Karina G. Zecchin

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.

Myofibroblasts in the stroma of oral squamous cell carcinoma are associated with poor prognosis

with lupus panniculitis. The core biopsy specimen, taken from the recurrent mass in 2003, demonstrated lymphoplasmacytic infiltrates similar to those seen in the previous excision specimen. Surgical intervention was deemed unnecessary and a conservative approach was employed. Lupus mastitis is a rare presentation of lupus profundus, usually following the clinical manifestations of SLE or chronic discoid lupus erythematosus (DLE). In some cases involvement of the fat precedes SLE for months. To our knowledge, only about 16 cases of lupus mastitis have been reported, occurring mainly in association with chronic DLE. The typical presentation of the elementary lesion is a deep subcutaneous nodule or swelling. The overlying skin can be normal or show typical features of discoid lupus. In the breast, fat necrosis commonly mimics malignancy clinically. A history of trauma is absent in most cases at this site. However, fat necrosis caused by trauma is common in the breast and differs from lupus profundus, as the latter process is centred around blood vessels and connective tissue, associated with heavy perivascular and periadnexal lymphocytic infiltrates and hyaline sclerosis of the dermal collagen. It is this feature which is responsible for the hard ‘carcinoma’like feel to lupus mastitis and its dense opacity on mammography. Fat necrosis in the breast is seen in other connective tissue disorders and relevant clinical information is essential when lupus profundus presents at this site. Our case illustrates the importance of providing relevant clinical information to histopathologists to make a diagnosis of lupus mastitis. Lupus mastitis should be suspected in a patient with SLE presenting with a recurrent painful mass in the breast. Equally, acknowledging the increased incidence of breast carcinoma in SLE patients and thus obtaining biopsy confirmation is essential. Lymphocytic vasculitis, sclerosis and hyaline panniculitis in the biopsy specimen would confirm the diagnosis of lupus mastitis and avoid unnecessary excisions which, in these patients, have been reported to lead to atrophic scarring or chronic ulcers. E Nigar K Contractor H Singhal R N Matin Departments of Pathology and Surgery and Dermatology, Northwick Park Hospital, North West London Hospitals NHS Trust, Harrow, UK 1. Irgang S. Lupus erythematosus profundus: report of an example with clinical resemblance to Darier-Roussy sarcoid. Arch. Derm. Syphilol. 1940; 42; 97–108. 2. Holland NW, McKnight K, Challa VR, Agudelo CA. Lupus panniculitis (profundus) involving the breast: report of two cases and review of the literature. J. Rheumatol. 1995; 22; 344–346. 3. Georgian-Smith D, Lawton TJ, Moe RE, Couser WG. Lupus mastitis: radiologic and pathologic features. AJR Am. J. Roentgenol. 2002; 178; 1233–1235. 4. Cernea SS, Kihara M, Sotto MN, Vilela Ma. Lupus mastitis. J. Am. Acad. Dermatol. 1993; 29; 343–346. 5. De Bandt M, Meyer O, Grossin M, Khan MF. Lupus mastitis heralding systemic lupus erythematosus with antiphospholipid syndrome. J. Rheumatol. 1993; 20; 1217–1220. 6. Ujiie H, Shimizu T, Ito M, Arita K, Shimizu H. Lupus erythematosus profundus successfully treated with dapsone: review of the literature. Arch. Dermatol. 2006; 142; 399–401. 7. Cerveira I, Costa Matos L, Garrido A et al. Lupus mastitis. Breast 2006; 15; 670–672. 8. Bernatsky S, Ramsey-Goldman R, Boivin JF et al. Do traditional Gail model risk factors account for increased breast cancer in women with lupus? J. Rheumatol. 2003; 30; 1505–1507.

Defective Insulin and Acetylcholine Induction of Endothelial Cell–Nitric Oxide Synthase Through Insulin Receptor Substrate/Akt Signaling Pathway in Aorta of Obese Rats

The actions of acetylcholine (ACh) on endothelium mainly are mediated through muscarinic receptors, which are members of the G protein–coupled receptor family. In the present study, we show that ACh induces rapid tyrosine phosphorylation and activation of Janus kinase 2 (JAK2) in rat aorta. Upon JAK2 activation, tyrosine phosphorylation of insulin receptor substrate (IRS)-1 is detected. In addition, ACh induces JAK2/IRS-1 and IRS-1/phosphatidylinositol (PI) 3-kinase associations, downstream activation of Akt/protein kinase B, endothelial cell–nitric oxide synthase (eNOS), and extracellular signal–regulated kinase (ERK)-1/2. The pharmacological blockade of JAK2 or PI 3-kinase reduced ACh-stimulated eNOS phosphorylation, NOS activity, and aorta relaxation. These data indicate a new signal transduction pathway for IRS-1/PI 3-kinase/Akt/eNOS activation and ERK1/2 by means of JAK2 tyrosine phosphorylation stimulated by ACh in vessels. Moreover, we demonstrate that in aorta of obese rats (high-fat diet), there is an impairment in the insulin- and ACh-stimulated IRS-1/PI 3-kinase pathway, leading to reduced activation with lower protein levels of eNOS associated with a hyperactivated ERK/mitogen-activated protein kinase pathway. These results suggest that in aorta of obese rats, there not only is insulin resistance but also ACh resistance, probably mediated by a common signaling pathway that controls the activity and the protein levels of eNOS.

