URTREGER Alejandro Jorge
congresos y reuniones científicas
The novel synthetic peptide CIGB-300 affects several signaling pathways involved in malignant progression of human and murine lung tumours
STÉFANO M. CIRIGLIANO; MARÍA INÉS DÍAZ BESSONE; CAROLINA FLUMIAN; DAMIAN E. BERARDI; SILVIO PEREA; ELISA D. BAL DE KIER JOFFE; HERNAN FARINA; LAURA B. TODARO; ALEJANDRO J. URTREGER
Congreso; 6 th Latin American Conference on Lung Cancer (LALCA); 2014
International Association for the Study of Lung Cancer (IASLC)
CK2 is a serine/threonine kinase involved in cell growth, survival and apoptosis. CIGB-300 is a synthetic peptide capable of binding to CK2 substrates thus preventing the enzyme activity. We have studied the effect of CIGB-300 on several signaling pathways involved in tumor progression, cell cycle, apoptosis, migration and spheroid growth, using a model of human and murine lung cancer cell lines. Previously, we have determined that CIGB-300 presented an inhibitory concentration 50 (IC50) of 119±2.4 uM in NCI-H125 human lung cancer cells. Throughout an Annexin V-FITC assay we could determine that CIGB-300 is a potent apoptosis inductor since the treatment with this drug for 60 minutes induced apoptosis at a level comparable with that observed with etoposide (10 uM). Concomitantly with cell death induction, we could observe caspase-3 activation and the decreased expression of c-myc, cyclin D1 and D2. In this work we analyzed whether CIGB-300 is able to alter the ability of lung cancer cells to grow in 3D and to modulate signaling pathways involved in tumor progression. Methods Cell lines: the murine 3LL (Lewis lung carcinoma clone) and the NCI-H125 human non-small-cell lung adenocarcinoma were used. Cytotoxicity assays: the response to CIGB-300 was evaluated measuring cell viability by MTS. Nuclear factor kB (NF-kB) Western blot (Wb): to evaluate NF-kB expression levels, we performed the separation of nuclear and cytoplasmic fractions using NPER kit, following vendor suggestions. Wound healing assay: migration of 3LL and NCI-H125 cells was evaluated in wound performed in cell monolayers in the presence of CIGB-300. Apoptosis: was assessed in cells treated with CIGB-300 or etoposide by fluorescence, using Annexin V/PI staining, according the manufacturer´s protocol (Invitrogen). Reporter genes assay: was performed by transfection with a plasmid containing the luciferase gene under the regulation of NF-kB promoter. Multicellular tumor spheroids: was done through the hanging-drop method. Results To analyze the effect of CIGB-300 on canonical Wnt signaling, this pathway was initially activated by incubating NCI-H125 cells with a conditioned media containing Wnt3a factor. This incubation led to a significant increase in the levels of cytoplasmic b-catenin. By Wb we could observe that the treatment with CIGB-300 blocked this increase, suggesting its role in the modulation of the Wnt canonical pathway. In order to analyze the NF-kB dependent pathway, this transcription factor was activated by a phorbol ester treatment (PMA, 15 nM). Short treatment (15 min) with CIGB-300 induced an important reduction in nuclear NF-kB levels. However, this inhibition was reverted 24 h later as determined by Wb and reporter gene expression assays. Next, we evaluated the effect CIGB-300 in the modulation of 3D growth capacity. NCI-H125 cells were able to developed stable spheroids in vitro. After 5 days of treatment, CIGB-300 induced a significant growth inhibition (volume: 4.07x107±0.70x107 um3 in control spheroids vs 2.13x107±0.35x107 um3 in spheroids treated with the IC50). Conclusion Our results indicate that treatment with CIGB-300 strongly induces apoptosis and negatively modulates several signaling pathways involved in malignant progression. Moreover, CIGB-300 would be involved in the reduction of lung cancer malignant progression since it alters migratory and three dimensional cell growth. Altogether indicates that CIGB-300 may become a new strategy for the treatment of lung cancer.