Phylogenetic analysis in the clinical risk management of an outbreak of hepatitis C virus infection among transfused thalassaemia patients in Italy.

BACKGROUND: Occurrence of hepatitis C virus (HCV) infection is reduced by effective risk management procedures, but patient-to-patient transmission continues to be reported in healthcare settings. AIM: To report the use of phylogenetic analysis in the clinical risk management of an HCV outbreak among 128 thalassaemia outpatients followed at a thalassaemia centre of an Italian hospital. METHODS: Epidemiological investigation and root-cause analysis were performed. All patients with acute hepatitis and known chronic infection were tested for HCV RNA, HCV genotyping, and NS3, NS5A, and NS5B HCV genomic region sequencing. To identify transmission clusters, phylogenetic trees were built for each gene employing Bayesian methods. FINDINGS: All patients with acute hepatitis were infected with HCV genotype 1b. Root-cause analysis, including a lookback procedure, excluded blood donors as the source of HCV transmission. The phylogenetic analysis, conducted on seven patients with acute infection and eight patients with chronic infection, highlighted four transmission clusters including at least one patient with chronic and one patient with acute HCV infection. All patients in the same cluster received a blood transfusion during the same day. Two patients with acute hepatitis spontaneously cleared HCV within four weeks and nine patients received ledipasvir plus sofosbuvir for six weeks, all achieving a sustained virological response. CONCLUSION: Combined use of root-cause analysis and molecular epidemiology was effective in ascertaining the origin of the HCV outbreak. Antiviral therapy avoided the chronic progression of the infection and further spread in care units and in the family environment.

Causal relationship of hepatic fat with liver damage and insulin resistance in nonalcoholic fatty liver.

BACKGROUND AND AIMS: Nonalcoholic fatty liver disease is epidemiologically associated with hepatic and metabolic disorders. The aim of this study was to examine whether hepatic fat accumulation has a causal role in determining liver damage and insulin resistance. METHODS: We performed a Mendelian randomization analysis using risk alleles in PNPLA3, TM6SF2, GCKR and MBOAT7, and a polygenic risk score for hepatic fat, as instruments. We evaluated complementary cohorts of at-risk individuals and individuals from the general population: 1515 from the liver biopsy cohort (LBC), 3329 from the Swedish Obese Subjects Study (SOS) and 4570 from the population-based Dallas Heart Study (DHS). RESULTS: Hepatic fat was epidemiologically associated with liver damage, insulin resistance, dyslipidemia and hypertension. The impact of genetic variants on liver damage was proportional to their effect on hepatic fat accumulation. Genetically determined hepatic fat was associated with aminotransferases, and with inflammation, ballooning and fibrosis in the LBC. Furthermore, in the LBC, the causal association between hepatic fat and fibrosis was independent of disease activity, suggesting that a causal effect of long-term liver fat accumulation on liver disease is independent of inflammation. Genetically determined hepatic steatosis was associated with insulin resistance in the LBC and SOS. However, this association was dependent on liver damage severity. Genetically determined hepatic steatosis was associated with liver fibrosis/cirrhosis and with a small increase in risk of type 2 diabetes in publicly available databases. CONCLUSION: These data suggest that long-term hepatic fat accumulation plays a causal role in the development of chronic liver disease.

PNPLA3 rs738409 I748M is associated with steatohepatitis in 434 non-obese subjects with hepatitis C.

