Inhibition of 17beta-hydroxysteroid dehydrogenase type 7 modulates breast cancer protein profile and enhances apoptosis by down-regulating GRP78.

17beta-hydroxysteroid dehydrogenase type 7 (17ß-HSD7) promotes breast cancer cell growth via dual-catalytic activity by modulating estradiol and DHT. Here, we clarified the expression pattern of 17ß-HSD7 in postmenopausal luminal A type breast cancer with The Cancer Genome Atlas (TCGA) cohort. The impact of 17ß-HSD7 inhibition on the proteome of MCF-7 cells was investigated and on cell apoptosis was revealed. MCF-7 cells were treated with an efficient inhibitor of 17ß-HSD7 (INH7) or with vehicle, and a differential proteomics study was performed using two-dimensional (2D) gel electrophoresis followed by mass spectrometry and ingenuity pathway analysis (IPA). Cell apoptosis was analyzed by flow cytometry, followed by reverse transcription quantitative real-time PCR (RT-qPCR) and Western blot to investigate the expression of apoptosis-related genes. Our data showed 17ß-HSD7 is amplified in primary and progressive breast cancer, inhibition of 17ß-HSD7 in MCF-7 cells modulated 104 proteins primarily involved in cell death/survival, cell growth and DNA processing. The expression of 78kDa glucose-regulated protein (GRP78) and anti-apoptosis factor Bcl-2 were significantly suppressed via 17ß-HSD7 inhibition with INH7, consequently induced MCF-7 cell apoptosis. However, INH7 treatment of T47D, another widely used epithelial ER+ breast cancer cell line, led to an up-regulation of GRP78 expression, resulting in a limited increase in apoptosis. These results suggest cell-specific effects of INH7 in the breast cancer, which is interesting for further study. An combinatory effect on apoptosis by INH7 and Letrozole (aromatase inhibitor) was further demonstrated in MCF-7. Down-regulation of GRP78 via 17ß-HSD7 inhibition enhances cell apoptosis in response to Letrozole. This study highlights GRP78 as a key regulator related to 17ß-HSD7 inhibition and effect. Taken together, results from the present study suggest a hypothesis that inhibition of 17ß-HSD7 would be a complementary strategy to Letrozole by suppression of GRP78 in ER+ breast cancer. However, from a research perspective, further studies have to be carried out with more breast cancer cell lines as well as in vivo model to assess the efficacy of inhibitor combination.

In vitro interactions between mammary fibroblasts (Hs 578Bst) and cancer epithelial cells (MCF-7) modulate aromatase, steroid sulfatase and 17ß-hydroxysteroid dehydrogenases.

Our objectives were to investigate the interactions between mammary cancer epithelial cells (MCF-7) and stromal cells (Hs-578Bst) at the level of the expression and inhibition of steroidogenesis enzymes by using monolayer and three dimensional co-culture models. Expressions of steroidogenesis enzymes and E2/DHT conversions in co-cultured MCF-7 and Hs-578Bst cells as well as the effects of aromatase inhibitor combined to steroid sulfatase (STS) and 17ß-hydroxysteroid dehydrogenases (17ßHSDs) inhibitors were evaluated. 17ß-HSD type 7 was mostly modulated in MCF-7 cells whereas aromatase was mostly regulated in Hs578Bst cells thereby increasing E2 conversion and MCF-7 cell growth. A combination of inhibitors toward aromatase, STS and 17ß-HSD7, was found to be the most significant treatment in decreasing E2 and elevating DHT thus inhibiting MCF-7 cell proliferation and spheroid-like cancer cell aggregation in collagen gel. The interactions between those cells modulated E2 formation in paracrine/intracrine manners by synergistically regulating aromatase, 17ß-HSD7 and STS. Among tumor-associated cells, stromal fibroblasts may participate in intratumoral E2 deposition; therefore promoting breast cancer cell growth.

Synergistic control of sex hormones by 17ß-HSD type 7: a novel target for estrogen-dependent breast cancer.

