Manoela Tiago

ARTIFICIAL SKIN IN PERSPECTIVE: CONCEPTS AND APPLICATIONS

Skin, the largest organ of the human body, is organized into an elaborate layered structure consisting mainly of the outermost epidermis and the underlying dermis. A subcutaneous adipose‐storing hypodermis layer and various appendages such as hair follicles, sweat glands, sebaceous glands, nerves, lymphatics, and blood vessels are also present in the skin. These multiple components of the skin ensure survival by carrying out critical functions such as protection, thermoregulation, excretion, absorption, metabolic functions, sensation, evaporation management, and aesthetics. The study of how these biological functions are performed is critical to our understanding of basic skin biology such as regulation of pigmentation and wound repair. Impairment of any of these functions may lead to pathogenic alterations, including skin cancers. Therefore, the development of genetically controlled and well characterized skin models can have important implications, not only for scientists and physicians, but also for manufacturers, consumers, governing regulatory boards and animal welfare organizations. As cells making up human skin tissue grow within an organized three‐dimensional (3D) matrix surrounded by neighboring cells, standard monolayer (2D) cell cultures do not recapitulate the physiological architecture of the skin. Several types of human skin recombinants, also called artificial skin, that provide this critical 3D structure have now been reconstructed in vitro. This review contemplates the use of these organotypic skin models in different applications, including substitutes to animal testing.

Proteasome inhibition and ROS generation by 4-nerolidylcatechol induces melanoma cell death.

Induction of apoptotic cell death in response to chemotherapy and other external stimuli has proved extremely difficult in melanoma, leading to tumor progression, metastasis formation and resistance to therapy. A promising approach for cancer chemotherapy is the inhibition of proteasomal activity, as the half‐life of the majority of cellular proteins is under proteasomal control and inhibitors have been shown to induce cell death programs in a wide variety of tumor cell types. 4‐Nerolidylcatechol (4‐NC) is a potent antioxidant whose cytotoxic potential has already been demonstrated in melanoma tumor cell lines. Furthermore, 4‐NC was able to induce the accumulation of ubiquitinated proteins, including classic targets of this process such as Mcl‐1. As shown for other proteasomal inhibitors in melanoma, the cytotoxic action of 4‐NC is time‐dependent upon the pro‐apoptotic protein Noxa, which is able to bind and neutralize Mcl‐1. We demonstrate the role of 4‐NC as a potent inducer of ROS and p53. The use of an artificial skin model containing melanoma also provided evidence that 4‐NC prevented melanoma proliferation in a 3D model that more closely resembles normal human skin.

Basic Red 51, a permitted semi-permanent hair dye, is cytotoxic to human skin cells: Studies in monolayer and 3D skin model using human keratinocytes (HaCaT).

The use of hair dyes is closely associated with the increase of cancer, inflammation and other skin disorders. The recognition that human skin is not an impermeable barrier indicates that there is the possibility of human systemic exposure. The carcinogenic potential of hair dye ingredients has attracted the attention of toxicologists for many decades, mainly due to the fact that some ingredients belong to the large chemical family of aromatic amines. Herein, we investigated the cytotoxicity of Basic Red 51 (BR51) in immortalized human keratinocytes (HaCaT). BR51 is a temporary hair dye that belongs to the azo group (NN); the cleavage of this bond may result in the release of toxic aromatic amines. The half maximal effective concentration (EC50) in HaCaT cells is 13μg/mL. BR51 induced a significant decrease on expression of p21 in a dose dependent manner. p53 was not affected, whereas BR51 decreased procaspase 8 and cleaved procaspase 9. These results proved that caspase 3 is fully involved in BR51-induced apoptosis. The dye was also able to stop this cell cycle on G2 in sub-toxic doses. Moreover, we reconstructed a 3D artificial epidermis using HaCaT cells; using this model, we observed that BR51 induced cell injury and cells were undergoing apoptosis, considering the fragmented nuclei. Subsequently, BR51 induced reactive oxygen species (ROS) leading to an increase on the levels of 8-oxo-dG. In conclusion, we provide strong evidence that consumer and/or professional exposure to BR51 poses risk to human health.

