Increased cytotoxicity of cisplatin in SK-MEL 28 melanoma cells upon down-regulation of melanoma inhibitor of apoptosis protein

BACKGROUND: Malignant melanoma is a highly metastatic cutaneous cancer and typically refractory to chemotherapy. Deregulated apoptosis has been identified as a major cause of cancer drug resistance, and upregulated expression of the inhibitor of apoptosis protein melanom, an inhibitor of apoptosis (ML-IAP) is frequent in melanoma. METHODS: Based on the conclusion that ML-IAP expression contributes to a malignant phenotype, we down-regulated the ML-IAP mRNA using sequence optimized antisense oligonucleotides. RESULTS: As measured by real-time PCR, oligonucleotides M706 and M711 inhibited ML-IAP mRNA expression by 47% and 52%, respectively in the highly metastatic and drug resistant SK-MEL28 cell line. Oligonucleotide M706, which was previously evaluated in G361 cells as the most efficient inhibitor of ML-IAP expression, was chosen to compare cell viability and drug sensitivity of these two melanoma cell lines with different p53 functionality. Protein expression was reduced by oligonucleotide M706 to 49% of the normal level and resulted in a dose-dependent specific reduction of cell viability with a maximum of 39% at 600 nM. Typical morphological changes showed that loss of viability was mainly due to cell death. In combination experiments, the use of oligonucleotide M706 resulted in a two-fold increase of cisplatin cytotoxicity at different concentrations of oligonucleotide and cisplatin (P<0.05). This is in line with our previous findings in G361 melanoma cell line, in which oligonucleotide M706 caused a 3-fold increase in cisplatin cytotoxicity. CONCLUSION: Our data suggest the use of ML-IAP antisense oligonucleotides to overcome drug resistance in metastatic melanoma, in spite of its p53 status.

The novel ATP-competitive inhibitor of the MET hepatocyte growth factor receptor EMD1214063 displays inhibitory activity against selected MET-mutated variants

The receptor tyrosine kinase MET is a prime target in clinical oncology due to its aberrant activation and involvement in the pathogenesis of a broad spectrum of malignancies. Similar to other targeted kinases, primary and secondary mutations seem to represent an important resistance mechanism to MET inhibitors. Here, we report the biologic activity of a novel MET inhibitor, EMD1214063, on cells that ectopically express the mutated MET variants M1268T, Y1248H, H1112Y, L1213V, H1112L, V1110I, V1206L, and V1238I. Our results demonstrate a dose-dependent decrease in MET autophosphorylation in response to EMD1214063 in five out of the eight cell lines (IC50 2-43nM). Blockade of MET by EMD1214063 was accompanied by a reduced activation of downstream effectors in cells expressing EMD1214063-sensitive mutants. In all sensitive mutant-expressing lines, EMD1214063 altered cell cycle distribution, primarily with an increase in G1 phase. EMD1214063 strongly influenced MET-driven biological functions, such as cellular morphology, MET-dependent cell motility and anchorage-independent growth. To assess the in vivo efficacy of EMD1214063, we used a xenograft tumor model in immunocompromised mice bearing NIH3T3 cells expressing sensitive and resistant MET mutated variants. Animals were randomized for the treatment with EMD1214063 (50mg/kg/day) or vehicle only. Remarkably, five days of EMD1214063 treatment resulted in a complete regression of the sensitive H1112L-derived tumors, while tumor growth remained unaffected in mice with L1213V tumors and in vehicle-treated animals. Collectively, the current data identifies EMD1214063 as a potent MET small molecule inhibitor with selective activity towards mutated MET variants.