17ß-hydroxysteroid dehydrogenase (17ß-HSD) type 1 is known as a critical target to block the final step of estrogen production in estrogen-dependent breast cancer. Recent confirmation of the role of dyhydroxytestosterone (DHT) in counteracting estrogen-induced cell growth prompted us to study the reductive 17ß-HSD type 7 (17ß-HSD7), which activates estrone while markedly inactivating DHT. The role of DHT in breast cancer cell proliferation is demonstrated by its independent suppression of cell growth in the presence of a physiological concentration of estradiol (E2). Moreover, an integral analysis of a large number of clinical samples in Oncomine datasets demonstrated the overexpression of 17ß-HSD7 in breast carcinoma. Inhibition of 17ß-HSD7 in breast cancer cells resulted in a lower level of E2 and a higher level of DHT, successively induced regulation of cyclinD1, p21, Bcl-2, and Bik, consequently arrested cell cycle in the G(0)/G(1) phase, and triggered apoptosis and auto-downregulation feedback of the enzyme. Such inhibition led to significant shrinkage of xenograft tumors with decreased cancer cell density and reduced 17ß-HSD7 expression. Decreased plasma E2 and elevated plasma DHT levels were also found. Thus, the dual functional 17ß-HSD7 is proposed as a novel target for estrogen-dependent breast cancer by regulating the balance of E2 and DHT. This demonstrates a conceptual advance on the general belief that the major role of this enzyme is in cholesterol metabolism.

Newly identified interfibrillar collagen crosslinking suppresses cell proliferation and remodelling.

Copper is becoming recognised as a key cation in a variety of biological processes. Copper chelation has been studied as a potential anti-angiogenic strategy for arresting tumour growth. Conversely the delivery of copper ions and complexes in vivo can elicit a pro-angiogenic effect. Previously we unexpectedly found that copper-stimulated intraperitoneal angiogenesis was accompanied by collagen deposition. Here, in hard tissue, not only was healing accelerated by copper, but again enhanced deposition of collagen was detected at 2 weeks. Experiments with reconstituted collagen showed that addition of copper ions post-fibrillogenesis rendered plastically-compressed gels resistant to collagenases, enhanced their mechanical properties and increased the denaturation temperature of the protein. Unexpectedly, this apparently interfibrillar crosslinking was not affected by addition of glucose or ascorbic acid, which are required for crosslinking by advanced glycation end products (AGEs). Fibroblasts cultured on copper-crosslinked gels did not proliferate, whereas those cultured with an equivalent quantity of copper on either tissue culture plastic or collagen showed no effect compared with controls. Although non-proliferative, fibroblasts grown on copper-cross-linked collagen could migrate, remained metabolically active for at least 14 days and displayed a 6-fold increase in Mmps 1 and 3 mRNA expression compared with copper-free controls. The ability of copper ions to crosslink collagen fibrils during densification and independently of AGEs or Fenton type reactions is previously unreported. The effect on MMP susceptibility of collagen and the dramatic change in cell behaviour on this crosslinked ECM may contribute to shedding some light on unexplained phenomena as the apparent benefit of copper complexation in fibrotic disorders or the enhanced collagen deposition in response to localised copper delivery.

Computational modeling of adherent cell growth in a hollow-fiber membrane bioreactor for large-scale 3-D bone tissue engineering.

The use of hollow-fiber membrane bioreactors (HFMBs) has been proposed for three-dimensional bone tissue growth at the clinical scale. However, to achieve an efficient HFMB design, the relationship between cell growth and environmental conditions must be determined. Therefore, in this work, a dynamic double-porous media model was developed to determine nutrient-dependent cell growth for bone tissue formation in a HFMB. The whole hollow-fiber scaffold within the bioreactor was treated as a porous domain in this model. The domain consisted of two interpenetrating porous regions, including a porous lumen region available for fluid flow and a porous extracapillary space filled with a collagen gel that contained adherent cells for promoting long-term growth into tissue-like mass. The governing equations were solved numerically and the model was validated using previously published experimental results. The contributions of several bioreactor design and process parameters to the performance of the bioreactor were studied. The results demonstrated that the process and design parameters of the HFMB significantly affect nutrient transport and thus cell behavior over a long period of culture. The approach presented here can be applied to any cell type and used to develop tissue engineering hollow-fiber scaffolds.

Modulatory effect of a complex fraction derived from colostrum on fibroblast contractibility and consequences on repair tissue.

