Laura Bratescu

Noncationic Peptides Obtained From Azurin Preferentially Enter Cancer Cells

Azurin, a member of the cupredoxin family of copper containing redox proteins, preferentially penetrates human cancer cells and exerts cytostatic and cytotoxic (apoptotic) effects with no apparent activity on normal cells. Amino acids 50 to 77 (p28) of azurin seem responsible for cellular penetration and at least part of the antiproliferative, proapoptotic activity of azurin against a number of solid tumor cell lines. We show by confocal microscopy and fluorescence-activated cell sorting that amino acids 50 to 67 (p18) are a minimal motif (protein transduction domain) responsible for the preferential entry of azurin into human cancer cells. A combination of inhibitors that interfere with discrete steps of the endocytotic process and antibodies for caveolae and Golgi-mediated transport revealed that these amphipathic, alpha-helical peptides are unique. Unlike the cationic cell-penetrating peptides, alpha-helical antennapedia-like, or VP22 type peptides, p18 and p28 are not bound by cell membrane glycosaminoglycans and preferentially penetrate cancer cells via endocytotic, caveosome-directed, and caveosome-independent pathways. Once internalized, p28, but not p18, inhibits cancer cell proliferation initially through a cytostatic mechanism. These observations suggest the azurin fragments, p18 and p28, account for the preferential entry of azurin into human cancer cells and a significant amount of the antiproliferative activity of azurin on human cancer cells, respectively.

Internalization of bacterial redox protein azurin in mammalian cells: entry domain and specificity.

Azurin is a member of a group of copper‐containing redox proteins called cupredoxins. Different cupredoxins are produced by different aerobic bacteria as agents of electron transfer. Recently, we demonstrated that azurin enters into J774 and several types of cancer cells leading to the induction of apoptosis. We now demonstrate that azurin is internalized in J774 or cancer cells in a temperature‐dependent manner. Azurin shows preferential entry into cancer compared with normal cells. An 28‐amino‐acid fragment of azurin fused to glutathione S‐transferase (GST) or the green fluorescent protein (GFP), which are incapable of entering mammalian cells by themselves, can be internalized in J774 or human melanoma or breast cancer cells at 37°C, but not at 4°C. Competition experiments as well as studies with inhibitors such as cytochalasin D suggest that azurin may enter cells, at least in part, by a receptor‐mediated endocytic process. The 28‐amino‐acid peptide therefore acts as a potential protein transduction domain (PTD), and can be used as a vehicle to transport cargo proteins such as GST and GST–GFP fusion proteins. Another member of the cupredoxin family, rusticyanin, that has also been shown to enter J774 and human cancer cells and exert cytotoxicity, does not demonstrate preferential entry for cancer cells and lacks the structural features characteristic of the azurin PTD.

A peptide fragment of azurin induces a p53-mediated cell cycle arrest in human breast cancer cells

We report that amino acids 50 to 77 of azurin (p28) preferentially enter the human breast cancer cell lines MCF-7, ZR-75-1, and T47D through a caveolin-mediated pathway. Although p28 enters p53 wild-type MCF-7 and the isogenic p53 dominant-negative MDD2 breast cancer cell lines, p28 only induces a G2-M-phase cell cycle arrest and apoptosis in MCF-7 cells. p28 exerts its antiproliferative activity by reducing proteasomal degradation of p53 through formation of a p28:p53 complex within a hydrophobic DNA-binding domain (amino acids 80-276), increasing p53 levels and DNA-binding activity. Subsequent elevation of the cyclin-dependent kinase inhibitors p21 and p27 reduces cyclin-dependent kinase 2 and cyclin A levels in a time-dependent manner in MCF-7 cells but not in MDD2 cells. These results suggest that p28 and similar peptides that significantly reduce proteasomal degradation of p53 by a MDM2-independent pathway(s) may provide a unique series of cytostatic and cytotoxic (apoptotic) chemotherapeutic agents. [Mol Cancer Ther 2009;8(10):2947–58]