Apoptosis through Bcl-2/Bax and Cleaved Caspase Up-Regulation in Melanoma Treated by Boron Neutron Capture Therapy

Boron neutron capture therapy (BNCT) is a binary treatment involving selective accumulation of boron carriers in a tumor followed by irradiation with a thermal or epithermal neutron beam. The neutron capture reaction with a boron-10 nucleus yields high linear energy transfer (LET) particles, alpha and 7Li, with a range of 5 to 9 µm. These particles can only travel very short distances and release their damaging energy directly into the cells containing the boron compound. We aimed to evaluate proliferation, apoptosis and extracellular matrix (ECM) modifications of B16F10 melanoma and normal human melanocytes after BNCT. The amounts of soluble collagen and Hsp47, indicating collagen synthesis in the ECM, as well as the cellular markers of apoptosis, were investigated. BNCT decreased proliferation, altered the ECM by decreasing collagen synthesis and induced apoptosis by regulating Bcl-2/Bax in melanoma. Additionally, BNCT also increased the levels of TNF receptor and the cleaved caspases 3, 7, 8 and 9 in melanoma. These results suggest that multiple pathways related to cell death and cell cycle arrest are involved in the treatment of melanoma by BNCT.

A Natural Bacterial-Derived Product, the Metalloprotease Arazyme, Inhibits Metastatic Murine Melanoma by Inducing MMP-8 Cross-Reactive Antibodies

The increased incidence, high rates of mortality and few effective means of treatment of malignant melanoma, stimulate the search for new anti-tumor agents and therapeutic targets to control this deadly metastatic disease. In the present work the antitumor effect of arazyme, a natural bacterial-derived metalloprotease secreted by Serratia proteomaculans, was investigated. Arazyme significantly reduced the number of pulmonary metastatic nodules after intravenous inoculation of B16F10 melanoma cells in syngeneic mice. In vitro, the enzyme showed a dose-dependent cytostatic effect in human and murine tumor cells, and this effect was associated to the proteolytic activity of arazyme, reducing the CD44 expression at the cell surface, and also reducing in vitro adhesion and in vitro/in vivo invasion of these cells. Arazyme treatment or immunization induced the production of protease-specific IgG that cross-reacted with melanoma MMP-8. In vitro, this antibody was cytotoxic to tumor cells, an effect increased by complement. In vivo, arazyme-specific IgG inhibited melanoma lung metastasis. We suggest that the antitumor activity of arazyme in a preclinical model may be due to a direct cytostatic activity of the protease in combination with the elicited anti-protease antibody, which cross-reacts with MMP-8 produced by tumor cells. Our results show that the bacterial metalloprotease arazyme is a promising novel antitumor chemotherapeutic agent.

Connections of Annexin A1 and Translocator Protein-18 KDa on Toll Like Receptor Stimulated BV-2 Cells