A complex compound (immune ('IM') fraction) from colostrum-derived whey was investigated for its potential wound healing properties. One of its most intriguing in vitro abilities was to significantly inhibit the contraction of collagen gel while fibroblast density remained as in control gels. This antagonist effect was dose dependent and fibroblasts in these gels did not exhibit any stress fibres. Subsequently, in vivo studies have been conducted in two wound models in guinea pigs. Daily application on full-thickness wounds of a liquid formulation of the IM fraction (first model) significantly delayed wound closure by contraction compared to what normally occurred in control wounds. In another wound model, a gel formulation of the IM fraction was applied on scar tissues, which resulted in a minimised residual scar on 5/8 wounds compared to corresponding wound areas seen prior to treatment. Conversely, most control wounds exhibited scar tissue from which 3/8 resembled hypertrophic scar tissue. Wound tissue treated with IM fraction covered a significantly larger area than in the control wounds, whereas the collagen deposition was unchanged as in the presence of α-smooth muscle actin. Thus, IM fraction may act by modulating the contraction rate and wound remodelling.

Effect of sterilization on non-woven polyethylene terephthalate fiber structures for vascular grafts.

Non-woven polyethylene terephthalate (PET) fibers produced via melt blowing and compounded into a 6 mm diameter 3D tubular scaffold were developed with artery matching mechanical properties. This work compares the effects of ethylene oxide (EtO) and low temperature plasma (LTP) sterilization on PET surface chemistry and biocompatibility. As seen through X-ray photoelectron spectroscopy (XPS) analysis, LTP sterilization led to an increase in overall oxygen content and the creation of new hydroxyl groups. EtO sterilization induced alkylation of the PET polymer. The in vitro cytotoxicity showed similar fibroblastic viability on LTP- and EtO-treated PET fibers. However, TNF-α release levels, indicative of macrophage activation, were significantly higher when macrophages were incubated on EtO-treated PET fibers. Subcutaneous mice implantation revealed an inflammatory response with foreign body reaction to PET grafts independent of the sterilization procedure.

Directional migration of endothelial cells towards angiogenesis using polymer fibres in a 3D co-culture system.

Development of an in vitro prevascularized scaffold is of great importance to produce vascularization in tissue-engineered devices and for other clinical purposes. To this aim, polymer fibres covered with human umbilical vein endothelial cells (HUVECs) were used to induce directional 'angiogenesis' in a 3D co-culture system. Gelatin or RGD peptides were immobilized on surface-modified polymer fibres [100 µm diameter poly(ethylene terephthalate) monofilaments] via N-hepthylamine plasma polymer and carboxy-methyl-dextran interlayers. Fibres fully covered with HUVECs were then embedded in a fibrin gel, following a parallel alignment pattern, in the presence of fibroblasts. Tube-like structures occurred along the fibres and a network was formed between neighbouring fibres. These events were promoted with increased incubation times. Biomolecule-grafted fibres created a guidance pathway that facilitated coated endothelial cells to form lumens and, from them, sprouting processes. However, there were no significant differences between the different surface modifications on fibres in terms of promoting tube-like structures. Thus, different stages of angiogenesis can be initiated and guided using HUVECs precovered polymer fibres embedded in a soft supportive matrix, such as fibrin, which can be further applied to the development of in vitro prevascularized tissue-engineered scaffolds.

Facilitating tissue infiltration and angiogenesis in a tubular collagen scaffold.

Among different strategies to provide blood supply to tissue-engineered devices and implants, the use of arteriovenous loops and bundles has been proposed. The aim of this study was to compare the vascularization and healing processes that took place in a one-end closed tubular collagen-based scaffold at different implantation sites in mice. These conditions were in the presence or absence of heparin and/or bone marrow cells. By 30 days, very few cell infiltrations were observed in the dorsal subcutaneous and peritoneal implants at any conditions; however, the presence of heparin and bone marrow cells improved cell infiltration toward an inflammatory reaction. The insertion of an arteriovenous bundle into the central cavity of the scaffold resulted in partial wound tissue infiltration in the control scaffolds implanted subcutaneously in the hind limb. In similar conditions, the presence of bone marrow cells and heparin resulted in dense wound tissue with numerous capillaries and a significant amount of newly deposited collagen fibers. The design of a central cavity in a porous scaffold with one closed end may facilitate invasion from the central part of the implant toward the implant wall. In addition, the presence of both a vascular component and stem/progenitor cells may lead to a vascularized implant while limiting the inflammatory reaction.