Nonanimal stabilized hyaluronic acid for tissue augmentation of the dorsal hands: a prospective study on 38 patients

BACKGROUND Often ignored, hands are one of the most telltale signs of aging. This prospective study was initiated to evaluate the effect of subcutaneous hyaluronic acid (HA) injections in aging hands, with special attention to complications and long-term outcomes. METHODS Between January 2010 and December 2010, a total of 38 patients with skin phototypes II-IV and between 58 and 76 years old were treated with HA injection for aging hands. The quantity of injection never exceeded 1.0-1.5 ml HA per hand. A clinical follow-up was performed at 2 weeks, 4 weeks, 3 months, and 6 months after injection. Complications were reviewed for the whole series. At the first follow-up, 2 weeks after the procedure, ultrasound was carried out to determine if additional filling material was required. At each follow-up, patients were asked to fill out a satisfaction questionnaire. RESULTS Nine patients developed slight ecchymosis that disappeared after 1 week. No other complications were seen in the series. Pain during the injection and discomfort after the procedure were minimal. At the 2-week follow-up, after ultrasound control, nine patients received a complementary injection. At each follow-up, overall patient satisfaction was high and was validated by clearance of rhytids, veins, bony prominences, and dermal and subcutaneous atrophy. CONCLUSION Skin revitalization with injectable HA can improve the clinical appearance of the back of the hands. However, this therapy requires knowledge of the possible complications and their remediation as well as knowledge and respect of injected doses. Moreover, despite excellent results at each follow-up, the results of our series are not as good after 6 months, and a longer follow-up would be needed to determine if this procedure provides long-lasting benefit. LEVEL OF EVIDENCE III This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Guineensine is a novel inhibitor of endocannabinoid uptake showing cannabimimetic behavioral effects in BALB/c mice

High-content screening led to the identification of the N-isobutylamide guineensine from Piper nigrum as novel nanomolar inhibitor (EC50 = 290 nM) of cellular uptake of the endocannabinoid anandamide (AEA). Noteworthy, guineensine did not inhibit endocannabinoid degrading enzymes fatty acid amide hydrolase (FAAH) or monoacylglycerol lipase (MAGL) nor interact with cannabinoid receptors or fatty acid binding protein 5 (FABP5), a major cytoplasmic AEA carrier. Activity-based protein profiling showed no inhibition of serine hydrolases. Guineensine also inhibited the cellular uptake of 2-arachidonoylglycerol (2-AG). Preliminary structure–activity relationships between natural guineensine analogs indicate the importance of the alkyl chain length interconnecting the pharmacophoric isobutylamide and benzodioxol moieties for AEA cellular uptake inhibition. Guineensine dose-dependently induced cannabimimetic effects in BALB/c mice shown by strong catalepsy, hypothermia, reduced locomotion and analgesia. The catalepsy and analgesia were blocked by the CB1 receptor antagonist rimonabant (SR141716A). Guineensine is a novel plant natural product which specifically inhibits endocannabinoid uptake in different cell lines independent of FAAH. Its scaffold may be useful to identify yet unknown targets involved in endocannabinoid transport.

Tissue properties of the human vertebral body sub-structures evaluated by means of microindentation

Purpose The better understanding of vertebral mechanical properties can help to improve the diagnosis of vertebral fractures. As the bone mechanical competence depends not only from bone mineral density (BMD) but also from bone quality, the goal of the present study was to investigate the anisotropic indentation moduli of the different sub-structures of the healthy human vertebral body and spondylophytes by means of microindentation. Methods Six human vertebral bodies and five osteophytes (spondylophytes) were collected and prepared for microindentation test. In particular, indentations were performed on bone structural units of the cortical shell (along axial, circumferential and radial directions), of the endplates (along the anterio-posterior and lateral directions), of the trabecular bone (along the axial and transverse directions) and of the spondylophytes (along the axial direction). A total of 3164 indentations down to a maximum depth of 2.5 µm were performed and the indentation modulus was computed for each measurement. Results The cortical shell showed an orthotropic behavior (indentation modulus, Ei, higher if measured along the axial direction, 14.6±2.8 GPa, compared to the circumferential one, 12.3±3.5 GPa, and radial one, 8.3±3.1 GPa). Moreover, the cortical endplates (similar Ei along the antero-posterior, 13.0±2.9 GPa, and along the lateral, 12.0±3.0 GPa, directions) and the trabecular bone (Ei= 13.7±3.4 GPa along the axial direction versus Ei=10.9±3.7 GPa along the transverse one) showed transversal isotropy behavior. Furthermore, the spondylophytes showed the lower mechanical properties measured along the axial direction (Ei=10.5±3.3 GPa). Conclusions The original results presented in this study improve our understanding of vertebral biomechanics and can be helpful to define the material properties of the vertebral substructures in computational models such as FE analysis.