A cell penetrating peptide derived from azurin inhibits angiogenesis and tumor growth by inhibiting phosphorylation of VEGFR-2, FAK and Akt

Amino acids 50–77 (p28) of azurin, a 128 aa cupredoxin isolated from Pseudomonas aeruginosa, is essentially responsible for azurin’s preferential penetration of cancer cells. We now report that p28 also preferentially penetrates human umbilical vein endothelial cells (HUVEC), co-localized with caveolin-1 and VEGFR-2, and inhibits VEGF- and bFGF-induced migration, capillary tube formation and neoangiogenesis in multiple xenograft models. The antiangiogenic effect of p28 in HUVEC is associated with a dose-related non-competitive inhibition of VEGFR-2 kinase activity. However, unlike other antiangiogenic agents that inhibit the VEGFR-2 kinase, p28 decreased the downstream phosphorylation of FAK and Akt that normally precedes cellular repositioning of the cytoskeletal (F-actin), focal adhesion (FAK and paxillin), and cell to cell junction protein PECAM-1, inhibiting HUVEC motility and migration. The decrease in pFAK and pAkt levels suggests that p28 induces a pFAK-mediated loss of HUVEC motility and migration and a parallel Akt-associated reduction in cell matrix attachment and survival. This novel, direct antiangiogenic effect of p28 on endothelial cells may enhance the cell cycle inhibitory and apoptotic properties of this prototype peptide on tumor cell proliferation as it enters a Phase II clinical trial.

Betulinic acid reduces ultraviolet-C-induced DNA breakage in congenital melanocytic naeval cells: evidence for a potential role as a chemopreventive agent.

Melanoma transformation progresses in a multistep fashion from precursor lesions such as congenital naevi. Exposure to ultraviolet (UV) light promotes this process. Betulinic acid (BA) was identified by our group as a selective inhibitor of melanoma that functions by inducing apoptosis. The present study was designed to investigate the effect of BA and UV-C (254 nm) on cultured congenital melanocytic naevi (CMN) cells, using the single-cell gel electrophoresis (comet) assay to detect DNA damage. Exposure to UV light induced a 1.7-fold increase in CMN cells (P = 0.008) when compared with controls. When a p53 genetic suppressor element that encodes a dominant negative polypeptide (termed GSE56) was introduced into the CMN cells, the transfected cells were more sensitive to UV-induced DNA breakage. This suggests that p53 can protect against UV-induced DNA damage and subsequent melanoma transformation. Pretreatment with BA (3 μm) for 48 h resulted in a 25.5% reduction in UV-induced DNA breakage in the CMN cells (P = 0.023), but no changes were observed in the transfected cells. However, Western blot analysis revealed no changes in the p53 or p21 levels in BA-treated cells, suggesting that BA might mediate its action via a non-p53 pathway. These data indicate that BA may have an application as a chemopreventive agent in patients with congenital naevi.

p28, A first in class peptide inhibitor of cop1 binding to p53

Background:A 28 amino-acid (aa) cell-penetrating peptide (p28) derived from azurin, a redox protein secreted from the opportunistic pathogen Pseudomonas aeruginosa, produces a post-translational increase in p53 in cancer cells by inhibiting its ubiquitination.Methods:In silico computational simulations were used to predict motifs within the p53 DNA-binding domain (DBD) as potential sites for p28 binding. In vitro direct and competitive pull-down studies as well as western blot and RT-PCR analyses were used to validate predictions.Results:The L1 loop (aa 112–124), a region within the S7–S8 loop (aa 214–236) and T140, P142, Q144, W146, R282 and L289 of the p53DBD were identified as potential sites for p28 binding. p28 decreased the level of the E3 ligase COP1 >80%, in p53wt and p53mut cells with no decrease in COP1 in p53dom/neg or p53null cells. Brief increases in the expression of the E3 ligases, TOPORS, Pirh2 and HDM2 (human double minute 2) in p53wt and p53mut cells were in response to sustained increases in p53.Conclusion:These data identify the specific motifs within the DBD of p53 that bind p28 and suggest that p28 inhibition of COP1 binding results in the sustained, post-translational increase in p53 levels and subsequent inhibition of cancer cell growth independent of an HDM2 pathway.