The stimulation of angiogenesis and collagen deposition by copper.

Copper is known to trigger endothelial cells towards angiogenesis. Different approaches have been investigated to develop vascularisation in biomaterials. The angiogenic and healing potential of copper ions in combination with two major angiogenic factors was examined. A 3D culture system in which, under stimulation by FGF-2 and to a lesser degree with VEGF, endothelial cells assembled into structures resembling to an angiogenic process was used. The combination of CuSO(4) with increasing doses of VEGF or FGF-2 enhanced the complexity of angiogenic networks in a significant manner. In vivo studies were also conducted by incorporating FGF-2 with CuSO(4) in a cylindrical collagen-based scaffold. CuSO(4) enhanced significantly the invasion of microvessel compared to control implants and to 20ng FGF-2+/-CuSO(4). Vascular infiltration was also significantly improved by combination of CuSO(4) with FGF-2, compared to FGF-2 alone (0.2 and 1microg). Nevertheless, in comparison with CuSO(4) alone, there was a significant increase only with 1microg of FGF-2 combined with CuSO(4). Significantly, collagen fiber deposition was enhanced following the combinatory loading in comparison to that with FGF-2 alone but not with CuSO(4) only. Thus, copper associated with growth factors may have synergistic effects which are highly attractive in the fields of tissue engineering (e.g., bone) and biomaterials.

Angiogenesis in calcium phosphate scaffolds by inorganic copper ion release.

Angiogenesis in a tissue-engineered device may be induced by incorporating growth factors (e.g., vascular endothelial growth factor [VEGF]), genetically modified cells, and=or vascular cells. It represents an important process during the formation and repair of tissue and is essential for nourishment and supply of reparative and immunological cells. Inorganic angiogenic factors, such as copper ions, are therefore of interest in the fields of regenerative medicine and tissue engineering due to their low cost, higher stability, and potentially greater safety compared with recombinant proteins or genetic engineering approaches. The purpose of this study was to compare tissue responses to 3D printed macroporous bioceramic scaffolds implanted in mice that had been loaded with either VEGF or copper sulfate. These factors were spatially localized at the end of a single macropore some 7 mm from the surface of the scaffold. Controls without angiogenic factors exhibited only poor tissue growth within the blocks; in contrast, low doses of copper sulfate led to the formation of microvessels oriented along the macropore axis. Further, wound tissue ingrowth was particularly sensitive to the quantity of copper sulfate and was enhanced at specific concentrations or in combination with VEGF. The potential to accelerate and guide angiogenesis and wound healing by copper ion release without the expense of inductive protein(s) is highly attractive in the area of tissue-engineered bone and offers significant future potential in the field of regenerative biomaterials.

A genome-wide shRNA screen identifies GAS1 as a novel melanoma metastasis suppressor gene.

Metastasis suppressor genes inhibit one or more steps required for metastasis without affecting primary tumor formation. Due to the complexity of the metastatic process, the development of experimental approaches for identifying genes involved in metastasis prevention has been challenging. Here we describe a genome-wide RNAi screening strategy to identify candidate metastasis suppressor genes. Following expression in weakly metastatic B16-F0 mouse melanoma cells, shRNAs were selected based upon enhanced satellite colony formation in a three-dimensional cell culture system and confirmed in a mouse experimental metastasis assay. Using this approach we discovered 22 genes whose knockdown increased metastasis without affecting primary tumor growth. We focused on one of these genes, Gas1 (Growth arrest-specific 1), because we found that it was substantially down-regulated in highly metastatic B16-F10 melanoma cells, which contributed to the high metastatic potential of this mouse cell line. We further demonstrated that Gas1 has all the expected properties of a melanoma tumor suppressor including: suppression of metastasis in a spontaneous metastasis assay, promotion of apoptosis following dissemination of cells to secondary sites, and frequent down-regulation in human melanoma metastasis-derived cell lines and metastatic tumor samples. Thus, we developed a genome-wide shRNA screening strategy that enables the discovery of new metastasis suppressor genes.