Levels of matrix metalloproteinase-9 within cerebrospinal fluid in a rabbit model of coccidioidal meningitis and vasculitis.

Matrix metalloproteinase (MMP)-9 is produced by the central nervous system and inflammatory cells in a variety of inflammatory conditions in both animals and humans. MMP-9 promotes inflammation, breakdown of the blood-brain barrier, and vasculitis. Because vasculitis is seen frequently in patients with coccidioidal meningitis (CM), this study evaluated the presence of MMP-9 within the cerebrospinal fluid (CSF) of rabbits infected intracisternally with Coccidioides immitis arthroconidia. Infected rabbits demonstrated systemic and neurological sequelae to infection, including CSF pleocytosis. Levels of MMP-9 within CSF were assayed by use of zymography and compared with MMP-2 levels, which served as an internal control. Elevated levels of MMP-9 were detectable by day 3, continued to increase through day 10, and declined by day 15 after infection. MMP-9 may contribute to inflammation and vasculitis in this animal model. Future work can focus on evaluation of MMP inhibitors, to gain a better perspective of the role of this MMP in CM.

Matrix metalloproteinase (MMP)-8 and MMP-9 in cerebrospinal fluid during bacterial meningitis: association with blood-brain barrier damage and neurological sequelae.

To evaluate the spectrum and regulation of matrix metalloproteinases (MMPs) in bacterial meningitis (BM), concentrations of MMP-2, MMP-3, MMP-8, and MMP-9 and endogenous inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were measured in the cerebrospinal fluid (CSF) of 27 children with BM. MMP-8 and MMP-9 were detected in 91% and 97%, respectively, of CSF specimens from patients but were not detected in control patients. CSF levels of MMP-9 were higher (P<.05) in 5 patients who developed hearing impairment or secondary epilepsy than in those who recovered without neurological deficits. Levels of MMP-9 correlated with concentrations of TIMP-1 (P<.001) and tumor necrosis factor-alpha (P=.03). Repeated lumbar punctures showed that levels of MMP-8 and MMP-9 were regulated independently and did not correlate with the CSF cell count. Therefore, MMPs may derive not only from granulocytes infiltrating the CSF space but also from parenchymal cells of the meninges and brain. High concentrations of MMP-9 are a risk factor for the development of postmeningitidal neurological sequelae.

Everolimus is a potent inhibitor of activated hepatic stellate cell functions in vitro and in vivo , while demonstrating anti-angiogenic activities

Progression of liver fibrosis to HCC (hepatocellular carcinoma) is a very complex process which involves several pathological phenomena, including hepatic stellate cell activation, inflammation, fibrosis and angiogenesis. Therefore inhibiting multiple pathological processes using a single drug can be an effective choice to curb the progression of HCC. In the present study, we used the mTOR inhibitor everolimus to observe its effect on the in vitro activation of hepatic stellate cells and angiogenesis. The results of the present study demonstrated that everolimus treatment blocked the functions of the immortalized human activated hepatic stellate cell line LX-2 without affecting the viability and migration of primary human stellate cells. We also observed that treatment with everolimus (20 nM) inhibited collagen production by activated stellate cells, as well as cell contraction. Everolimus treatment was also able to attenuate the activation of primary stellate cells to their activated form. Angiogenesis studies showed that everolimus blocked angiogenesis in a rat aortic ring assay and inhibited the tube formation and migration of liver sinusoidal endothelial cells. Finally, everolimus treatment reduced the load of tumoral myofibroblasts in a rat model of HCC. These data suggest that everolimus targets multiple mechanisms, making it a potent blocker of the progression of HCC from liver fibrosis.