Establishment and characterization of four Sinclair Swine cutaneous malignant melanoma cell lines

Cutaneous malignant melanoma of Sinclair Swine (SSCM) is a heritable, congenital neoplasm which either proves fatal to the neonatal animal or undergoes spontaneous regression. Four SSCM cell lines, UISO-SSCM-433, UISO-SSCM-438, UISO-SSCM-5052, and UISO-SSCM-8093, were derived from biopsy specimens of primary tumors removed from swine at 26, 8, and 8 weeks of age, and 15 weeks gestation, respectively. Morphologic features, DOPA oxidase staining, and abnormal karyotype were suggestive of malignant melanoma. Each cell line was morphologically heterogeneous in culture with dendritic, spindle- and cuboidal-shaped cells. Pigmented melanosomes and DOPA oxidase activity were present in all cell lines at passages 20-22. UISO-SSCM-433 and UISO-SSCM-5052 contained hypodiploid and hypotetraploid sublines whereas UISO-SSCM-438 and UISO-SSCM-8093 were hypodiploid and hypotetraploid, respectively. At later passages, all cell lines presented evolutionary, karyotypic changes; the same chromosomes were involved in the alterations, however. Chromosomes 2, 6, 13, and 14 were the most affected, exhibiting numerical and structural alterations in all four cell lines. Despite the presence of multiple chromosomal anomalies in all cell lines, each with a unique set of chromosomal markers, clonal growth was not detected in soft agar, nor were any of the lines tumorigenic following s.c. inoculation in athymic mice. This suggests that the loss of malignant potential in SSCM may be inherent.

Bexarotene Induces Cellular Senescence in MMTV-Neu Mouse Model of Mammary Carcinogenesis

Previous studies have shown that retinoids and rexinoids can prevent breast cancer in animal models and in women with increased risk of developing the disease. The cellular effects of these vitamin A analogues have been primarily associated with induction of differentiation and inhibition of proliferation. In this study, we tested the hypothesis that bexarotene (LGD1069, Targretin), a rexinoid, can not only inhibit cell proliferation but also induce cellular senescence in mammary epithelial cells, premalignant lesions, and tumors of the MMTV-Neu model of mammary carcinogenesis, which develops estrogen receptor–negative tumors. Mice with palpable mammary tumors were treated for 4 weeks with bexarotene at 80 or 40 mg/kg body weight, and senescent cells were determined by SA-β-Gal assay. Bexarotene decreased in a dose-dependent manner the multiplicity of premalignant lesions and tumors, and this was associated with inhibition of cell proliferation and induction of cellular senescence and apoptosis. By double labeling of senescent cells, first by SA-β-Gal and then by antibodies against genes related to cellular senescence, we found that p21, p16, and RARβ, but not p53, were upregulated by bexarotene in mammary tumors and in breast cancer cell lines, suggesting involvement of multiple signaling pathways in mediating the senescence program of rexinoids. These findings indicate that, in addition to cell proliferation and apoptosis, cellular senescence could be used as a potential biomarker of response in breast cancer prevention and therapy studies with rexinoids and possibly with other antitumor agents. Cancer Prev Res; 6(4); 299–308. ©2013 AACR.