Protein tyrosine phosphatase inhibition induces anti-tumor activity: evidence of Cdk2/p27 kip1 and Cdk2/SHP-1 complex formation in human ovarian cancer cells.

The protein tyrosine phosphatase (PTP) superfamily of enzymes functions with protein tyrosine kinases to regulate a broad spectrum of fundamental physiological processes. Addition of the PTP inhibitor potassium bisperoxo(1,10-phenanthroline)oxo-vanadate(V) [bpV(phen)] to the culture medium of human ovarian cancer cells (OVCAR-3) resulted in a dose-dependent decrease in the formation of tumors in a 3-D culture system. An evaluation of the potency of bpV(phen) in vivo confirmed the anti-tumor activity. Further study of the mechanism of action revealed a 40% decrease in Cdk2 kinase activity, an elevated level of Cdk2/p27(kip1), and the appearance of Cdk2/SHP-1 complexes. Therefore, a cytostatic dose of a PTP inhibitor increases the intracellular levels of Cdk2/p27(kip) and Cdk2/SHP-1 complexes, which indicate the presence of additional mechanisms underlying the anti-tumor activity.

Adipogenesis in nonadherent and adherent bone marrow stem cells grown in fibrin gel and in the presence of adult plasma.

Bone marrow-derived mesenchymal stem cells (i.e., adherent cells) are known to differentiate into fat tissue in the presence of adipogenic supplements in cultures. Induction of adipogenesis has not been investigated within the nonadherent cell fraction that includes predominantly hematopoietic cells. In the present study, murine nonadherent bone marrow-derived stem cells (96% CD45+ cells) were seeded and then grown in fibrin gel to form cell clusters in which most cells were positive to DiI-acetylated low-density lipoprotein uptake. Amongst different culture media supplemented either in fetal bovine serum, horse serum, murine plasma, human plasma or adipogenic supplements, a subpopulation of nonadherent stem cells within clusters differentiated into adipocytes, specifically in the presence of adult syngeneic plasma. This was confirmed by the observation and quantification of oil red O-positive cells, the measurement of glycerol-3-phosphate dehydrogenase activity and peroxisome proliferator-activated receptor-gamma mRNA expression. Similarly, adipogenesis was also observed in the presence of murine plasma with adherent mesenchymal stem cells and 3T3-L1 preadipocytes which were grown either in monolayer plastic cultures or in fibrin gel. Thus, it is possible that nonadherent cells, once in a 3-dimensional environment, can further differentiate towards adipogenesis.

Osteoconduction and osteoinduction of low-temperature 3D printed bioceramic implants.

Rapid prototyping is a valuable implant production tool that enables the investigation of individual geometric parameters, such as shape, porosity, pore size and permeability, on the biological performance of synthetic bone graft substitutes. In the present study, we have employed low-temperature direct 3D printing to produce brushite and monetite implants with different geometries. Blocks predominantly consisting of brushite with channels either open or closed to the exterior were implanted on the decorticated lumbar transverse processes of goats for 12 weeks. In addition, similar blocks with closed channel geometry, consisting of either brushite or monetite were implanted intramuscularly. The design of the channels allowed investigation of the effect of macropore geometry (open and closed pores) and osteoinduction on bone formation orthotopically. Intramuscular implantation resulted in bone formation within the channels of both monetite and brushite, indicating osteoinductivity of these resorbable materials. Inside the blocks mounted on the transverse processes, initial channel shape did not seem to significantly influence the final amount of formed bone and osteoinduction was suggested to contribute to bone formation.

Biocompatibility and light transmission of liposomal lenses.