Tissue metabolomics of hepatocellular carcinoma: Tumor energy metabolism and the role of transcriptomic classification

Hepatocellular carcinoma (HCC) is one of the commonest causes of death from cancer. A plethora of metabolomic investigations of HCC have yielded molecules in biofluids that are both up- and down-regulated but no real consensus has emerged regarding exploitable biomarkers for early detection of HCC. We report here a different approach, a combined transcriptomics and metabolomics study of energy metabolism in HCC. A panel of 31 pairs of HCC tumors and corresponding nontumor liver tissues from the same patients was investigated by gas chromatography-mass spectrometry (GCMS)-based metabolomics. HCC was characterized by ∼2-fold depletion of glucose, glycerol 3- and 2-phosphate, malate, alanine, myo-inositol, and linoleic acid. Data are consistent with a metabolic remodeling involving a 4-fold increase in glycolysis over mitochondrial oxidative phosphorylation. A second panel of 59 HCC that had been typed by transcriptomics and classified in G1 to G6 subgroups was also subjected to GCMS tissue metabolomics. No differences in glucose, lactate, alanine, glycerol 3-phosphate, malate, myo-inositol, or stearic acid tissue concentrations were found, suggesting that the Wnt/β-catenin pathway activated by CTNNB1 mutation in subgroups G5 and G6 did not exhibit specific metabolic remodeling. However, subgroup G1 had markedly reduced tissue concentrations of 1-stearoylglycerol, 1-palmitoylglycerol, and palmitic acid, suggesting that the high serum α-fetoprotein phenotype of G1, associated with the known overexpression of lipid catabolic enzymes, could be detected through metabolomics as increased lipid catabolism. Conclusion: Tissue metabolomics yielded precise biochemical information regarding HCC tumor metabolic remodeling from mitochondrial oxidation to aerobic glycolysis and the impact of molecular subtypes on this process.

The sub-lethal effects and tissue concentration of the human pharmaceutical atenolol in rainbow trout (Oncorhynchus mykiss).

Atenolol is a highly prescribed anti-hypertensive pharmaceutical and a member of the group of β-blockers. It has been detected at concentrations ranging from ng L(-1) to low μg L(-1) in waste and surface waters. The present study aimed to assess the sub-lethal effects of atenolol on rainbow trout (Oncorhynchus mykiss) and to determine its tissue-specific bioconcentration. Juvenile rainbow trout were exposed for 21 and 42 days to three concentration levels of atenolol (1 μg L(-1) - environmentally relevant concentration, 10 μg L(-1), and 1000 μg L(-1)). The fish exposed to 1 μg L(-1) atenolol exhibited a higher lactate content in the blood plasma and a reduced haemoglobin content compared with the control. The results show that exposure to atenolol at concentrations greater than or equal to 10 μg L(-1) significantly reduces both the haematocrit value and the glucose concentration in the blood plasma. The activities of the studied antioxidant enzymes (catalase and superoxide dismutase) were not significantly affected by atenolol exposure, and only the highest tested concentration of atenolol significantly reduced the activity of glutathione reductase. The activities of selected CYP450 enzymes were not affected by atenolol exposure. The histological changes indicate that atenolol has an effect on the vascular system, as evidenced by the observed liver congestion and changes in the pericardium and myocardium. Atenolol was found to have a very low bioconcentration factor (the highest value found was 0.27). The bioconcentration levels followed the order liver>kidney>muscle. The concentration of atenolol in the blood plasma was below the limit of quantification (2.0 ng g(-1)). The bioconcentration factors and the activities of selected CYP450 enzymes suggest that atenolol is not metabolised in the liver and may be excreted unchanged.

