Viviana C. Blank

Lysosomal and mitochondrial permeabilization mediates zinc(II) cationic phthalocyanine phototoxicity.

In order to find a novel photosensitizer to be used in photodynamic therapy for cancer treatment, we have previously showed that the cationic zinc(II) phthalocyanine named Pc13, the sulfur-linked dye 2,9(10),16(17),23(24)-tetrakis[(2-trimethylammonium) ethylsulfanyl]phthalocyaninatozinc(II) tetraiodide, exerts a selective phototoxic effect on human nasopharynx KB carcinoma cells and induces an apoptotic response characterized by an increase in the activity of caspase-3. Since the activation of an apoptotic pathway by chemotherapeutic agents contributes to the elimination of malignant cells, in this study we investigated the molecular mechanisms underlying the antitumor action of Pc13. We found that after light exposure, Pc13 induced the production of reactive oxygen species (ROS), which are mediating the resultant cytotoxic action on KB cells. ROS led to an early permeabilization of lysosomal membranes as demonstrated by the reduction of lysosome fluorescence with acridine orange and the release of lysosomal proteases to cytosol. Treatment with antioxidants inhibited ROS generation, preserved the integrity of lysosomal membrane and increased cell proliferation in a concentration-dependent manner. Lysosome disruption was followed by mitochondrial depolarization, cytosolic release of cytochrome C and caspases activation. Although no change in the total amount of Bax was observed, the translocation of Bax from cytosol to mitochondria, the cleavage of the pro-apoptotic protein Bid, together with the decrease of the anti-apoptotic proteins Bcl-XL and Bcl-2 indicated the involvement of Bcl-2 family proteins in the induction of the mitochondrial pathway. It was also demonstrated that cathepsin D, but not caspase-8, contributed to Bid cleavage. In conclusion, Pc13-induced cell photodamage is triggered by ROS generation and activation of the mitochondrial apoptotic pathway through the release of lysosomal proteases. In addition, our results also indicated that Pc13 induced a caspase-dependent apoptotic response, being activation of caspase-8, -9 and -3 the result of a post-mitochondrial event.

STAT1, STAT3 and p38MAPK are involved in the apoptotic effect induced by a chimeric cyclic interferon-α2b peptide

In the search of mimetic peptides of the interferon-alpha2b molecule (IFN-alpha2b), we have previously designed and synthesized a chimeric cyclic peptide of the IFN-alpha2b that inhibits WISH cell proliferation by inducing an apoptotic response. Here, we first studied the ability of this peptide to activate intracellular signaling pathways and then evaluated the participation of some signals in the induction of apoptosis. Stimulation of WISH cells with the cyclic peptide showed tyrosine phosphorylation of Jak1 and Tyk2 kinases, tyrosine and serine phosphorylation of STAT1 and STAT3 transcription factors and activation of p38 MAPK pathway, although phosphorylation levels or kinetics were in some conditions different to those obtained under IFN-alpha2b stimulus. JNK and p44/42 pathways were not activated by the peptide in WISH cells. We also showed that STAT1 and STAT3 downregulation by RNA interference decreased the antiproliferative activity and the amount of apoptotic cells induced by the peptide. Pharmacological inhibition of p38 MAPK also reduced the peptide growth inhibitory activity and the apoptotic effect. Thus, we demonstrated that the cyclic peptide regulates WISH cell proliferation through the activation of Jak/STAT signaling pathway. In addition, our results indicate that p38 MAPK may also be involved in cell growth regulation. This study suggests that STAT1, STAT3 and p38 MAPK would be mediating the antitumor and apoptotic response triggered by the cyclic peptide in WISH cells.