BACKGROUND: The PNPLA3/Adiponutrin rs738409 C/G single nucleotide polymorphism is associated with the severity of steatosis, steatohepatitis and fibrosis in patients with non-alcoholic fatty liver disease, as well as the severity of steatosis and fibrosis in patients with chronic hepatitis C (CHC). AIM: To test in genotype 1(G1)-CHC patients, the putative association between the PNPLA3 variant and histological features of steatohepatitis, as well as their impact on the severity of fibrosis. METHODS: Four hundred and thirty-four consecutively biopsied Caucasian G1-CHC patients were genotyped for PNPLA3 rs738409, its effect evaluated by using an additive model. Histological features of steatohepatitis in CHC were assessed using the Bedossa classification. Hepatic expression of PNPLA3 mRNA was evaluated in 63 patients. RESULTS: The prevalence of steatohepatitis increased from 16.5% in patients with PNPLA3 CC, to 23.2% in CG and 29.2% in the GG genotype (P = 0.02). By multiple logistic regression, PNPLA3 genotype (OR 1.54, 95% CI 1.03-2.30, P = 0.03), together with age (OR 1.03, 95% CI 1.00-1.05, P = 0.02), BMI ≥ 30 (OR 2.06, 95% CI 1.04-4.10, P = 0.03) and homoeostasis model assessment (HOMA, OR 1.18, 95% CI 1.04-1.32, P = 0.006) were independently linked to steatohepatitis. When stratifying for obesity, PNPLA3 was associated with NASH in non-obese patients only (12.0% in CC vs. 18.3% in CG vs. 27.3% in GG, P = 0.01), including after correction for metabolic confounders (OR 2.06, 95% CI 1.26-3.36, P = 0.004). We showed an independent association between steatohepatitis (OR 2.05, 95% CI 1.05-4.02, P = 0.003) and severe fibrosis. Higher liver PNPLA3 mRNA was associated both with the severity of steatosis (adjusted P = 0.03) and steatohepatitis after adjusting for gender, age, BMI and HOMA (P = 0.002). CONCLUSION: In patients with genotype 1 hepatitis C, the PNPLA3 G variant is associated with a higher risk of steatosis severity and steatohepatitis, particularly among non-obese subjects.

Association of vitamin D serum levels and its common genetic determinants, with severity of liver fibrosis in genotype 1 chronic hepatitis C patients.

Lower 25-hydroxyvitamin D [25(OH)D] serum levels have been associated with the severity of liver fibrosis in genotype 1 chronic hepatitis C patients (G1CHC). In addition, a recent genome-wide study identified genetic variants (rs12785878, near dehydrocholesterol reductase, DHCR7; rs10741657, near CYP2R1; and rs7041, near vitamin D-binding protein, GC) affecting 25(OH)D serum levels in healthy populations. We aimed to assess the association between vitamin D serum levels and its genetic determinants, with the severity of liver fibrosis. Two hundred and sixty patients with biopsy-proven G1CHC were consecutively evaluated. The 25(OH)D serum levels were measured by high-pressure liquid chromatography. All patients were genotyped for DHCR7 rs12785878, CYP2R1 rs10741657 and GC rs7041 single nucleotide polymorphisms. DHCR7 GG genotype (P = 0.003) and the severity of fibrosis (P = 0.03) were independent factors associated with lower 25(OH)D serum levels in multiple linear regression analysis. Interestingly, 53.8% (7/13) of patients with DHCR7 GG genotype had severe liver fibrosis, compared to 27.1% (67/247) of those with DHCR7 TT/TG genotype (P = 0.03). By multivariate logistic regression analysis, severe fibrosis was independently associated with older age (OR, 1.056; 95% CI, 1.023-1.089, P = 0.001), low cholesterol (OR, 0.984; 95% CI, 0.974-0.994, P = 0.002), high triglycerides (OR, 1.008; 95% CI, 1.002-1.015, P = 0.01), low 25(OH)D (OR, 0.958; 95% CI, 0.919-0.999, P = 0.04), DHCR7 GG genotype (OR, 4.222; 95% CI, 1.106-16.120; P = 0.03), moderate-severe steatosis (OR, 2.588; 95% CI, 1.355-4.943; P = 0.004) and moderate-severe necroinflammatory activity (grading) (OR, 2.437; 95% CI, 1.307-4.763; P = 0.001). No associations were found between liver fibrosis and both CYP2R1 and GC genotypes. In patients with G1CHC, GG homozygosis for DHCR7 gene and lower 25(OH)D levels are independently associated with the severity of liver fibrosis.