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.

Inhibition of fatty acid synthase in melanoma cells activates the intrinsic pathway of apoptosis

Fatty acid synthase (FASN) is the metabolic enzyme responsible for the endogenous synthesis of the saturated long-chain fatty acid, palmitate. In contrast to most normal cells, FASN is overexpressed in a variety of human cancers, including cutaneous melanoma, in which its levels of expression are associated with tumor invasion and poor prognosis. We have previously shown that FASN inhibition with orlistat significantly reduces the number of spontaneous mediastinal lymph node metastases following the implantation of B16-F10 mouse melanoma cells in the peritoneal cavity of C57BL/6 mice. In this study, we investigate the biological mechanisms responsible for the FASN inhibition-induced apoptosis in B16-F10 cells. Both FASN inhibitors, cerulenin and orlistat, significantly reduced melanoma cell proliferation and activated the intrinsic pathway of apoptosis, as demonstrated by the cytochrome c release and caspase-9 and -3 activation. Further, apoptosis was preceded by an increase in both reactive oxygen species production and cytosolic calcium concentrations and independent of p53 activation and mitochondrial permeability transition. Taken together, these findings demonstrate the mitochondrial involvement in FASN inhibition-induced apoptosis in melanoma cells.

Simvastatin inducing PC3 prostate cancer cell necrosis mediated by calcineurin and mitochondrial dysfunction

In the present study we analyzed the mechanisms of simvastatin toxicity for the PC3 human prostate cancer cell line. At 10 μM, simvastatin induced principally apoptosis, which was prevented by mevalonic acid but not by cyclosporin A, the inhibitor of calcineurin and mitochondrial permeability transition (MPT). At 60 μM, simvastatin induced the necrosis of PC3 cells insensitive to mevalonic acid. Cell necrosis was preceded by a threefold increase in cytosolic free Ca2+ concentration and a significant decrease in both respiration rate and mitochondrial membrane potential. Both mitochondrial dysfunction and necrosis were sensitive to the compounds cyclosporin A and bongkrekic acid, as well as the calcineurin inhibitor FK506. We have concluded that simvastatin-induced PC3 cells apoptosis is dependent on 3-hydroxy-3-methylglutaryl coenzyme-A reductase inhibition and independent of MPT, whereas necrosis is dependent on mitochondrial dysfunction caused, at least in part, by calcineurin.

Mitochondrial ATP-sensitive K + channels as redox signals to liver mitochondria in response to hypertriglyceridemia

We have recently demonstrated that hypertriglyceridemic (HTG) mice present both elevated body metabolic rates and mild mitochondrial uncoupling in the liver owing to stimulated activity of the ATP-sensitive potassium channel (mitoK(ATP)). Because lipid excess normally leads to cell redox imbalance, we examined the hepatic oxidative status in this model. Cell redox imbalance was evidenced by increased total levels of carbonylated proteins, malondialdehydes, and GSSG/GSH ratios in HTG livers compared to wild type. In addition, the activities of the extramitochondrial enzymes NADPH oxidase and xanthine oxidase were elevated in HTG livers. In contrast, Mn-superoxide dismutase activity and content, a mitochondrial matrix marker, were significantly decreased in HTG livers. Isolated HTG liver mitochondria presented lower rates of H(2)O(2) production, which were reversed by mitoK(ATP) antagonists. In vivo antioxidant treatment with N-acetylcysteine decreased both mitoK(ATP) activity and metabolic rates in HTG mice. These data indicate that high levels of triglycerides increase reactive oxygen generation by extramitochondrial enzymes that promote mitoK(ATP) activation. The mild uncoupling mediated by mitoK(ATP) increases metabolic rates and protects mitochondria against oxidative damage. Therefore, a biological role for mitoK(ATP) as a redox sensor is shown here for the first time in an in vivo model of systemic and cellular lipid excess.

