Mechanisms of cellular uptake, intracellular transportation, and degradation of CIGB-300, a Tat-conjugated peptide, in tumor cell lines.

CIGB-300 is a cyclic synthetic peptide that induces apoptosis in malignant cells, elicits antitumor activity in cancer animal models, and shows tumor reduction signs when assayed in first-in-human phase I trial in patients with cervical tumors. CIGB-300 impairs phosphorylation by casein kinase 2 through targeting the substrate's phosphoacceptor domain. CIGB-300 was linked to the cell penetrating peptide Tat to facilitate the delivery into cells. Previously, we showed that CIGB-300 had a differential antiproliferative behavior in different tumor cell lines. In this work, we studied differential antiproliferative behavior in terms of cellular uptake, intracellular transportation, and degradation in tumor cell lines with dissimilar sensitivity to CIGB-300. The internalization of CIGB-300 was studied in different malignant cell lines. We found that the cell membrane heparan sulfate proteoglycans act as main receptors for extracellular CIGB-300 uptake. The most sensitive tumor cell lines showed higher intracellular incorporation of CIGB-300 in comparison to less sensitive cell lines. Furthermore, CIGB-300 uptake is time- and concentration-dependent in all studied cell lines. It was shown that CIGB-300 has the ability to penetrate cells mainly by direct membrane translocation. However, a minor proportion of the peptide uses an energy-dependent endocytic pathway mechanism to gain access into cells. CIGB-300 is internalized and transported into cells preferentially by caveolae-mediated endocytosis. Lysosomes are involved in CIGB-300 degradation; highly sensitive cell lines showed degradation at earlier times compared to low sensitive cells. Altogether, our data suggests a mechanism of internalization, vesicular transportation, and degradation for CIGB-300 in tumor cells.

CIGB-300, a proapoptotic peptide, inhibits angiogenesis in vitro and in vivo.

We have previously demonstrated that a proapoptotic cyclic peptide CIGB-300, formerly known as P15-Tat delivered into the cells by the cell-penetrating peptide Tat, was able to abrogate the CK2-mediated phosphorylation and induce tumor regression when injected directly into solid tumors in mice or by systemic administration. In this work, we studied the role of CIGB-300 on the main events that take place in angiogenesis. At non-cytotoxic doses, CIGB-300 was able to inhibit adhesion, migration, and tubular network formation induced by human umbilical vein endothelial cells (HUVEC) growing upon Matrigel in vitro. Likewise, we evaluated the cellular penetration and localization into the HUVEC cells of CIGB-300. Our results confirmed a quick cellular penetration and a cytoplasmic accumulation in the early minutes of incubation and a translocation into the nuclei beginning at 12h of treatment, with a strong presence in the perinuclear area. A microarray analysis was used to determine the genes affected by the treatment. We observed that CIGB-300 significantly decreased four genes strongly associated with tubulogenesis, growth, and differentiation of endothelial cells. The CIGB-300 was tested in vivo on chicken embryo chorioallantoic membranes (CAM), and a large number of newly formed blood vessels were significantly regressed. The results suggested that CIGB-300 has a potential as an antiangiogenic treatment. The mechanism of action may be associated with partial inhibition of VEGF and Notch pathways.

CIGB-300, an anti-CK2 peptide, inhibits angiogenesis, tumor cell invasion and metastasis in lung cancer models.

OBJECTIVES: Casein kinase 2 (CK2) is overexpressed in several types of cancer. It has more than 300 substrates mainly involved in DNA reparation and replication, chromatin remodeling and cellular growth. In recent years CK2 became an interesting target for anticancer drug development. CIGB-300 is a peptidic inhibitor of CK2 activity, designed to bind to the phospho-acceptor domain of CK2 substrates, impairing the correct phosphorylation by the enzyme. The aim of this work was to explore the antitumor effects of this inhibitor in preclinical lung cancer models. MATERIALS AND METHODS: Human H125 and murine 3LL Lewis lung carcinoma cell lines were used to evaluate the effect of CIGB-300 treatment in vitro. For this purpose, adhesion, migration and invasion capabilities of cancer cells were tested. Proteolytic activity of tumor cell-secreted uPA and MMP after CIGB-300 incubation was also analyzed. In vivo anticancer efficacy of the peptide was evaluated using experimental and spontaneous lung colonization assays in C57BL/6 mice. Finally, in order to test the effect of CIGB-300 on tumor cell-induced angiogenesis, a modified Matrigel plug assay was conducted. RESULTS AND CONCLUSION: We demonstrate that treatment with low micromolar concentrations of CIGB-300 caused a drastic reduction of adhesion, migration and invasion of lung cancer cells. Reduced invasiveness after CIGB-300 incubation was associated with decreased proteolytic activity of tumor cell-conditioned medium. In vivo, intravenous administration of CIGB-300 (10mg/kg) markly decreased lung colonization and metastasis development of 3LL cells. Interestingly, after 5days of systemic treatment with CIGB-300, tumor cell-driven neovascularization was significantly reduced in comparison to control group. Altogether our data suggest an important role of CK2 in lung tumor development, suggesting a potential use of CIGB-300 as a novel therapeutic agent against lung cancer.