In vivo liver expression of TLR2, TLR3 and TLR7 in chronic hepatitis C.

The role of innate immune response mediated by Toll-like receptors in HCV infection, is not yet well understood and there is a lack of data regarding liver tissue expression of these molecules in chronic hepatitis C (CHC). Our study is aimed to investigate ex vivo, liver expression of TLR2, TLR3 and TLR7, which are more involved in the immune-pathogenesis of CHC, and to explore possible correlations with features of disease. We obtained liver biopsies and collected peripheral blood mononuclear cells (PBMC) from 23 consecutive patients with CHC and from 6 patients of control, without liver disease, undergoing surgery for cholecystectomy. The levels of TLRs mRNA in the samples were determined using a real-time reverse transcription quantitative PCR (RT-qPCR). We found a significant high expression of TLR3 in the liver of CHC patients respect to controls (also higher than expression in the PBMC). Conversely no differences emerged in the TLR2 and TLR7 levels between cases and controls. Also we found a correlation of TLR2 and TLR7 levels with the grade of necro-inflammation in the liver. Furthermore TLR7 hepatic levels resulted related to a more advanced stage of liver fibrosis. Ours is the first study to provide data on tissue expression of TLRs during chronic hepatitis C and we believe that it could lead to a better understanding of the role of these molecules in the HCV-mediated liver damage.

Role of IL-28B and inosine triphosphatase polymorphisms in efficacy and safety of Peg-Interferon and ribavirin in chronic hepatitis C compensated cirrhosis with and without oesophageal varices.

Genetic factors can influence the outcome of antiviral therapy in chronic hepatitis C (HCV). We evaluated the role of interleukin-28B single nucleotide polymorphisms (SNPs) and inosine triphosphatase (ITPA) gene variants in HCV cirrhosis treated with Peg-Interferon and ribavirin. A prospective cohort of 233 patients with compensated cirrhosis received 1-1.5 µg/kg/week of Peg-Interferon alpha-2b plus 1000-1200 mg/day of RBV for 48 weeks. A sustained virologic response (SVR) was achieved in 27% of patients. On multivariate logistic analysis, the absence of oesophageal varices (OR 3.64 CI 95% 1.27-10.44 P = 0.016), infection with genotype 2 or 3 (OR 4.06, CI 95% 1.08-15.26, P = 0.038), C/C alleles of rs12979860 SNP (OR 7.04, CI 95% 2.40-20.72, P < 0.001) and rapid virologic response (RVR) (OR 78.29, CI 95% 16.07-381.29, P < 0.001) were independently associated with SVR. Patients who experienced post-treatment relapse received lower total doses of Peg-Interferon (52.0 ± 15.8 µg/kg vs 65.7 ± 13.3 µg/kg, P < 0.001) and lower total dose of RBV (3829 ± 1210 mg vs 4709 ± 954 mg, P < 0.001) than patients who achieved an SVR. ITPA variants predictive of high ITPase activity were associated with reduction of haemoglobin ≥3 g/dL in the first 4 weeks (P < 0.001), and with reduction of haemoglobin <10 g/dL (P = 0.03) on treatment. In conclusion, combination therapy with Peg-Interferon and RBV in patients with HCV cirrhosis must be guided by virus genotype, severity of portal hypertension, favourable IL-28B polymorphisms and ITPA variants, and RVR on treatment.

Superficial venous thrombosis: role of inherited deficiency of natural anticoagulants in extension to deep veins.

