Stefania Grimaudo

Transmembrane 6 superfamily member 2 gene variant disentangles nonalcoholic steatohepatitis from cardiovascular disease

Excess hepatic storage of triglycerides is considered a benign condition, but nonalcoholic steatohepatitis (NASH) may progress to fibrosis and promote atherosclerosis. Carriers of the TM6SF2 E167K variant have fatty liver as a result of reduced secretion of very‐low‐density lipoproteins (VLDLs). As a result, they have lower circulating lipids and reduced risk of myocardial infarction. In this study, we aimed to assess whether TM6SF2 E167K affects liver damage and cardiovascular outcomes in subjects at risk of NASH. Liver damage was evaluated in 1,201 patients who underwent liver biopsy for suspected NASH; 427 were evaluated for carotid atherosclerosis. Cardiovascular outcomes were assessed in 1,819 controls from the Swedish Obese Subjects (SOS) cohort. Presence of the inherited TM6SF2 E167K variant was determined by TaqMan assays. In the liver biopsy cohort, 188 subjects (13%) were carriers of the E167K variant. They had lower serum lipid levels than noncarriers (P < 0.05), had more‐severe steatosis, necroinflammation, ballooning, and fibrosis (P < 0.05), and were more likely to have NASH (odds ratio [OR]: 1.84; 95% confidence interval [CI]: 1.23‐2.79) and advanced fibrosis (OR, 2.08; 95% CI: 1.20‐3.55), after adjustment for age, sex, body mass index, fasting hyperglycemia, and the I148M PNPLA3 risk variant. However, E167K carriers had lower risk of developing carotid plaques (OR, 0.49; 95% CI: 0.25‐0.94). In the SOS cohort, E167K carriers had higher alanine aminotransferase ALT and lower lipid levels (P < 0.05), as well as a lower incidence of cardiovascular events (hazard ratio: 0.61; 95% CI: 0.39‐0.95). Conclusions: Carriers of the TM6SF2 E167K variant are more susceptible to progressive NASH, but are protected against cardiovascular disease. Our findings suggest that reduced ability to export VLDLs is deleterious for the liver. (Hepatology 2015;61:506‐514)

Statin use and non-alcoholic steatohepatitis in at risk individuals

BACKGROUND & AIMS Excess hepatic free cholesterol contributes to the pathogenesis of non-alcoholic steatohepatitis, and statins reduce cholesterol synthesis. Aim of this study was to assess whether statin use is associated with histological liver damage related to steatohepatitis. METHODS The relationship between statin use, genetic risk factors, and liver damage was assessed in a multi-center cohort of 1201 European individuals, who underwent liver biopsy for suspected non-alcoholic steatohepatitis. RESULTS Statin use was recorded in 107 subjects, and was associated with protection from steatosis, NASH, and fibrosis stage F2-F4, in a dose-dependent manner (adjusted p<0.05 for all). In 100 treated patients matched 1:1 for modality of recruitment, gender, presence of IFG or type 2 diabetes, PNPLA3 I148M risk alleles, TM6SF2 E167K variant, age, and BMI, statin use remained associated with protection from steatosis (OR 0.09, 95% C.I. 0.01-0.32; p=0.004), steatohepatitis (OR 0.25, 95% C.I. 0.13-0.47; p<0.001), and fibrosis stage F2-F4 (OR 0.42, 95% C.I. 0.20-0.8; p=0.017). Results were confirmed in a second analysis, where individuals were matched within recruitment center (p<0.05 for all). The protective effect of statins on steatohepatitis was stronger in subjects not carrying the I148M PNPLA3 risk variant (p=0.02 for interaction), as statins were negatively associated with steatohepatitis in patients negative (p<0.001), but not in those positive for the I148M variant (p=n.s.). CONCLUSIONS Statin use was associated with protection towards the full spectrum of liver damage in individuals at risk of non-alcoholic steatohepatitis. However, the I148M PNPLA3 risk variant limited this beneficial effect.

Design, synthesis, and biological evaluation of thiophene analogues of chalcones

Chalcones are characterized by possessing an enone moiety between two aromatic rings. A series of chalcone-like agents, in which the double bond of the enone system is embedded within a thiophene ring, were synthesized and evaluated for antiproliferative activity and inhibition of tubulin assembly and colchicine binding to tubulin. The replacement of the double bond with a thiophene maintains antiproliferative activity and therefore must not significantly alter the relative conformation of the two aryl rings. The synthesized compounds were found to inhibit the growth of several cancer cell lines at nanomolar to low micromolar concentrations. In general, all compounds having significant antiproliferative activity inhibited tubulin polymerization with an IC(50)<2microM. Several of these compounds caused K562 cells to arrest in the G2/M phase of the cell cycle.