Smad7 Blocks Transforming Growth Factor-β1–Induced Gingival Fibroblast–Myofibroblast Transition via Inhibitory Regulation of Smad2 and Connective Tissue Growth Factor

BACKGROUND Transforming growth factor-β1 (TGF-β1), its downstream signaling mediators (Smad proteins), and specific targets, including connective tissue growth factor (CTGF), play important roles in tissue remodeling and fibrosis via myofibroblast activation. We investigated the effect of overexpression of Smad7, a TGF-β1 signaling inhibitor, on transition of gingival fibroblast to myofibroblast. Moreover, we analyzed the participation of CTGF on TGF-β1-mediated myofibroblast transformation. METHODS To study the inhibitory effect of Smad7 on TGF-β1/CTGF-mediating gingival fibroblast transition into myofibroblasts, we stably overexpressed Smad7 in normal gingival fibroblasts and in myofibroblasts from hereditary gingival fibromatosis (HGF). Myofibroblasts were characterized by the expression of the specific marker isoform α of the smooth muscle actin (α-SMA) by Western blot, flow cytometry, and immunofluorescence. Enzyme-linked immunosorbent assay for type I collagen was performed to measure myofibroblast activity. CTGFs role on myofibroblast transformation was examined by enzyme-linked immunosorbent assay and small interference RNA. RESULTS TGF-β1 induced the expression of α-SMA and CTGF, and small interference RNA-mediating CTGF silencing prevented fibroblast-myofibroblast switch induced by TGF-β1. In Smad7-overexpressing fibroblasts, ablation of TGF-β1-induced Smad2 phosphorylation marked decreased α-SMA, CTGF, and type I collagen expression. Similarly, HGF transfectants overexpressing Smad7 demonstrated low levels of α-SMA and phospho-Smad2 and significant reduction on CTGF and type I collagen production. CONCLUSIONS CTGF is critical for TGF-β1-induced gingival fibroblast-myofibroblast transition, and Smad7 overexpression is effective in the blockage of myofibroblast transformation and activation, suggesting that treatments targeting myofibroblasts by Smad7 overexpression may be clinically effective in gingival fibrotic diseases, such as HGF.

Ovariectomy Reduces the Gelatinolytic Activity and Expression of Matrix Metalloproteinases and Collagen in Rat Molar Extraction Wounds

Osteoporosis is commonly associated with estrogen deficiency. However, the mechanisms by which the lack of this hormone causes bone loss are poorly understood. The bone structure of the oral cavity seems to be affected by estrogen deficiency, since a delayed healing process after tooth extraction has been observed after ovariectomy in rats. The aim of this study was to describe the effect of the absence of estrogen on the expression and activity of matrix metalloproteinases (MMC)-2 and -9 and expression of types I and III collagens in the alveolar granulation tissue of young female rats after tooth extraction. Sixty-six, four-week-old female rats underwent bilateral ovariectomies (OVX) or sham operations. Three weeks later, both first and second mandibular molars were extracted and the animals were killed by cervical dislocation 3, 5, or 7 days after tooth extraction. The granulation tissues were collected from the extracted alveolar sockets and used for zymographic, Western blot, or reverse transcription polymerase chain reaction (RT-PCR) analysis. There was a gradual increase on the expression of all studied proteins as well as MMP-2 and -9 activities in the periods after surgery. In contrast, OVX animals showed a significant decrease in the gelatinolytic activities and expression of MMP-2 and -9 and types I and III collagens. The results presented here in suggest that the absence of estrogen may possibly contribute to the delayed alveolar wound healing by interfering with the extracellular matrix turnover.

Fatty acid synthase inhibitors induce apoptosis in non-tumorigenic melan-a cells associated with inhibition of mitochondrial respiration.

The metabolic enzyme fatty acid synthase (FASN) is responsible for the endogenous synthesis of palmitate, a saturated long-chain fatty acid. In contrast to most normal tissues, a variety of human cancers overexpress FASN. One such cancer is cutaneous melanoma, in which the level of FASN expression is associated with tumor invasion and poor prognosis. We previously reported that two FASN inhibitors, cerulenin and orlistat, induce apoptosis in B16-F10 mouse melanoma cells via the intrinsic apoptosis pathway. Here, we investigated the effects of these inhibitors on non-tumorigenic melan-a cells. Cerulenin and orlistat treatments were found to induce apoptosis and decrease cell proliferation, in addition to inducing the release of mitochondrial cytochrome c and activating caspases-9 and -3. Transfection with FASN siRNA did not result in apoptosis. Mass spectrometry analysis demonstrated that treatment with the FASN inhibitors did not alter either the mitochondrial free fatty acid content or composition. This result suggests that cerulenin- and orlistat-induced apoptosis events are independent of FASN inhibition. Analysis of the energy-linked functions of melan-a mitochondria demonstrated the inhibition of respiration, followed by a significant decrease in mitochondrial membrane potential (ΔΨm) and the stimulation of superoxide anion generation. The inhibition of NADH-linked substrate oxidation was approximately 40% and 61% for cerulenin and orlistat treatments, respectively, and the inhibition of succinate oxidation was approximately 46% and 52%, respectively. In contrast, no significant inhibition occurred when respiration was supported by the complex IV substrate N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD). The protection conferred by the free radical scavenger N-acetyl-cysteine indicates that the FASN inhibitors induced apoptosis through an oxidative stress-associated mechanism. In combination, the present results demonstrate that cerulenin and orlistat induce apoptosis in non-tumorigenic cells via mitochondrial dysfunction, independent of FASN inhibition.