RESUMEN
BACKGROUND: Cell-free DNA (cfDNA) is a source for liquid biopsy used for cancer diagnosis, therapy selection, and disease monitoring due to its non-invasive nature and ease of extraction. However, cfDNA also participates in cancer development and progression by horizontal transfer. In humans, cfDNA circulates complexed with extracellular vesicles (EV) and macromolecular complexes such as nucleosomes, lipids, and serum proteins. The present study aimed to demonstrate whether cfDNA not associated with EV induces cell transformation and tumorigenesis. METHODS: Supernatant of the SW480 human colon cancer cell line was processed by ultracentrifugation to obtain a soluble fraction (SF) and a fraction associated with EV (EVF). Primary murine embryonic fibroblast cells (NIH3T3) underwent passive transfection with these fractions, and cell proliferation, cell cycle, apoptosis, cell transformation, and tumorigenic assays were performed. Next, cfDNA was analyzed by electronic microscopy, and horizontal transfer was assessed by human mutant KRAS in recipient cells via PCR and recipient cell internalization via fluorescence microscopy. RESULTS: The results showed that the SF but not the EVF of cfDNA induced proliferative and antiapoptotic effects, cell transformation, and tumorigenesis in nude mice, which were reduced by digestion with DNAse I and proteinase K. These effects were associated with horizontal DNA transfer and cfDNA internalization into recipient cells. CONCLUSIONS: The results suggest pro-tumorigenic effects of cfDNA in the SF that can be offset by enzyme treatment. Further exploration of the horizontal tumor progression phenomenon mediated by cfDNA is needed to determine whether its manipulation may play a role in cancer therapy.
Asunto(s)
Ácidos Nucleicos Libres de Células , Humanos , Animales , Ratones , Ácidos Nucleicos Libres de Células/genética , Ratones Desnudos , Células 3T3 NIH , Carcinogénesis , ADNRESUMEN
Infliximab is therapeutic monoclonal antibody (mAb) against TNF-α employed in the treatment of immunoinflammatory diseases. The development of biosimilar mAbs is a global strategy to increase drug accessibility and reduce therapy-associated costs. Herein we compared key physicochemical characteristics and biological activities produced by infliximab and infliximab-Probiomed in order to identify functionally relevant differences between the mAbs. Binding of infliximab-Probiomed to TNF-α was specific and had kinetics comparable to that of the reference product. Both mAbs had highly similar neutralizing efficacy in HUVEC cell cultures stimulated with TNF-α. In vitro induction of CDC and ADCC were also similar between the evaluated products. In vivo comparability was assessed using a transgenic mouse model of arthritis that expresses human TNF-α in a 13-week multiple-administration study. Infliximab and infliximab-Probiomed showed comparable efficacy, safety, and pharmacokinetic profiles. Our results indicate that infliximab-Probiomed has highly similar activities to infliximab in preclinical models, warranting a clinical evaluation of its biosimilarity.
Asunto(s)
Antirreumáticos , Biosimilares Farmacéuticos , Infliximab , Animales , Antirreumáticos/farmacocinética , Antirreumáticos/farmacología , Antirreumáticos/uso terapéutico , Artritis/tratamiento farmacológico , Artritis/metabolismo , Biosimilares Farmacéuticos/farmacocinética , Biosimilares Farmacéuticos/farmacología , Biosimilares Farmacéuticos/uso terapéutico , Células CHO , Células Cultivadas , Cricetulus , Citocinas/genética , Citocinas/metabolismo , Selectina E/metabolismo , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Humanos , Infliximab/farmacocinética , Infliximab/farmacología , Infliximab/uso terapéutico , Molécula 1 de Adhesión Intercelular/metabolismo , Masculino , Ratones , Ratones Transgénicos , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Coumarin in vivo has antitumor activity in various types of cancer. In vitro, coumarin and 7-hydroxycoumarin, its major biotransformation product in humans, inhibit the proliferation of several human tumor cell lines. The molecular mechanisms of these effects are unknown. To gain information about these mechanisms, we studied the effects of coumarin and 7-hydroxycoumarin in the human lung adenocarcinoma cell line A-427 on the inhibition of: (i) cell proliferation; (ii) cell cycle progression; and (iii) expression of cyclins D1, E and A. The inhibitory concentrations 50 (IC(50)) of both compounds were estimated by cytostatic assays of tetrazolium (MTT) reduction. The effects on cell cycle progression were assayed with propidium iodide and BrdU using DNA histograms and multiparametric flow cytometry. The percentages of cells expressing cyclins D1, E, and A were estimated by means of bivariate flow cytometry using propidium iodide, and FITC-conjugated monoclonal antibodies for each cyclin. The IC(50) (+/-S.E.M. n=3) of 7-hydroxycoumarin and coumarin at 72 h exposure, were 100+/-4.8 and 257+/-8.8 microg/ml, respectively. 7-Hydroxycoumarin at the concentration of 160 microg/ml (1 mM), inhibited the G(1)/S transition of the cell cycle, an action consistent with the cytostatic effect. No significant decreases of cyclins E and A were observed. In contrast, cyclin D1 significantly decreased, which appears to indicate an action of 7-hydroxycoumarin in early events of phase G(1). However, messenger RNA of cyclin D1, assayed by RT-PCR, did not change. This suggests a posttranscriptional effect. The effects of coumarin were not significant. Cyclin D1 is overexpressed in many types of cancer, and its inhibition has been proposed as a pharmacological and therapeutic target for novel antitumor agents. Knowledge of the decrease of cyclin D1 by 7-hydroxycoumarin may lead to its use in cancer therapy, as well as to the development of more active compounds.
Asunto(s)
Adenocarcinoma/patología , Antineoplásicos/farmacología , Ciclo Celular/efectos de los fármacos , Cumarinas/farmacología , Ciclina D1/biosíntesis , Regulación Neoplásica de la Expresión Génica , Neoplasias Pulmonares/patología , Umbeliferonas/farmacología , Biotransformación , División Celular , Relación Dosis-Respuesta a Droga , Humanos , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células Tumorales CultivadasRESUMEN
Integrins are receptors that mediate cell adhesion and the formation of signaling complex. Changes in the expression of integrins are required during the following steps in the generation of metastases: a) angiogenesis; b) detachment from the primary tumor; c) tumor cell-platelet interaction; d) adhesion to vascular endothelium and e) proliferation. There is a correlation between invasive capability and changes in the expression of some proteins that are clustered in focal adhesion sites, as FAK, CD82, CD9 or CD63. Both, integrin blocking (using antibodies or RGD containing peptides), as well as induced changes in the expression of integrin-associated molecules, are able to inhibit formation of metastases. Discovery and characterization of molecules that regulate the adhesive capability of tumor cells, will lead to development of antimetastasic therapies. In the search of tumor dissemination inhibitors, integrins and some integrin-associated molecules are important pharmacological targets.