Synthesis and biological evaluation of 1-methyl-2-(3 ',4 ',5 '-trimethoxybenzoyl)-3-aminoindoles as a new class of antimitotic agents and tubulin inhibitors

The 2-(3,4,5-trimethoxybenzoyl)-2-aminoindole nucleus was used as the fundamental structure for the synthesis of compounds modified with respect to positions C-4 to C-7 with different moieties (chloro, methyl, or methoxy). Additional structural variations concerned the indole nitrogen, which was alkylated with small alkyl groups such as methyl or ethyl. We have identified 1-methyl-2-(3,4,5-trimethoxybenzoyl)-3-amino-7-methoxyindole as a new highly potent antiproliferative agent that targets tubulin at the colchicine binding site and leads to apoptotic cell death.

IL28B and PNPLA3 polymorphisms affect histological liver damage in patients with non-alcoholic fatty liver disease

BACKGROUND & AIMS Genetic background may affect liver damage in patients with non-alcoholic fatty liver disease (NAFLD). The main outcomes of the study were to assess whether IL28B rs12979860 and rs8099917 polymorphisms, together with PNPLA3 rs738409 C>G polymorphism, are associated with lobular inflammation and fibrosis, in NAFLD patients. METHODS One hundred sixty consecutive NAFLD patients were assessed by liver biopsy (Kleiner score); anthropometric, and biochemical and metabolic features were included. IL28B rs12979860 C>T, IL28B rs8099917 G>C, and PNPLA3 rs738409 C>G single nucleotide polymorphisms were tested. RESULTS Seventy-four (46.2%) patients had IL28B rs12979860 CC polymorphism, compared with 72 (45%) and 14 (8.8%) with TC and TT variants, respectively. PNPLA3 rs738409 CC polymorphism was present in 47 (29.4%) patients, compared with 79 (49.4%) and 34 (21.3%) with CG and GG variants, respectively. Multivariate logistic regression analysis showed that age (OR 1.043, 95% CI 1.012-1.075, p=0.007), triglycerides (OR 1.005, 95% CI 1.000-1.010, p=0.04), hyperuricemia (OR 5.027, 95% CI 1.839-13.742, p=0.002), IL28B rs12979860 TT/TC (OR 0.219, 95% CI 0.101-0.472, p<0.001), and steatosis grade (OR 1.704, 95% CI 1.048-2.773, p=0.03) were independently linked to moderate-severe lobular inflammation. Finally, IL28B rs12979860 CC was associated with severe fibrosis (F3-F4) on univariate analysis, even if only older age (OR 1.064, 95% CI 1.026-1.104, p=0.001), high HOMA (OR 1.213, 95% CI 1.068-1.377, p=0.003), and lobular inflammation (OR 3.181, 95% CI 1.438-7.036, p=0.004), remained associated in multivariate logistic regression analysis. CONCLUSIONS In NAFLD patients, IL28B rs12979860 CC genotype, together with PNPLA3 rs738409 GG, is associated with the severity of liver damage.

Anti-inflammatory effects of chemically modified tetracyclines by the inhibition of nitric oxide and interleukin-12 synthesis in J774 cell line

We investigated the effects of chemically modified tetracyclines (CMTs) on the production of nitric oxide (NO) and on the synthesis of some cytokines: tumour necrosis factor alpha (TNF-alpha), interleukin(IL)-10 and IL-12 in lipopolysaccharide (LPS)-treated J774 cell line. Furthermore, we studied the ability of these drugs to modify the viability in LPS-stimulated J774 macrophages. CMTs decreased, in a dose-dependent manner, inducible NO synthase (iNOS) activity and, consequently, nitrite formation in J774 cultures. The CMT-induced decrease in NO production is due to the inhibition of enzyme activity rather than to a direct effect on enzyme expression. The absence of the inhibition in mRNA accumulation indicates that the inhibiting activity is mainly post-transcriptional. CMTs were unable to modulate TNF-alpha and IL-10 synthesis and they were not effective in modifying the transcription of relative mRNA in J774 macrophages. On the contrary, IL-12 mRNA expression was significantly increased by CMT-1 and CMT-8 with LPS activation. Since IL-12 protein secretion was inhibited by CMTs, these compounds interfere in the blocking of post-transcriptional events. The studies on cell viability showed that various CMTs induced a dose-dependent decrease in J774 macrophage viability. The cytotoxic activity was present even though NO production was inhibited by CMTs. These compounds appear to be able to activate apoptosis in aNO-independent way. Altogether, these results indicate that CMTs can exert anti-inflammatory effects by inhibiting NO synthesis, and they are able to modify cell viability by exerting a strong apoptotic activity.