Synthesis and biological properties of chimeric interferon-α2b peptides

We have previously reported the antiproliferative activity of synthetic sequences 29-35 and 122-139 of the interferon-alpha2b (IFN-alpha2b), both probably representing a common receptor recognition domain. In the search of new peptidic agonists, we designed and synthesized the linear peptide (Gly)2-122-137-Gly138-Gly29-30-35-(Gly)2, in which Gly residues replaced the 138 and 29 Cys bound through a disulfide bridge in the native cytokine. Additionally, a cyclic analog was obtained by reaction of the N- and C-terminal ends of the linear fragment. Thus, the distance that separates residues 122 and 35 in the crystalline structure of the IFN-alpha2b was maintained through a (Gly)4 bridge. When the influence of chimeric peptides on the proliferation of WISH cells was studied, it was shown that both derivatives significantly diminished cell growth. A more evident inhibitory effect on (125)I-IFN-alpha2b binding to WISH cell-membrane receptors was observed for both peptides. Results indicated that chimeric IFN-alpha2b peptides behaved as partial agonists of the IFN-alpha2b molecule and may be of interest for drug design purposes.

2'-Nitroflavone induces apoptosis and modulates mitogen-activated protein kinase pathways in human leukaemia cells.

The cytotoxic activity of 2′-nitroflavone was evaluated in different haematological cancer cell lines and its mechanism of action was further studied in HL-60 cells. 2′-Nitroflavone arrested the cell cycle at the G2/M phase and induced an apoptotic response characterized by an increase in the sub-G1 fraction of cells, a typical DNA ladder fragmentation, chromatin condensation and the detection of cells stained with Annexin V. Apoptosis was dependent on the activation of at least caspase-8, caspase-9 and caspase-3. The involvement of the death receptor pathway was indicated by the upregulation of both the tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and its death receptor (DR5). We also showed that 2′-nitroflavone increased the expression levels of Bax and induced the release of cytochrome C to cytosol, suggesting the participation of the mitochondria-dependent pathway. When mitogen-activated protein kinases pathways were studied, it was found that p38 and c-Jun NH2-terminal kinase (JNK) pathways were activated by 2′-nitroflavone in HL-60 cells, whereas the phosphorylation levels of extracellular signal-regulated kinases (ERK) 1/2 decreased significantly. In addition, whereas both pharmacological inhibition of JNK and downregulation of JNK expression by RNA interference reduced the nitroflavone growth-inhibitory activity and the apoptotic effect, contrasting results were obtained when the ERK1/2 pathway was inhibited, and no effect was observed in the presence of a specific inhibitor of p38 mitogen-activated protein kinase. These findings show for the first time the antitumour action of 2′-nitroflavone in haematological cancer cell lines and suggest that both JNK and ERK1/2 cascades are involved in the apoptotic response induced by 2′-nitroflavone in HL-60 cells.

A cyclic chimeric interferon-α2b peptide induces apoptosis in tumor cells

Interferons alpha (IFNsα) are a family of related proteins exhibiting antiviral, antiproliferative and immunoregulatory activities. Although IFNsα have been widely employed for the pharmacological treatment of different types of cancer, the therapeutic efficacy occasionally can be diminished by the appearance of side effects, neutralizing antibodies or tumor resistance. In the search of mimetic peptides of the IFN-α2b molecule, we have recently synthesized a chimeric cyclic peptide that inhibits IFN-α2b binding to its receptor and exerts an IFN-like antiproliferative activity. In order to study the mechanism of growth inhibition of the cyclic chimera, we evaluated its ability to induce cell cycle arrest or apoptosis in WISH cells. We found that the chimeric peptide did not cause a cell cycle arrest, although the entire IFN-α2b molecule did modify cell cycle by increasing the number of S-phase cells. In spite of this difference, both molecules were able to induce apoptosis through the activation of caspases 8 and 9, indicating the involvement of death receptor and mitochondrial pathways. In addition, both peptidic derivative and IFN-α2b altered the expression of Bcl-2 family proteins and induced the release of cytochrome C to cytosol, supporting the participation of mitochondrial pathway in the induction of apoptosis. In conclusion, we demonstrated that the chimeric cyclic peptide behaved as a potent inducer of apoptosis and it could be a potentially useful agent for the treatment of certain malignancies.