PURPOSE: To validate the biocompatibility and transmittance properties of contact lenses bearing intact liposomes. These liposomal lenses loaded with therapeutics can be used as ophthalmic drug delivery systems. METHODS: The biocompatibility of soft contact lenses, coated with liposomes was evaluated through in vitro direct and indirect cytocompatibility assays on human corneal epithelial cells, on reconstructed human corneas and on ex vivo rabbit corneas. The direct and indirect transmission spectra of liposome-covered lenses were also evaluated to test if they transmit all wavelengths of the ultraviolet-visible spectrum, to thereby fulfill their optical function, without gross alteration of the colors perception and with a minimum of light dispersion. RESULTS: Contact lenses bearing layers of stable liposomes did not induce any significant changes in cell viability and in cell growth, compared with lenses bearing no liposome. Elution assays revealed that no cytotoxic compound leaks from the lenses whether bearing liposomes or not. Histological analyses of reconstructed human corneas and ex vivo rabbit corneas directly exposed to liposomal lenses revealed neither alteration to the cell nor to the tissue structures. Contact lenses bearing layers of liposomes did not significantly affect light transmission compared with control lenses without liposome at the wavelength of maximal photopic sensitivity, i.e., 550 nm. In addition, the contact lenses afford more eye protection in the ultraviolet spectrum, compared with the control lenses. CONCLUSIONS: Liposomal contact lenses are biocompatible and their transmittance properties are not affected in the visible light range.

Angiogenic response of endothelial cells seeded dispersed versus on beads in fibrin gels.

Induction of an inter-connected microvessel network in a tissue-engineered construct prior to implantation may be an alternative to improve the success rate of cell/tissue survival and wound integration. Conditions of endothelial cell-seeding density and distribution were investigated in two 3-D angiogenesis culture systems. Endothelial cells were either seeded dispersed in a fibrin gel, or subconfluent on micro-beads (Cytodex) prior to being embedded in fibrin. Human fibroblasts and growth factors were introduced to optimize angiogenesis. A density higher than 4 x 10(4) cells/ml of fibrin was necessary to induce angiogenic-like structures (i.e., sprouting, cord-, lumen-like structures) by 14 days in the dispersed cell model. Endothelial cells on micro-beads also exhibited angiogenic-like structures that were inter-connected to those on neighboring beads. The sizes of the angiogenic-like structures were larger on beads compared to those found in the dispersed cell model. High cell density was needed in angiogenesis when cells were seeded separately, whereas the association of endothelial cells on bead surfaces significantly reduced the cell density used. Moreover, increasing bead density was not necessary to facilitate further angiogenic formation. Micro-spheres may represent a potential support for endothelial cells in microvessel networking, with subsequent applications in the pre-vascularization of bio-implants.

Perfluorocarbon emulsions cytotoxic effects on human fibroblasts and effect of aging on particle size distribution.

The purpose of this study was to characterize emulsion preparations made of perfluorooctyl bromide (PFOB) and egg yolk phospholipid (EYP) and their cytotoxicity. Dynamic light scattering and transmission electron microscopy revealed that freshly prepared emulsions stored at different temperatures for a 24-h period have a unimodal particle size distribution with an average particle size of ca. 200 nm. The emulsion displayed a broader particle size distribution following a 14-day storage. Primary human fibroblasts exposure to PFOB/EYP emulsions permanently inhibited cell proliferation and decreased mitochondrial activity. Scanning electron microscopy pictures reveal the presence of spherical particles on the fibroblasts following exposure to the emulsions after thorough rinsing with culture media.

Bioactive polymer fibers to direct endothelial cell growth in a three-dimensional environment.

This study reports the fabrication of bioactive polymer fibers onto which signaling molecules can control and direct cell responses. To encourage and control directional biological responses, GRGDS peptides were immobilized onto the surface of 100 microm diameter poly(ethylene terephtalate) (PET) fibers (monofilaments). PET fiber surfaces were first coated with a thin polymeric interfacial bonding layer bearing amine groups by plasma polymerization. Carboxy-methyl-dextran (CMD) was covalently grafted onto the surface amine groups using water-soluble carbodiimide chemistry. GRGDS were covalently immobilized onto CMD-coated fiber surfaces. X-ray photoelectron spectroscopy (XPS) analyses enabled characterization of the multilayer fabrication steps. Human umbilical vein endothelial cells were seeded and grown on fibers to investigate cell patterning behavior (i.e., adhesion, spreading, cytoskeleton organization, and cell orientation). Cell adhesion was reduced on CMD-coated fibers, whereas amine- and GRGDS-coated fibers promoted cell adhesion and spreading. Cell adhesion was enhanced as the GRGDS concentration increased. Epifluorescence microscopic visualization of cells on RGD-coated substrates showed well-defined stress fibers and sharp spots of vinculin, typical of focal adhesions. In comparison to plasticware commonly used in cell cultures, fiber curvature promoted cell orientation along the fiber axis.