mTOR inhibitor everolimus reduces invasiveness of melanoma cells

The mammalian target of rapamycin (mTOR) plays a key role in several cellular processes: proliferation, survival, invasion, and angiogenesis, and therefore, controls cell behavior both in health and in disease. Dysregulation of the mTOR signaling is involved in some of the cancer hallmarks, and thus the mTOR pathway is an important target for the development of a new anticancer therapy. The object of this study is recognition of the possible role of mTOR kinase inhibitors-everolimus single and in combination with selected downstream protein kinases inhibitors: LY294002 (PI3 K), U0126 (ERK1/2), GDC-0879 (B-RAF), AS-703026 (MEK), MK-2206 (AKT), PLX-4032 (B-RRAF) in cell invasion in malignant melanoma. Treatment of melanoma cells with everolimus led to a significant decrease in the level of both phosphorylated: mTOR (Ser2448) and mTOR (Ser2481) as well as their downstream effectors. The use of protein kinase inhibitors produced a significant decrease in metalloproteinases (MMPs) activity, as well as diminished invasion, especially when used in combination. The best results in the inhibition of both MMPs and cell invasiveness were obtained for the combination of an mTOR inhibitor- everolimus with a B-RAF inhibitor-PLX-4032. Slightly less profound reduction of invasiveness was obtained for the combinations of an mTOR inhibitor-everolimus with ERK1/2 inhibitor-U126 or MEK inhibitor-AS-703026 and in the case of MMPs activity decrease for PI3 K inhibitor-LY294002 and AKT inhibitor-MK-2206. The simultaneous use of everolimus or another new generation rapalog with selected inhibitors of crucial signaling kinases seems to be a promising concept in cancer treatment

mTOR inhibitor Everolimus-induced apoptosis in melanoma cells

Melanoma is the most aggressive, therapy-resistant skin cancer. The mammalian target of rapamycin (mTOR), the serine/ threonine kinase which integrates both intracellular and extracellular signals, plays a crucial role in coordinating the balance between the growth and death of cells. The object of this study is a comparison of the influence of mTOR inhibitor everolimus in the concentration range between 20 nM and 10 μM, used individually and in combination with selected downstream protein kinases inhibitors: LY294002 (PI3K), U0126 (ERK1/2), AS-703026 (MEK) and MK-2206 (AKT) on the expression of prosurvival proteins: p-Bcl-2 (S70), p-Bcl-2 (T56), Bcl-2, Bcl-xL, Mcl-1, activity of caspase-3, proliferation and induction of apoptosis in melanoma cells. Current results clearly show that the nanomolar concentration of the mTOR inhibitor everolimus in combination with the inhibitor of MAP kinase (AS-703026) or AKT kinase (MK-2206) is effective in inducing apoptosis and reducing proliferation of melanoma cells. The herein research results confirm the hypothesis on the important role of mTOR signaling in cancer progression, and gives hope that implementation of successful combination of its inhibitors will find recognition and application in cancer treatment in the near future

Integrin linked kinase regulates endosomal recycling of N-cadherin in melanoma cells

Malignant transformation is characterized by a phenotype "switch" from E- to N-cadherin - a major hallmark of epithelial to mesenchymal transition (EMT). The increased expression of N-cadherin is commonly followed by a growing capacity for migration as well as resistance to apoptosis. Integrin Linked Kinase (ILK) is a key molecule involved in EMT and progression of cancer cells. ILK is known as a major signaling mediator involved in cadherin switch, but the specific mechanism through which ILK modulates N-cadherin expression is still not clear. Studies were carried out on human melanoma WM793 and 1205Lu cell lines. Expression of proteins was analyzed using PCR and Western Blot; siRNA transfection was done for ILK. Analysis of cell signaling pathways was monitored with phospho-specific antibodies. Subcellular localization of protein was studied using the ProteoExtract Subcellular Kit and Western blot analysis. Our data show that ILK knockdown by siRNA did suppress N-cadherin expression in melanoma, but only at the protein level. The ILK silencing-induced decrease of N-cadherin membranous expression in melanoma highlights the likely crucial role of ILK in the coordination of membrane trafficking through alteration of Rab expression. It is essential to understand the molecular mechanism of increased N-cadherin expression in cancer to possibly use it in the search of new therapeutic targets

Iatrogenic injuries to the trachea and main bronchi

INTRODUCTION: Iatrogenic tracheobronchial injuries are rare. AIM: To analyse the mechanism of injury, symptoms and treatment of these patients. MATERIAL AND METHODS: Retrospective analysis of hospital records of all patients treated for main airway injuries between 1990 and 2012 was performed. RESULTS: There were 24 patients, including 21 women and 3 men. Mean time between injury and initiation of treatment was 12 hours (range: 2-48). In 16 patients the injury occurred during tracheal intubation, in 1 during rigid bronchoscopy, in 1 during rigid oesophagoscopy, in 1 during mediastinoscopy and in 5 during open surgery. Mean length of airway tear was 3.8 cm (range: 1.5-8). In 1 patient there was an injury to the cervical trachea and in the remaining 23 in the thoracic part of the airway. The treatment included repair of the membranous part of the trachea performed via right thoracotomy in 10 patients (in 1 patient additionally coverage with a pedicled intercostal muscle flap was used), a self-expanding metallic stent in 1 patient, suture of the right main bronchus and the oesophagus in 1, left upper sleeve lobectomy in 1, right upper lobectomy in 1, implantation of a silicone Y stent in 3, mini-tracheostomy in 1, and conservative treatment in 5 patients. CONCLUSIONS: Intubation is the most frequent cause of iatrogenic main airway injuries. Patients with these life-threatening complications require an individualised approach and treatment in a reference centre

Neuropathic alterations of the myenteric plexus neurons following subacute intraperitoneal administration of salsolinol

Introduction. Impairment of the enteric nervous system has been suggested to occur within the pathogenesis of neurodegenerative diseases. Thus, in the current study, we consider salsolinol (1-methyl-6,7-dihydroxy-1,2,3,4-tetrahydroisoquinoline, SAL) as a substance that can potentially induce myenteric neurodegen­eration. Material and methods. Male Wistar rats were subjected to continuous intraperitoneal dosing of salsolinol (200 mg/kg in total) with osmotic mini-pumps for either two or four weeks. An equivalent group of rats served as the control. Jejunal myenteric neurons were subjected to immunofluorescence staining to detect neuron specific protein — protein gene product (pan-neuronal marker, PGP 9.5), nitric oxide synthase (NOS), choline acetyltransferase (ChAT), Bax-protein and alpha-synuclein. In search of any functional impairment within the gastrointestinal tract, gut motility was assessed by determining the residual solid food contents in the stomach and the small and large intestine transit. Results. The myenteric neuron count, the mean size of the neuron body, the area of ganglia and the diameter of nerve strands were decreased in both of the salsolinol-treated groups compared with the controls. The number of NOS-positive cells was lower in the salsolinol-treated groups, while the number of ChAT-positive cells remained unchanged in comparison with the controls. Neurons expressing the pro-apoptotic Bax protein and alpha-synuclein deposits were observed among the myenteric neurons of the salsolinol-treated rats. Conclusions. Salsolinol evokes enteric neuronal cell death via initiation of apoptosis and leads to the formation of pathological aggregates of alpha-synuclein. Impairment of myenteric neurons, mainly the inhibitory motor neurons, might be responsible for the abnormal intestinal transit. Thus, salsolinol might be regarded as a suitable compound for inducing experimental enteric neurodegeneration in rats