Positive cooperative effects between receptors induced by an anti-human growth hormone allosteric monoclonal antibody

Monoclonal antibodies (MAb) anti-human growth hormone (hGH) termed MAb AE5, AC8 and F11 recognize a cluster of epitopes left exposed after hormone binding to receptors. Since these MAb were able to produce either positive (MAb AE5) or negative (MAb AC8 and F11) allosteric effects on hGH binding, the purpose of this work was to further characterize MAb behavior. Results indicated a straight correlation between MAb allosteric effects and affinity constant values for binding of different hGH:MAb complexes to lactogenic receptors from rat liver. Affinity of hGH:MAb AE5 as well as hGH:Fab AE5 complexes enhanced proportionally to the fraction of occupied receptors and Hill coefficients higher than 1 were obtained, suggesting the induction of positive cooperative effects between membrane-bound receptors. On the other hand, hGH:MAb AC8 and hGH:MAb F11 complexes binding affinity to lactogenic sites could not be related to receptor occupancy degree. It is proposed that binding of hGH:MAb AE5 complexes to receptors would elicit a conformational change on adjacent receptor molecules leading to an increase of their affinity to bind subsequent hGH:MAb AE5 complexes.

Granulocyte colony-stimulating factor (G-CSF) upregulates β1 integrin and increases migration of human trophoblast Swan 71 cells via PI3K and MAPK activation.

Multiple cytokines and growth factors expressed at the fetal-maternal interface are involved in the regulation of trophoblast functions and placental growth, but the role of G-CSF has not been completely established. Based on our previous study showing that G-CSF increases the activity of matrix metalloproteinase-2 and the release of vascular endothelial growth factor in Swan 71 human trophoblast cells, in this work we explore the possible contribution of G-CSF to cell migration and the G-CSF-triggered signaling pathway. We found that G-CSF induced morphological changes on actin cytoskeleton consistent with a migratory cell phenotype. G-CSF also up-regulated the expression levels of β1 integrin and promoted Swan 71 cell migration. By using selective pharmacological inhibitors and dominant negative mutants we showed that PI3K, Erk 1/2 and p38 pathways are required for promoting Swan 71 cell motility. It was also demonstrated that PI3K behaved as an upstream regulator of Erk 1/2 and p38 MAPK. In addition, the increase of β1 integrin expression was dependent on PI3K activation. In conclusion, our results indicate that G-CSF stimulates β1 integrin expression and Swan 71 cell migration by activating PI3K and MAPK signaling pathways, suggesting that G-CSF should be considered as an additional regulatory factor that contributes to a successful embryo implantation and to the placenta development.

In vitro anticancer activity and SAR studies of triazolyl aminoacyl(peptidyl) penicillins

A library of triazolyl aminoacyl(peptidyl) penicillins was designed, synthesized, and evaluated for their antiproliferative activity against HeLa and B16-F0 cell lines. Structure–activity relationship studies were carried out, and minimal structural requirements were determined. Among the tested compounds, derivatives 7f, 7p and 7m demonstrated the highest anticancer activity and a promising selectivity profile against these two cell lines.

A solid- and solution-phase-based library of 2β-methyl substituted penicillin derivatives and their effects on growth inhibition of tumor cell lines

We described the design, synthesis and antiproliferative properties of a series of twenty 2β-methyl substituted penicillin derivatives. This analysis includes evaluation against HeLa and MCF-7 human tumor cell lines and LM3 and B16-F0 murine tumor cell lines. The epithelial cell line derived from the normal mammary gland of mice (NMuMG) and the mouse embryo fibroblastcell line (3T3) were used as controls (non-cancer cells).

A chimeric cyclic interferon-α2b peptide induces apoptosis by sequential activation of phosphatidylinositol 3-kinase, protein kinase Cδ and p38 MAP kinase

We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells.