Débora Campanella Bastos

Fatty acid synthase inhibition with Orlistat promotes apoptosis and reduces cell growth and lymph node metastasis in a mouse melanoma model

Fatty acid synthase (FASN) is the enzyme responsible for the endogenous synthesis of the saturated fatty acid palmitate. In contrast to most normal cells, malignant cells depend on FASN activity for growth and survival. In fact, FASN is overexpressed in a variety of human cancers including cutaneous melanoma, in which its levels of expression are associated with a poor prognosis and depth of invasion. Here, we show that the specific inhibition of FASN activity by the antiobesity drug Orlistat or siRNA is able to significantly reduce proliferation and promote apoptosis in the mouse metastatic melanoma cell line B16‐F10. These results prompted us to verify the effect of FASN inhibition on the metastatic process in a model of spontaneous melanoma metastasis, in which B16‐F10 cells injected in the peritoneal cavity of C57BL/6 mice metastasize to the mediastinal lymph nodes. We observed that mice treated with Orlistat 48 hr after the inoculation of B16‐F10 cells exhibited a 52% reduction in the number of mediastinal lymph node metastases, in comparison with the control animals. These results suggest that FASN activity is essential for B16‐F10 melanoma cell proliferation and survival while its inactivation by Orlistat significantly reduces their metastatic spread. The chemical inhibition of FASN activity could have a potential benefit in association with the current chemotherapy for melanoma.

The Fatty Acid Synthase Inhibitor Orlistat Reduces the Growth and Metastasis of Orthotopic Tongue Oral Squamous Cell Carcinomas

Fatty acid synthase (FASN) is the biosynthetic enzyme responsible for the endogenous synthesis of fatty acids. It is downregulated in most normal cells, except in lipogenic tissues such as liver, lactating breast, fetal lung, and adipose tissue. Conversely, several human cancers, including head and neck squamous cell carcinomas (HNSCC), overexpress FASN, which has been associated with poor prognosis and recently suggested as a metabolic oncoprotein. Orlistat is an irreversible inhibitor of FASN activity with cytotoxic properties on several cancer cell lines that inhibits tumor progression and metastasis in prostate cancer xenografts and experimental melanomas, respectively. To explore whether the inhibition of FASN could impact oral tongue squamous cell carcinoma (OTSCC) metastatic spread, an orthotopic model was developed by the implantation of SCC-9 ZsGreen LN-1 cells into the tongue of BALB/c nude mice. These cells were isolated through in vivo selection, show a more invasive behavior in vitro than the parental cells, and generate orthotopic tumors that spontaneously metastasize to cervical lymph nodes in 10 to 15 days only. SCC-9 ZsGreen LN-1 cells also exhibit enhanced production of MMP-2, ERBB2, and CDH2. The treatment with orlistat reduced proliferation and migration, promoted apoptosis, and stimulated the secretion of VEGFA165b by SCC-9 ZsGreen LN-1 cells. In vivo, the drug was able to decrease both the volume and proliferation indexes of the tongue orthotopic tumors and, importantly, reduced the number of metastatic cervical lymph nodes by 43%. These results suggest that FASN is a potential molecular target for the chemotherapy of patients with OTSCC. Mol Cancer Ther; 13(3); 585–95. ©2013 AACR.

Effects of fatty acid synthase inhibitors on lymphatic vessels: an in vitro and in vivo study in a melanoma model

Fatty acid synthase (FASN) is responsible for the endogenous production of fatty acids from acetyl-CoA and malonyl-CoA. Its overexpression is associated with poor prognosis in human cancers including melanomas. Our group has previously shown that the inhibition of FASN with orlistat reduces spontaneous lymphatic metastasis in experimental B16-F10 melanomas, which is a consequence, at least in part, of the reduction of proliferation and induction of apoptosis. Here, we sought to investigate the effects of pharmacological FASN inhibition on lymphatic vessels by using cell culture and mouse models. The effects of FASN inhibitors cerulenin and orlistat on the proliferation, apoptosis, and migration of human lymphatic endothelial cells (HDLEC) were evaluated with in vitro models. The lymphatic outgrowth was evaluated by using a murine ex vivo assay. B16-F10 melanomas and surgical wounds were produced in the ears of C57Bl/6 and Balb-C mice, respectively, and their peripheral lymphatic vessels evaluated by fluorescent microlymphangiography. The secretion of vascular endothelial growth factor C and D (VEGF-C and -D) by melanoma cells was evaluated by ELISA and conditioned media used to study in vitro lymphangiogenesis. Here, we show that cerulenin and orlistat decrease the viability, proliferation, and migration of HDLEC cells. The volume of lymph node metastases from B16-F10 experimental melanomas was reduced by 39% in orlistat-treated animals as well as the expression of VEGF-C in these tissues. In addition, lymphatic vessels from orlistat-treated mice drained more efficiently the injected FITC-dextran. Orlistat and cerulenin reduced VEGF-C secretion and, increase production of VEGF-D by B16-F10 and SK-Mel-25 melanoma cells. Finally, reduced lymphatic cell extensions, were observed following the treatment with conditioned medium from cerulenin- and orlistat-treated B16-F10 cells. Altogether, our results show that FASN inhibitors have anti-metastatic effects by acting on lymphatic endothelium and melanoma cells regardless the increase of lymphatic permeability promoted by orlistat.