AIM: Superficial venous thrombosis (SVT) has been considered for a long time a limited clinical condition of low importance, but this approach has changed in recent years, when several studies demonstrated that extension to deep veins occurs in 7.3 to 44% of patients, with high prevalence of pulmonary embolism. The aim of this study was to evaluate the prevalence of inherited deficiency of natural coagulation inhibitors in patients suffering from SVT in both normal and varicose veins, and to understand their role in extension to deep veins. METHODS: The study included 83 patients with SVT, without clinically obvious risk factors. Ultrasound examination was performed, and deficiencies of Protein C, Protein S and Antithrombin (AT) were investigated. RESULTS: In the patients where SVT occurred in normal veins, coagulation inhibitor deficiencies were 6.45% in the absence of extension and 62.5% in patients with extension to deep veins. In the patients with varicose vein SVT, the presence of these factors was less evident, but their prevalence was considerably higher in those with extension to deep veins (36.3%) than in non-extension (6.06%). CONCLUSIONS: Present data confirm the role of inherited thrombophilic states related to inhibitor deficiency, considering them as risk factors for SVT in normal veins. Furthermore, an association has been found between their presence and the progression of SVT to deep veins.

Home therapy for deep vein thrombosis and pulmonary embolism in cancer patients.

Outpatient treatment of deep vein thrombosis (DVT) has become a common practice in uncomplicated patients. Few data are still present in patients with comorbidity (such as cancer) or concomitant symptomatic pulmonary embolism. Cancer patients with DVT are often excluded from home treatment because they have a higher risk of both bleeding and recurrent DVT. We tested the feasibility and safety of the Home Treatment (HT) program for acute DVT a PE in cancer patients. Patients were treated as outpatients unless they required admission for other medical problems, were actively bleeding or had pain that requires parenteral narcotics. Outpatient treatment was with low molecular weight heparin (LMWH) followed by warfarin or with LMWH alone. An educational program for patients was implemented. Two-hundred and seven patients with cancer were evaluated, 36 (17.4%) of whom had metastatic disease. Treatment with LMWH and warfarin was prescribed to 106 (51.2%) and LMWH alone to 102 (48.8%). One hundred and twenty-seven patients (61.3%) were entirely treated at home. There were no differences between patients treated at home and hospitalized patients with regard to gender, mean age, site of cancer, presence of metastases, and treatment. After 6 months, recurrent thrombo-embolism occurred in 8.7% of patients treated at home and in 5.6% of hospitalized patients (P=0.58); major bleeding in 2.0% and 1.5%, respectively (P=0.06). Twenty-seven patients (33%) in the hospitalized, and 33 (26%) in the home-treatment group, died after a follow-up of 6 months. These results indicate that, regarding cancer patients with acute DVT and/or PE, there is no difference between hospitalised and home-treated patients in terms of major outcomes.

Prolonged prothrombin time, Factor VII and activated FVII levels in chronic liver disease are partly dependent on Factor VII gene polymorphisms.

BACKGROUND: Prothrombin time is a benchmark for functional assessment in cirrhosis and Factor VII levels (FVII), crucial in determining the prothrombin time, are genetically determined. METHODS: We have evaluated the prothrombin time, a number of haemostatic variables synthesised by the liver (FII, FV, FVII and activated FVII, AT and fibrinogen) and two polymorphisms of the FVII gene (5'F7 and 353R/Q) in: (a) patients with liver cirrhosis (n=118), (b) patients with chronic hepatitis (n=102) and (c) controls (n=100). RESULTS: By one-way analyses of variance, the prothrombin time and the mean levels of the FII, FV, FVIIc, FVIIa, and AT were statistically different between cirrhotics, chronic hepatitis patients and controls. The allele frequency of the FVII polymorphisms did not differ between the three groups. Those rare patients (4.6%) who were homozygous for the type 2 alleles had markedly reduced FVIIc and FVIIa levels. The analysis carried out taking into account Child class versus FVII genotype showed that the mean FVIIc levels were comparable for different genotypes within each Child's class, with the exception of the patients homozygous for the type 1 allele. CONCLUSION: Our findings help to explain the not infrequent finding of a severely prolonged prothrombin time in patients who are otherwise in a good functional class.

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.

Heterocycle-containing retinoids. Discovery of a novel isoxazole arotinoid possessing potent apoptotic activity in multidrug and drug-induced apoptosis-resistant cells.

