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-ß-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-ß-Gal and then by antibodies against genes related to cellular senescence, we found that p21, p16, and RARß, 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.

Early in vitro passages of breast cancer cells are differentially susceptible to retinoids and differentially express RARß isoforms.

The effect of retinoids on breast cancer has been predominantly studied in vitro, on established cell lines, which in biology differ significantly from primary tumor cells. Little is known on whether early in vitro passages of breast cancer cells (EPBCCs) are differentially sensitive to retinoids and differentially express retinoid acid receptors (RARs) and retinoid X receptors (RXRs). We have previously identified a novel RARß isoform (RARß5) and hypothesized that it may serve as a potential target of retinoids in EPBCCs. Breast cancer cells isolated from primary tumors were cultured in vitro for 6-12 passages (EPBCCs) and their epithelial origin was confirmed by a cocktail of antibodies against cytokeratins. EPBCCs were treated for 4 days with 1.0 µM of all-trans retinoic acid (atRA), 9-cis retinoic acid (9cRA) or 4-hydroxy-phenylretinamide (4-HPR) and their viability determined by MTT assay. Among nine EPBCCs consistently grown in vitro, three were resistant to the above retinoids, five were susceptible to atRA, four to 4-HPR and two to 9cRA, suggesting that patients with breast carcinomas may differentially respond to various retinoids. All EPBBCs differentially expressed RARα, RARγ, RXRα, RXRß proteins and RARß5 and RARß2 mRNAs. However, only one EPBCC (BCA-2) expressed RARß5 at mRNA and protein level and it was resistant to retinoids, both in vitro and in a xenograft tumor assay. RARß5 suppression by siRNA in BCA-2 cells increased their susceptibility to atRA. No correlation was found between sensitivity of EPBCCs to the above retinoids and RARß5 and RARß2 mRNA expression. atRA reduced RARß expression in most EPBCCs suggesting that this retinoid receptor is most probably the prime target of retinoids in breast cancer. These data may have clinical implication in selecting patients with breast cancer that would benefit the most from clinical trials with retinoids.

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.

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 G(2)-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.

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 degrees C, but not at 4 degrees 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.

Retinoids suppress premalignant MCF10AT but not malignant MCF10CA1a breast epithelial cells in vivo. Role of retinoic acid receptor beta2 expression.

Recently we found that retinoic acid receptors (alpha, beta, gamma) and retinoid X receptors (alpha, beta, gamma) are variably expressed in MCF10A model of breast cancer development and progression. Here we employed this model to assess both in vitro and in vivo sensitivity of cells to retinoids and the role of RARbeta2 in mediating the antitumor potential of retinoids. In vitro, we found that transformation of the benign MCF10A cells into premalignant MCF10AT and malignant MCF10CA1a cells increased their sensitivity to 4-(Hydroxyphenyl)retinamide (4-HPR) and all-trans-retinoic acid (atRA) but not to 9-cis-retinoic acid (9cRA) and LGD1069 and this was associated with loss of induction of RARbeta2 by retinoids. RARbeta2 expression in premalignant MCF10AT cells decreased their proliferating activity and increased their sensitivity to atRA. In vivo, when transplanted into the mammary fat pads of nude mice, MCF10A cells did not grow, MCF10AT cells formed slow-growing tumor nodules, and MCF10CA1a cells were highly malignant, grew quickly and infiltrated the surrounding tissues. Of the retinoids used, only 4-HPR suppressed the growth of slow-growing hyperplastic and premalignant MCF10AT but not of the malignant MCF10CA1a tumor nodules. These data may have clinical implication in selecting women with hyperplastic and premalignant breast lesions that may benefit the most from clinical trials with retinoids.