitation and dephosphorylation of tyrosine 1176 determined by immunoblot. LRs are isolated by sucrose density gradient, and the distribution of L1 in LRs is determined. RESULTS: EtOH at all doses reduced the relative amount of Y1176 dephosphorylation as well as the relative amount of L1 phosphorylated on other tyrosines. The proportion of L1 present in LRs is significantly increased in pups who received 6 g/kg EtOH compared to intubated controls. CONCLUSIONS: L1 is a target for EtOH developmental neurotoxicity in vivo.