In a search for retinoic acid (RA) receptor ligands endowed with potent apoptotic activity, a series of novel arotinoids were prepared. Because the stereochemistry of the C9-alkenyl portion of natural 9-cis-RA and the olefinic moiety of the previously synthesized isoxazole retinoid 4 seems to have particular importance for their apoptotic activity, novel retinoid analogues with a restricted or, vice versa, a larger flexibility in this region were designed and prepared. The new compounds were evaluated in vitro for their ability to activate natural retinoid receptors and for their differentiation-inducing activity. Cytotoxic and apoptotic activities were, in addition, evaluated. In general, these analogues showed low cytotoxicity, with the restricted structures being slightly more active than the more flexible ones. As an exception, however, the isoxazole retinoid 15b proved to be particularly able to induce apoptosis at concentrations <5 microM, showing a higher activity than the classical retinoids such as all-trans-RA, 13-cis-RA, and 9-cis-RA and the previously described synthetic retinoid 4. 15b also exhibited a good affinity for the retinoid receptors. Interestingly, another important property of 15b was its ability to induce apoptosis in the HL60R multidrug-resistant (MDR) cell line, at the same concentration as is effective in HL60. Therefore, 15b represents a new retinoid possessing high apoptotic activity in an MDR cell line. The ability of 15b to act on K562 and HL60R cells suggests that this compound may have important implications in the treatment of different leukemias, and its structure could offer an interesting model for the design of new compounds endowed with apoptotic activity on MDR- and retinoid-resistant malignancies.

Effects of chemically modified tetracyclines (CMTs) in sensitive, multidrug resistant and apoptosis resistant leukaemia cell lines.

Recently discovered chemically modified tetracyclines (CMTs) have shown in vitro and in vivo anti-proliferative and anti-tumour activities. Here, we evaluated in vitro the anti-proliferative and apoptotic activity of six different dedimethylamino chemically modified tetracyclines (CMT-1, CMT-3, CMT-5, CMT-6, CMT-7 and CMT-8) in sensitive and multidrug resistant myeloid leukaemia cells (HL60 and HL60R) in vitro. Three of these compounds (CMT-5, CMT-6, CMT-7) showed low cytotoxic activity both in sensitive and in resistant cells, CMT-3 was endowed with a high anti-proliferative activity only in sensitive cells and was moderately effective as apoptosis inducing agent, with an activity similar to that shown by doxycycline. On the contrary, CMT-1 and CMT-8 were very effective as programmed cell death inducing agents. The apoptotic pathway activated by these compounds involved the activation of caspases, especially caspase-9 and, for CMT-1, also the activation of FAS: Interestingly CMT-8, but not CMT-1, was able to induce apoptosis in multidrug resistant HL60R and in Fas-ligand resistant HUT78B1 cell lines. These properties, together with others previously described (e.g. anti-metastatic and anti-osteolytic activities), suggest that CMT-8 may have important applications in the clinical management of cancer. The comparative analysis of structure-activity relationship of CMT-8 and doxycycline suggests that the C-5 hydroxy moiety may play an important role in conferring activity in multidrug resistant cells. These findings appear to support the hypothesis that CMT-8 may represent an interesting lead for the development of a new class of potent apoptosis inducer agents active in multidrug resistant and Fas-ligand resistant malignancies.

Apoptotic effects of thiazolobenzimidazole derivatives on sensitive and multidrug resistant leukaemic cells.