Suppression of MAGE-A10 alters the metastatic phenotype of tongue squamous cell carcinoma cells

MAGE-A10 is a member of the MAGE protein family (melanoma associated antigen) which is overexpressed in cancer cells. Although MAGE-A10 has been characterized for some time and is generally associated to metastasis its function remains unknown. Here we describe experiments using as models oral squamous cell carcinoma (OSCC) cell lines displaying increasing metastatic potential (LN1 and LN2). These cell lines were transduced with lentivirus particles coding for short hairpin against MAGE-A10 mRNA. Repression of MAGE-A10 expression in LN2 cells altered their morphology and impaired growth of LN1 and LN2 cell lines. Furthermore, repression of MAGE-A10 expression increased cell-cell and cell matrix adhesion. Furthermore shMAGEA10 cells were shown to assemble aberrantly on a 3D culture system (microspheroids) when compared to cells transduced with the control scrambled construct. Cell migration was inhibited in knocked down cells as revealed by two different migration assays, wound healing and a phagokinetic track motility assay. In vitro invasion assay using a leiomyoma tissue derived matrix (myogel) showed that shMAGEA10 LN1 and shMAGEA10 LN2 cells displayed a significantly diminished ability to penetrate the matrices. Concomitantly, the expression of E-cadherin, N-cadherin and vimentin genes was analyzed. shMAGEA10 activated the expression of E-cadherin and repression N-cadherin and vimentin transcription. Taken together the results indicate that MAGE-A10 exerts its effects at the level of the epithelial-mesenchymal transition (EMT) presumably by regulating the expression of adhesion molecules.

Tooth Loss Related to Root Perforation: Legal Approach in Endodontic Practice

ABSTRACT: Most of the techniques used in endodontic treatment are limited to the internal space to the tooth,avoiding reaching the periapical region. However, some accidents can happen, such as fracture of endodontic instruments,perforation, leakage of material, tooth fracture among others, depending on the complexityof each case. The aim of thisstudy was to report a case involving a tooth loss caused by root perforation. In this case, the patient filed a lawsuit against theformer professional who had performed endodontic treatment requiring compensation for moral and material damages. Inconclusion, the practice endodontic the clarification of the risks is crucial, considering the particularities of each case. Th us,it is suggested that the verbal and written information should be offered to patients.˚ KEY WORDS: radicular perforation, accident, endodontic treatment, legal odontology. INTRODUCTION ˚˚˚Despite technological advances in dentalinstrumentation and in the development of dentalmaterials, endodontic incidents are frequently found indental practice. Unfortunately, accidental rootperforations during access preparation, canalinstrumentation or preparation of post space are notuncommon. Instrument fractures during filing or reaming,as well as root fractures during condensation are stillfrequent (Motamedi, 2006). In addition, perforations canbe caused by artificial and involuntary injuries as rootresorption and caries promoting communication betweenthe pulp cavity and the periodontal ligament.˚Considering that the anatomy of root canal isextremely variable and dental roots can show abnormalcurvatures, these accidents are, mostly, difficult to avoidspecially during the root access, cleaning andpreparation. However, these incidents can delayendodontic therapy and increase risk of treatmentfailure, especially in teeth with necrotic pulps andperiradicular lesions (Motamedi). According to Arens& Torabinejad (1996) the lack of knowledge in dentalanatomy, atypical tooth position in the arch, presenceof prosthetic crowns, calcified pulp chamber andinappropriate use of endodontic instruments are themost common causes of iatrogenic perforations. Sincethe root perforation and its clinical complications canstrongly affect the patient with the possibility of toothloss and the dentist in ethical and legal implications,the aim of this study is to report a case involving atooth loss caused by root perforation, which resultedin a judgment against˚ the professional in a civilprocedure, highlighting the importance of explainingthe risks of the endodontic treatment to the patient.

Análise dos efeitos de diferentes inibidores da enzima ácido graxo síntase (FASN) em células de carcinoma espino celular oral (SCC-9)

Resumo A enzima ácido graxo sintase (FASN) é responsável pela síntese endógena de ácidos graxos. FASN é superexpressa e associada ao prognóstico do carcinoma espinocelular (CEC) oral. A inibição da FASN com Orlistat (ORL), Triclosan (TCS) ou C75, que inibem diferentes domínios da FASN, resulta em efeitos antiproliferativos em diversas linhagens de neoplasias malignas. O objetivo deste estudo foi comparar o efeito da inibição farmacológica de diferentes domínios catalíticos da FASN sobre o ciclo celular e apoptose associando com as alterações morfológicas nas SCC-9. A inibição dos diferentes domínios catalíticos da FASN com os inibidores farmacológicos C75, TCS e ORL, induziu efeitos diversos na morfologia de células SCC-9 e promoveu alterações na progressão do ciclo celular e indução de apoptose.