Antiangiogenic activity of the penicillin derivative TAP7f in melanoma.

Previously , we demonstrated that the non-antibiotic penicillin derivative TAP7f inhibited melanoma metastasis in vitro and in vivo through the downregulation of ß-catenin and integrin αVß3. As angiogenesis is required for tumor growth and metastasis, we decided to explore the possible antiangiogenic effect of TAP7f. We found that TAP7f inhibited proliferation, migration, tube formation, and actin cytoskeleton organization of human endothelial cells. In a gel plug assay, an in vivo model for angiogenesis, TAP7f also blocked vascular formation induced by fibroblast growth factor 2. Furthermore, when murine B16-F10 melanoma cells pre-treated with TAP7f were injected intradermally in mice, we observed a decrease in the number and thickness of the capillaries surrounding the tumor. Additionally, TAP7f downregulated vascular endothelial growth factor (VEGF) and platelet-derived growth factor-B (PDGF-B) expression in B16-F10 cells and VEGF receptor expression in HMEC-1 endothelial cells. When the antitumor effect of TAP7f was studied in C57BL/6 J mice challenged with B16-F10 melanoma cells, a significant reduction of tumor growth was observed. Furthermore, a decreased expression of VEGF, PDGF-B, and the endothelial cell marker CD34 was observed in tumors from TAP7f-treated mice. Together, our results suggest that the antiangiogenic activity of TAP7f contributes to its antitumor and antimetastatic action and positions this penicillin derivative as an alternative or complementary agent for the treatment of melanoma. KEY MESSAGES: • TAP7f inhibits proliferation, migration, tube formation, and actin cytoskeleton organization of endothelial cells. • TAP7f downregulates VEGF receptor expression in endothelial cells. • TAP7f downregulates VEGF and PDGF expression in melanoma cells. • TAP7f inhibits angiogenesis in vivo.

Contribution of endoplasmic reticulum stress, MAPK and PI3K/Akt pathways to the apoptotic death induced by a penicillin derivative in melanoma cells.

We have previously examined the in vitro and in vivo antitumor action of TAP7f, a synthetic triazolylpeptidyl penicillin, on murine melanoma cells. In this work, we explored the signal transduction pathways modulated by TAP7f in murine B16-F0 and human A375 melanoma cells, and the contribution of some intracellular signals to the apoptotic cell death. TAP7f decreased ERK1/2 phosphorylation and increased phospho-p38, phospho-JNK and phospho-Akt levels. ERK1/2 blockage suppressed cell growth, while inhibition of p38, JNK and PI3K-I pathways reduced the antitumor effect of TAP7f. Pharmacological inhibition of p38 and JNK, or blockage of PI3K-I/Akt cascade with a dominant negative PI3K-I mutant diminished Bax expression levels and PARP-1 cleavage, indicating the involvement of these pathways in apoptosis. PI3K-I/Akt inhibition also favored an autophagic response, as evidenced by the higher expression levels of Beclin-1 and LC3-II detected in transfected cells exposed to TAP7f. However, although PI3K-I/Akt blockage promoted an autophagic survival response, this mechanism appears not to be critical for TAP7f antitumor action. It was also shown that TAP7f induced ER stress by enhancing the expression of ER stress-related genes and proteins. Downregulation of CHOP protein with specific siRNA increased cell growth and decreased cleavage of PARP-1, supporting its role in apoptosis. Furthermore, it was found that activation of p38, JNK and Akt occurred downstream ER perturbation. In summary, our results showed that TAP7f triggers an apoptotic cell death in melanoma cells through induction of ER stress and activation of p38, JNK and PI3K-I/Akt pathways.

Design, synthesis and cytotoxic evaluation of peptoid analogs of an anticancer active triazolylpeptidyl penicillin.

Aim: Encouraged by the antitumor activity exhibited by triazolylpeptidyl penicillins, we decided to synthesize and evaluate a library of peptoid analogs. Results: The replacement of the dipeptide unit of the reference compound, TAP7f, was investigated. In addition, the effect of the triazole linking group on the biological activity of these new derivatives was evaluated, exchanging it with a glycine spacer. The cytotoxic effect of the library compounds was determined in the B16-F0 cell line and compared with the effects on normal murine mammary gland cells. Conclusion: Among the tested compounds, peptoid 4e exhibited the highest antiproliferative activity.

Design, synthesis and cytotoxic evaluation of a library of oxadiazole-containing hybrids.

The development of hybrid compounds led to the discovery of new pharmacologically active agents for some of the most critical diseases, including cancer. Herein, we describe a new series of oxadiazole-containing structures designed by a molecular hybridization approach. Penicillin derivatives and amino acids were linked to amino acid and aromatic moieties through the formation of a 1,2,4-oxadiazole ring. Alternatively, condensation between amino acid-derived hydrazides and an activated penicillanic acid led to a series of 1,3,4-oxadiazole penicillin-containing hybrids and non-cyclized diacylhydrazides. From the cytotoxicity assays it is highlighted that two 1,2,4-oxadiazoles and one 1,3,4-oxadiazole connecting a penicillin and aliphatic amino acids displayed a high degree of cytotoxic selectivity, ranging between being three and four times more potent against tumor cells than normal cells. The results give a very interesting perspective suggesting that these hybrid compounds can offer a novel antitumor scaffold with promising cytotoxicity profiles.

A Penicillin Derivative Exerts an Anti-Metastatic Activity in Melanoma Cells Through the Downregulation of Integrin αvß3 and Wnt/ß-Catenin Pathway.