Background: Annexin A1 (ANXA1) and Translocator Protein‐18KDa (TSPO) down‐regulate neuroinflammation. We investigated the role of recombinant ANXA1 (rANXA) on TSPO functions on Toll Like Receptor (TLR) activated microglia. Methods: BV‐2 cells (murine microglia), were stimulated by E. coli Lipopolysaccharide (LPS) and treated with rANXA1 in order to measure TSPO expression and inflammatory parameters. Anti‐sense ANXA1 and TLR4 and TSPO shRNA, as well as pharmacological treatments, were employed to assess the mechanisms involved. Results: LPS‐stimulated BV‐2 cells caused overexpression of TSPO, which was inhibited by: pharmacological blockade of TLR4 or TLR4 mRNA silencing; inhibition of myeloid differentiation primary response gene 88 (MyD88) dimerization; or blocking of nuclear factor &kgr;B (NF‐&kgr;B) activation. rANXA1 treatment impaired LPS‐induced TSPO upregulation by down‐modulating MyD88 and NF‐&kgr;B signaling; the effect was abolished by WRW4, an antagonist of formyl peptide receptor 2 (FPR2). rANXA1 treatment also downregulated interleukin 1&bgr; (IL‐1&bgr;) and tumor necrosis factor‐&agr; (TNF&agr;) secretion in LPS‐stimulated BV‐2 cells. TSPO knockdown in BV‐2 cells augmented LPS‐induced TNF&agr; secretion and abolished the inhibitory effect of rANXA1 on TNF&agr; secretion evoked by LPS. Conclusions: exogenous ANXA1 down‐modulates LPS‐induced TSPO via MyD‐88/NF‐&kgr;B pathways, and constitutive TSPO is pivotal for the control of ANXA1 on TNF&agr; secretion. TSPO actions may be involved with the mechanisms of ANXA1 on inflammatory brain diseases. HIGHLIGHTSLPS stimulates TSPO expression via TLR4/MyD88/NF&kgr;B pathway in BV2 cells.ANXA1 reverses LPS‐induced TSPO expression by down‐modulating TLR4/MyD88/NF&kgr;B pathways via interaction with FPR2 in BV2 cells.TSPO is a down modulator and mediator of ANXA1 on LPS‐induced TNF&agr; secretion in BV2 cells.

Three-Dimensional Systems in Polybutylcyanoacrylate Nanoparticles Safety Evaluation

It is generally accepted that three-dimensional (3D) cell culture systems better represent cell physiology and morphology than two-dimensional (monolayer) cell cultures. The aim of this work was to develop a 3D model that could efficiently evaluate polymeric materials biocompatibility using polybutylcyanoacrylate nanoparticles (PBCA-NP). Normal human fibroblasts and melanoma cells were cultured both in monolayer and on type I collagen matrix. Trypan blue and MTT assays were used to evaluate PBCA-NP cytotoxicity, which was shown to be dose- and time-dependent to all cell types. These results were confirmed by cell death analysis with flow cytometry. There is evidence that treatment with PBCA-NP in concentrations close to the IC50 inhibit autophagy in fibroblasts and melanoma cells when cultured in monolayer, but this response could not be observed in the 3D system. These findings strongly suggest that PBCA-NP can modify cell response depending on the concentration used and the conditions of cell culture. GRAPHICAL ABSTRACT

Molecular effects of 1-naphthyl-methylcarbamate and solar radiation exposures on human melanocytes.

Carbaryl (1-naphthyl-methylcarbamate), a broad-spectrum insecticide, has recently been associated with the development of cutaneous melanoma in an epidemiological cohort study with U.S. farm workers also exposed to ultraviolet radiation, the main etiologic factor for skin carcinogenesis. We hypothesized that carbaryl exposure may increase deleterious effects of UV solar radiation on skin melanocytes. This study aimed to characterize human melanocytes after individual or combined exposure to carbaryl (100μM) and solar radiation (375mJ/cm2). In a microarray analysis, carbaryl, but not solar radiation, induced an oxidative stress response, evidenced by the upregulation of antioxidant genes, such as Hemeoxygenase-1 (HMOX1), and downregulation of Microphtalmia-associated Transcription Factor (MITF), the main regulator of melanocytic activity; results were confirmed by qRT-PCR. Carbaryl and solar radiation induced a gene response suggestive of DNA damage and cell cycle alteration. The expression of CDKN1A, BRCA1/2 and MDM2 genes was notably more intense in the combined treatment group, in a synergistic manner. Flow cytometry assays demonstrated S-phase cell cycle arrest, reduced apoptosis levels and faster induction of cyclobutane pyrimidine dimers (CPD) lesions in carbaryl treated groups. Our data suggests that carbaryl is genotoxic to human melanocytes, especially when associated with solar radiation.