A cysteine protease inhibitor of plasmodium berghei is essential for exo-erythrocytic development

Plasmodium parasites express a potent inhibitor of cysteine proteases (ICP) throughout their life cycle. To analyze the role of ICP in different life cycle stages, we generated a stage-specific knockout of the Plasmodium berghei ICP (PbICP). Excision of the pbicb gene occurred in infective sporozoites and resulted in impaired sporozoite invasion of hepatocytes, despite residual PbICP protein being detectable in sporozoites. The vast majority of these parasites invading a cultured hepatocyte cell line did not develop to mature liver stages, but the few that successfully developed hepatic merozoites were able to initiate a blood stage infection in mice. These blood stage parasites, now completely lacking PbICP, exhibited an attenuated phenotype but were able to infect mosquitoes and develop to the oocyst stage. However, PbICP-negative sporozoites liberated from oocysts exhibited defective motility and invaded mosquito salivary glands in low numbers. They were also unable to invade hepatocytes, confirming that control of cysteine protease activity is of critical importance for sporozoites. Importantly, transfection of PbICP-knockout parasites with a pbicp-gfp construct fully reversed these defects. Taken together, in P. berghei this inhibitor of the ICP family is essential for sporozoite motility but also appears to play a role during parasite development in hepatocytes and erythrocytes.

Pneumomediastinum and soft tissue emphysema of the neck in postmortem CT and MRI; a new vital sign in hanging?

Spontaneous pneumomediastinum commonly occurs in healthy young men or parturient women in whom an increased intra-alveolar pressure (Valsalva maneuver, asthma, cough, emesis) leads to the rupture of the marginal pulmonary alveoli. The air ascends along the bronchi to the mediastinum and the subcutaneous space of the neck, causing cervico-fascial subcutaneous emphysema in 70-90% of cases. Ninety-five forensic cases, including five cases of hanging, were examined using postmortem multi-slice computed tomography (MSCT) and magnetic resonance imaging (MRI) prior to autopsy until December 2003. This paper describes the findings of pneumomediastinum and cervical emphysema in three of five cases of hanging. The mechanism of its formation is discussed based on these results and a review of the literature. In conclusion, when putrefaction gas can be excluded the findings of pneumomediastinum and cervical soft tissue emphysema serve as evidence of vitality of a hanged person. Postmortem cross-sectional imaging is considered a useful visualization tool for emphysema, with a great potential for examination and documentation.

Discovery and characterization of a novel non-competitive inhibitor of the divalent metal transporter DMT1/SLC11A2

Divalent metal transporter-1 (SLC11A2/DMT1) uses the H+ electrochemical gradient as the driving force to transport divalent metal ions such as Fe2+, Mn2+ and others metals into mammalian cells. DMT1 is ubiquitously expressed, most notably in proximal duodenum, immature erythroid cells, brain and kidney. This transporter mediates H+-coupled transport of ferrous iron across the apical membrane of enterocytes. In addition, in cells such as to erythroid precursors, following transferrin receptor (TfR) mediated endocytosis; it mediates H+-coupled exit of ferrous iron from endocytic vesicles into the cytosol. Dysfunction of human DMT1 is associated with several pathologies such as iron deficiency anemia hemochromatosis, Parkinson's disease and Alzheimer's disease, as well as colorectal cancer and esophageal adenocarcinoma, making DMT1 an attractive target for drug discovery. In the present study, we performed a ligand-based virtual screening of the Princeton database (700,000 commercially available compounds) to search for pharmacophore shape analogs of recently reported DMT1 inhibitors. We discovered a new compound, named pyrimidinone 8, which mediates a reversible linear non-competitive inhibition of human DMT1 (hDMT1) transport activity with a Ki of ∼20 μM. This compound does not affect hDMT1 cell surface expression and shows no dependence on extracellular pH. To our knowledge, this is the first experimental evidence that hDMT1 can be allosterically modulated by pharmacological agents. Pyrimidinone 8 represents a novel versatile tool compound and it may serve as a lead structure for the development of therapeutic compounds for pre-clinical assessment.