The synthetic triazolylpeptidyl penicillin derivative, named TAP7f, has been previously characterized as an effective antitumor agent in vitro and in vivo against B16-F0 melanoma cells. In this study, we investigated the anti-metastatic potential of this compound on highly metastatic murine B16-F10 and human A375 melanoma cells. We found that TAP7f inhibited cell adhesion, migration and invasion in a dose-dependent manner. Additionally, we demonstrated that TAP7f downregulated integrin αvß3 expression and Wnt/ß-catenin pathway, a signaling cascade commonly related to tumor invasion and metastasis. Thus, TAP7f reduced both the enzymatic activity and the expression levels of matrix-metalloproteinases-2 and -9 in a time dependent manner. Moreover, TAP7f inhibited the expression of the transcription factor Snail and the mesenchymal markers vimentin, and N-cadherin, and up-regulated the expression of the epithelial marker E-cadherin, suggesting that the penicillin derivative affects epithelial-mesenchymal transition. Results obtained in vitro were supported by those obtained in a B16-F10-bearing mice metastatic model, that showed a significant TAP7f inhibition of lung metastasis. These findings suggest the potential of TAP7f as a chemotherapeutic agent for the treatment of metastatic melanoma.

Granulocyte colony-stimulating factor (G-CSF) upregulates ß1 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 ß1 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 ß1 integrin expression was dependent on PI3K activation. In conclusion, our results indicate that G-CSF stimulates ß1 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.

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.

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 G(2)/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 NH(2)-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.

STAT1, STAT3 and p38MAPK are involved in the apoptotic effect induced by a chimeric cyclic interferon-alpha2b 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.

2'-Nitroflavone induces cell cycle arrest and apoptosis in HeLa human cervical carcinoma cells.

The mechanism of antitumor action of a synthetic nitroflavone derivative, 2'-nitroflavone, was evaluated in vitro in HeLa human cervix adenocarcinoma cells. We showed that the nitroflavone derivative slowed down the cell cycle at the S phase and increase the population of cells at the G2/M phase after 24h of incubation. The treatment with 2'-nitroflavone also induced an apoptotic response, characterized by an increase of the sub-G1 fraction of cells, by cells with chromatin condensation and membrane blebbing, by a typical ladder of DNA fragmentation and by detection of apoptotic cells stained with Annexin V. The observed apoptosis was regulated by caspase-8 and -9, both contributing to the activation of the effector caspase-3. In addition, inhibitors of caspase-8 or -9 partially protected HeLa cells from 2'-nitroflavone-induced cell death. We also found that 2'-nitroflavone did not affect the total amount of Bax and Bcl-2 proteins, although a translocation of Bax from cytosol to mitochondria was evident after 6h of exposure. Furthermore, 2'-nitroflavone decreased the expression of the anti-apoptotic Bcl-XL protein, induced the release of cytochrome C to cytosol and increased the levels of Fas and Fas-L. Our results indicated that both death receptor and mitochondria-dependent pathways are involved in the apoptotic cell death triggered by 2'-nitroflavone and suggest that this derivative could be a potentially useful agent for the treatment of certain malignancies.

A cyclic chimeric interferon-alpha2b peptide induces apoptosis in tumor cells.

Interferons alpha (IFNsalpha) are a family of related proteins exhibiting antiviral, antiproliferative and immunoregulatory activities. Although IFNsalpha 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-alpha2b molecule, we have recently synthesized a chimeric cyclic peptide that inhibits IFN-alpha2b 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-alpha2b 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-alpha2b 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.

Antitumor activity of some natural flavonoids and synthetic derivatives on various human and murine cancer cell lines.

The effect of various natural flavonoids, cinnamic acid derivatives, and a series of synthetic flavones on cell proliferation was evaluated in vitro in a panel of established human and murine tumor cell lines. The most potent antiproliferative agents were caffeic acid n-butyl ester (12) > 2'-nitroflavone (26) > caffeic acid ethyl ester (11) approximately = 2',6-dinitroflavone (27) > apigenin (3) > 3'-bromoflavone (20) approximately = 2'-fluoro-6-bromoflavone (31). Some compounds showed a moderate effect, the order of cytotoxic activities being chrysin (2) > 2'-fluoro-6-chloroflavone (30) approximately = 2'-chlorochrysin (32) > alpha-naphthoflavone (7) > beta-naphthoflavone (8) approximately = 6-chloroflavone (14) approximately = 6-bromoflavone (15) approximately = 4'-nitroflavone (23). A structure-activity relationship analysis of each group of compounds was performed. None of the natural or synthetic compounds tested affected the proliferation of epithelial cells derived from normal mammary gland of mice or fibroblastic cells from mouse embryo, suggesting a selective action against tumor cells.

Synthesis and biological properties of chimeric interferon-alpha2b 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.

Antiproliferative activity of various flavonoids and related compounds: additive effect of interferon-alpha2b.

The antiproliferative activity of several natural and synthetic flavonoids and some related compounds was evaluated in vitro against a cell line derived from a human cervical carcinoma (WISH cells). According to their activities, the most potent derivatives were 2'-nitroflavone (14), 2',6-dinitroflavone (15) and the n-buthyl ester of caffeic acid (29). When these compounds were tested in the presence of recombinant human interferon-alpha2b (rhIFN-alpha2b), a cytokine exhibiting an antimitogenic action on WISH cells, an additive effect on cell growth inhibition was observed. Time course studies of the antiproliferative action exerted by the active derivatives or the rhIFN-alpha2b suggested that these compounds induced cell death.