Chromosome abnormalities in metastatic melanoma

Primary cutaneous melanoma develops from an abnormal proliferation of melanocytes. The mechanism(s) that trigger and control this abnormal proliferation are not fully understood. Many other malignancies display recurring sites of chromosome changes (3,23,25,28,30,36). These nonrandom chromosomal abnormalities are useful in localizing and identifying genes that are central to the process of malignant transformation. Several cytogenetic studies of malignant melanoma, performed on advanced lesions (mainly metastasis) and established cell lines, have also reported nonrandom chromosomal abnormalities (8-11,14,29,32), Most of these studies reported a high level of aneuploidy in ceils, along with multiple rearrangements in chromosomes. The most frequently rearranged chromosomes found in these studies were chromosomes 1, 6, and 7. In the present investigation, ultrastructure and cytogenetic analy~es were performed in 10 cell lines derived from biopsies obtained from patients with metastatic melanoma. Our results show that the cell lines are mainly composed of melanoma cells with no fibroblasts, and karyotyping confirmed previous observations on the multiple chromosomal rearrangements involving chromosome 1, 6, and 7. However, in contrast to previous reports, we found a high frequency of changes involving chromosome 3. In 2 of the 10 cell

Abstract 2870: p28, a cell penetrating peptide fragment of azurin, inhibits COP1 mediated ubiquitination of wild type and mutated p53, but does not alter intracellular levels of c-jun

Amino acids (aa) 50-77 of azurin, p28, a 128 aa member of the cupredoxin family of copper containing redox proteins, isolated from the opportunistic pathogen Pseudomonas aeruginosa is a cell penetrating peptide (CPP) that preferentially penetrates a wide variety of human cancer cells. Exposure of p53 wt, mut cancer cells to p28 produces a post translational increase in the level of p53, inhibition of the cell cycle at G 2 /M and apoptosis (Yamada et. al., Mol. Can. Therap., 8:2947-58, 2009). Computer simulation (ClusPro, GROMACS) and domain specific antibodies to p53 suggest p28 binds to motifs that span aa 110–146 in flexible L 1 loop and 80 and 276 of the p53 DNA binding domain (DBD), respectively. Western and RT-PCR analyses of human breast cancer, MCF-7 (p53 wt ), MCF-7 derived MDD2 (p53 dom/neg ), MDA-MB-231 (p53 mut ), and melanoma cells, Mel-29 (p53 wt ), Mel-23 (p53 mut ) and Mel-6 (p53 null ) exposed to p28 for 72 hrs showed a significant increase in the levels of p53 and p21 in MCF-7, MDA-MB-231, Mel-29 and Mel-23 cells, while significantly reducing the level of FoxM1 over a 72hr exposure. The level of STMN1 was not altered suggesting the block at G 2 /M does not involve microtubules. p28 significantly decreased the level of the E3 ligase COP1 in p53 wt, mut MCF-7, MDA-MB-231, Mel-29 and Mel-23, but not in p53 null Mel-6 cells. In addition, p28 did not significantly decrease the level of E3 ligases TOPORS, Pirh2 or HDM2 which also ubiquitinate p53. The levels of p53, p21, FoxM 1, STMN1, COP1, TOPORS and Pirh2 either remained essentially at control levels (p53, p21) or were elevated (FoxM1, STMN1, COP1, TOPORS, Pirh2) at some point during a 72 hr exposure of p53 dom/neg MDD2 cells to p28. In contrast, COP1 has also been reported to negatively regulate cell proliferation in a c-jun-dependent manner and not regulate intracellular levels of p53 (Migliorini et. al., J. Clin. Invest., 121: 1329-43, 2011). The p28 induced decrease in the level of COP1 in p53 wt, mut cancer cells was not accompanied by a sustained increase in c-jun, in any cancer cell line, suggesting that COP1 regulates the level of p53 in cancer cells independent of any effect on c-jun. These data also suggest that COP1, but not TOPORS or Pirh2, also binds within this region of p53, and that COP1 is a major regulator of p53 activity. In sum, these data suggest that p28 binds to a specific motif within the p53 DBD where it inhibits COP1 mediated binding to and ubiquitination of p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2870. doi:1538-7445.AM2012-2870