Albert Green

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.

Inhibition of growth and induction of differentiation of metastatic melanoma cells in vitro by genistein : chemosensitivity is regulated by cellular p53

We have investigated the effect of the soybean isoflavone genistein on the growth and differentiation of human melanoma cells. Four human melanoma cell lines, either completely lacking or containing different levels of wild-type p53, were treated with genistein in vitro in culture. It has been found that genistein significantly inhibited cell growth and that the chemosensitivity might depend on cellular p53 content. Specifically, the data suggest that high levels of wild-type p53 expression make cells resistant to genisteins growth-inhibitory action. Further support for this observation came from the stable transfection studies in which p53 transfectants expressing high levels of wild-type p53 became resistant to genistein. With respect to cell differentiation, our study showed that genistein increased melanin content and tyrosinase activity and caused the cells to form dendrite-like structures. Cells lacking p53 responded more than cells with p53 to dendrite-like structure formation. We also observed that genistein-induced differentiation involved an increase in tyrosinase mRNA level; the mechanisms by which genistein increases tyrosinase transcripts remain to be elucidated. Genistein treatment of the melanoma cell lines resulted in cell cycle arrest at G2/M check point and no significant apoptosis was observed.

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.

Dehydroepiandrosterone inhibits the progression phase of mammary carcinogenesis by inducing cellular senescence via a p16-dependent but p53-independent mechanism.

IntroductionDehydroepiandrosterone (DHEA), an adrenal 17-ketosteroid, is a precursor of testosterone and 17β-estradiol. Studies have shown that DHEA inhibits carcinogenesis in mammary gland and prostate as well as other organs, a process that is not hormone dependent. Little is known about the molecular mechanisms of DHEA-mediated inhibition of the neoplastic process. Here we examine whether DHEA and its analog DHEA 8354 can suppress the progression of hyperplastic and premalignant (carcinoma in situ) lesions in mammary gland toward malignant tumors and the cellular mechanisms involved.MethodsRats were treated with N-nitroso-N-methylurea and allowed to develop mammary hyperplastic and premalignant lesions with a maximum frequency 6 weeks after carcinogen administration. The animals were then given DHEA or DHEA 8354 in the diet at 125 or 1,000 mg/kg diet for 6 weeks. The effect of these agents on induction of apoptosis, senescence, cell proliferation, tumor burden and various effectors of cellular signaling were determined.ResultsBoth agents induced a dose-dependent decrease in tumor multiplicity and in tumor burden. In addition they induced a senescent phenotype in tumor cells, inhibited cell proliferation and increased the number of apoptotic cells. The DHEA-induced cellular effects were associated with increased expression of p16 and p21, but not p53 expression, implicating a p53-independent mechanism in their action.ConclusionWe provide evidence that DHEA and DHEA 8354 can suppress mammary carcinogenesis by altering various cellular functions, inducing cellular senescence, in tumor cells with the potential involvement of p16 and p21 in mediating these effects.

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.

Cell proliferation, apoptosis, and expression of cyclin D1 and cyclin E as potential biomarkers in tamoxifen-treated mammary tumors.

Tamoxifen has been widely used for treatment, and more recently, for the prevention of breast cancer. Since breast carcinomas are composed of heterogeneous populations of estrogen receptor-positive (ER+) cells, we hypothesized that tamoxifen may suppress tumor growth by differentially affecting cell proliferation and apoptosis. ER+ mammary tumors were induced in Sprague–Dawley rats by N-methyl-N-nitrosourea (MNU) and when they became palpable, the animals were treated for 5, 10, or 20 days with tamoxifen, 1.0 mg/kg body weight. Tamoxifen induced a time-dependent decrease in proliferating (BrdU-labeled) cells, arrested the cells in G1/0 phase, and differentially decreased the cyclin E and cyclin D1 expression at mRNA and protein levels. In the same tumors, apoptotic cells increased during the first 10 days of treatment, but their number remained unchanged with extension of the treatment to 20 days. Thus, we provide data that tamoxifen may differentially affect cell proliferation and apoptosis in mammary tumors and that the expression levels of cyclin D1 and cyclin E might also be considered potential intermediate biomarkers of response of mammary tumors to tamoxifen and possibly to other selective estrogen receptor modulators (SERMs).

Cellular responses of mammary carcinomas to aromatase inhibitors: effects of vorozole.

Vorozole (Vz) is a competitive non-steroidal inhibitor of aromatase, which has been used to treat breast cancer in postmenopausal women and in various chemoprevention pre-clinical studies. Recently, we assessed the inhibitory effect of Vz on Mnu-induced mammary carcinogenesis (Lubet et al., 1994), as well as on the progression of mammary tumors (Lubet et al., 1998). In this study we evaluated the effects of Vz on tumor growth, serum estradiol, cell proliferation, apoptotic and non-apoptotic cell death to determine whether any of these surrogate’ markers might reflect the efficacy of various doses of Vz. Vz at doses of 2.5 (Hi), 0.32 (Md), and 0.08 (Lo) mg/kg body weight induced complete (100%), 60%, and 20% regression of mammary tumors, respectively. Vz at Hi and Md doses caused a decrease in serum estradiol within the first two days of treatment, and the estradiol values remained low with additional treatment for 4 and 10 days. When Vz was administered to animals bearing palpable tumors a time and dose-dependent decrease in the proliferating cells (BrdU-LI) was observed. The percentage of apoptotic cells (Al) sharply increased 2 days after initiation of Vz treatment and then decreased followed by an increase in non-apoptotic dead cells. Interestingly even the Lo dose of Vz, which was only moderately effective in suppressing tumor growth, decreased cell proliferation and increased cell death in the peripheral tumor areas at 4 and 10 days after initiation of treatment. The time- and dose-dependent alterations in various cell parameters suggest two different phases of Vz-induced cellular responses: (1) an early phase (2–4 days of treatment) with a sharp increase in apoptotic cells and decrease in proliferating cells, and (2) a later phase (10 days) with disintegration of tumor parenchyma, increase in non-apoptotic dead cells, and decrease in apoptotic cells. The dose-dependent decrease in proliferating cells and increase in apoptotic and non-apoptotic cell death in Vz-treated animals suggest that these biomarkers might be used as potential surrogate endpoints for efficacy in breast cancer chemoprevention and therapy studies with aromatase inhibitors.

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.