The CD95/CD95 ligand system is not the major effector in anticancer drug-mediated apoptosis

Many anticancer drugs are able to induce apoptosis in tumor cells but the mechanisms underlying this phenomenon are poorly understood. Some authors reported that the p53 tumor suppressor gene may be responsible for drug-induced apoptosis; however, chemotherapy-induced apoptosis can also be observed in p53 negative cells. Recently, doxorubicin (DXR) was reported to induce CD95L expression to mediate apoptosis through the CD95/CD95L system. Thus, an impairment of such a system may be involved in drug resistance. We evaluated the in vitro antitumor activity of several cytotoxic drugs on two human p53-negative T-cell lymphoma cell lines, the HUT78-B1 CD95L-resistant cell line and the HUT78 parental CD95L-sensitive cell line. We demostrated by Western blotting assay that DXR and etoposide (VP-16) were able to induce CD95L expression after 4 h of treatment. In contrast, they were unable to induce the expression of p53. DXR, at concentrations ranging from 0.001–1 μg/ml, and VP16, at concentrations ranging from 0.05–1 μg/ml, were equally cytotoxic and induced apoptosis in both cell lines as assessed by fluorescence microscopy and flow cytometry analyses. Although we observed a slightly reduced percentage of apoptotic cells in HUT78B1 when compared with the parental HUT78 cells after few hours of drug exposure, this difference was no longer evident at 48 or 72 h. Similarly, the exposure of HUT78 cells to a CD95-blocking antibody partially reduced early apoptosis (24 h) without affecting the long-term effects of the drugs including cytotoxicity. Furthermore, as observed with DXR and VP-16, both the CD95L-sensitive and the CD95L-resistant cell lines resulted equally sensitive to the cytotoxic effects of a number of different cytotoxic drugs (vincristine, camptothecin, 5-fluorouracil and methotrexate). The treatment with the Caspase-3 tetrapeptide aldehyde inhibitor, Ac-DEVD-CHO, did not affect the DXR-induced apoptosis whereas it only modestly inhibited apoptosis and cytotoxicity of VP-16, while Z-VAD.FMK, a Caspase inhibitor that prevents the processing of Caspase-3 to its active form, was able to block DXR-induced apoptosis at 24 h but not at 48 h. Thus, our results do not confirm a crucial role for the CD95/CD95L system in drug-induced apoptosis and suggest the involvement of alternative p53-independent pathways at least in this experimental model system.

Identification of Biphenyl-Based Hybrid Molecules Able To Decrease the Intracellular Level of Bcl-2 Protein in Bcl-2 Overexpressing Leukemia Cells

With the aim of enhancing the structural complexity and diversity of an existing collection of bi- and terphenyl compounds, we synthesized hybrid molecules comprising of spirocyclic ketones (a complexity-bearing core) and bi/terphenyls (privileged fragments). Compounds 1, 3, 4, and 6 showed well-defined activity on apoptosis and differentiation, making them potential leads for development as new anticancer agents and chemical probes to study signaling networks in neoplastic cells.

Glucokinase Regulatory Protein Gene Polymorphism Affects Liver Fibrosis in Non-Alcoholic Fatty Liver Disease

Background and Aims Variant in glucokinase regulatory protein (GCKR), associated with lipid and glucose traits, has been suggested to affect fatty liver infiltration. We aimed to assess whether GCKR rs780094 C→T SNP influences the expression of steatosis, lobular inflammation and fibrosis in NAFLD patients, after correction for PNPLA3 genotype. Methods In 366 consecutive NAFLD patients (197 from Sicily, and 169 from center/northern Italy), we assessed anthropometric, biochemical and metabolic features; liver biopsy was scored according to Kleiner. PNPLA3 rs738409 C>G and GCKR rs780094 C>T single nucleotide polymorphisms were also assessed. Results At multivariate logistic regression analysis in the entire NAFLD cohort, the presence of significant liver fibrosis (>F1) was independently linked to high HOMA (OR 1.12, 95% CI 1.01–1.23, p = 0.02), NAFLD activity score ≥5 (OR 4.09, 95% CI 2.45–6.81, p<0.001), and GCKR C>T SNP (OR 2.06, 95% CI 1.43–2.98, p<0.001). Similar results were observed considering separately the two different NAFLD cohorts. GCKR C>T SNP was also associated with higher serum triglycerides (ANOVA, p = 0.02) in the entire cohort. Conclusions In patients with NAFLD, GCKR rs780094 C>T is associated with the severity of liver fibrosis and with higher serum triglyceride levels.

Retinoic acid and analogs as potent inducers of differentiation and apoptosis. New promising chemopreventive and chemotherapeutic agents in oncology

In this report we will describe the preparation and the biological activity of a novel class of heterocyclic arotinoids endowed with potent cytotoxic and apoptotic acitivity. Structureactivity relationship studies revealed that the different stereochemistry at the C9 double bond of retinoids seems associated with a different biological activity: potent apoptotic activity for the cis-isomers, whereas differentiating activity for the trans structures. An interesting modified Wittig procedure that allows easily to arotinoids will also be described. The substitution of the alkenyl portion with a more flexible oxymethyl or aminomethyl moiety gave compounds with poor activity, whereas isoxazole-bridged arotinoids allowed compounds active also on multidrug-resistant (MDR) leukemia cell lines.