We investigated the cytotoxic activity of eight thiazolobenzimidazole derivatives on sensitive HL60 and multidrug-resistant (MDR) (HL60R) leukaemia cell lines. The antitumour effects of these compounds were compared with those of RS-TBZ, a thiazolobenzimidazole derivative, previously described in our reports, that was able to induce apoptosis more markedly in MDR cells than in the parental sensitive cell lines. Only two compounds in this study proved to have interesting effects: (a) the S-enantiomer of TBZ, that was able to induce apoptosis in MDR cells in a slightly more selective manner than TBZ (racemic form); and (b) TBZ-4-OCH3 (TBZ-4-OCH3), that showed cytotoxic and apoptotic effects on sensitive and resistant leukaemia cells greater than TBZ, without cytotoxic effects on normal haemopoietic progenitor cells. Moreover, we observed that TBZ-4-OCH3 was also active in cells expressing Bcr-Abl, an oncogene that confers resistance to apoptosis induced by several stimuli, including cytotoxic agents. The inhibition of caspase-9 and caspase-3 by specific polypeptide inhibitors decreased the apoptotic effects of TBZ-4-OCH3 in HL60 cells indicating that apoptosis induced by this compound was, at least partly, caspase-mediated. On the contrary, the blocking of FL-associated cell surface antigen (Fas) using a specific Fas-blocking monoclonal antibody did not affect the level of apoptosis induced by TBZ-4-OCH3 suggesting that the Fas pathway was not involved. In addition, the caspase 8 inhibitor was unable to inhibit the apoptotic activity of TBZ-4-OCH3. The very low toxicity shown by TBZ-4-OCH3 in normal haemopoietic progenitor cells and its high activity in sensitive and MDR neoplastic cells suggest a possible clinical use for this new compound.

Factor V Leiden and prothrombin gene mutation in inflammatory bowel disease in a Mediterranean area.

BACKGROUND: Thromboembolism has been reported to be associated with inflammatory bowel disease. AIM: To evaluate the association of factor V Leiden and prothrombin gene mutation with inflammatory bowel disease in a population of patients with thromboembolic events and inflammatory bowel disease and in a control population of patients with inflammatory bowel disease without thromboembolic events. PATIENTS AND METHODS: A series of 18 patients with inflammatory bowel disease and a history of arterial or venous thrombosis and 45 patients with inflammatory bowel disease without thromboembolic events were evaluated for the presence of factor V Leiden and prothrombin gene mutation. Frequency of gene mutation was compared with its occurrence in 100 healthy controls. RESULTS: One patient with inflammatory bowel disease without thromboembolic events was heterozygous for factor V Leiden mutation. whereas no patient with a thromboembolic event had factor V Leiden mutation. No patients (either cases or controls) had prothrombin gene mutation. In the healthy population the frequency of factor V Leiden and prothrombin mutation was 5% and 2%, respectively. CONCLUSIONS: Data emerging from the present study do not support any role of factor V Leiden and prothrombin gene mutation as the cause of thromboembolism in inflammatory bowel disease.

Programmed cell death (PCD) associated with the stilbene motif of arotinoids: discovery of novel apoptosis inducer agents possessing activity on multidrug resistant tumor cells.

Considering that the stereochemistry of the C9-C10 alkenyl portion of natural 9-cis-RA, as the one of the olefinic moiety of the previously described isoxazole retinoid 4, seems of particular importance for their apoptotic activity, we prepared a novel class of TTNPB analogues bearing both the cis or trans configuration of the alkenyl portion. The compounds were evaluated in vitro for their cytotoxic and apoptotic activities. We discovered that the cis-TTNPB 9c possesses apoptotic activity comparable with that of the retinoid 4. Moreover, the amino arotinoid 16c showed potent apoptotic activity in HL60 promyelocytic leukemia cells. Interestingly, 16c proved to be a particularly potent apoptosis-inducing agent active in multidrug resistant (MDR) cell lines. Therefore, to the best of our knowledge, 16c may represent the first known aminoarotinoid endowed with potent apoptotic activity in MDR cells. Taken together, these results seem to point out that the cis-stilbene motif of arotinoids may be at least an important feature in conferring cytotoxic and apoptotic activity to this class of compounds.

Structure-activity relationship studies of novel heteroretinoids: induction of apoptosis in the HL-60 cell line by a novel isoxazole-containing heteroretinoid.

In a search for retinoic acid receptor (RAR and RXR)-selective ligands, a series of isoxazole retinoids was synthesized and evaluated in vitro in transcriptional activation and competition binding assays for RARs and RXRs. In addition, these compounds were evaluated for their differentiating, cytotoxic, and apoptotic activities. In general, these derivatives showed scarcely any binding affinity and were not active in the transcriptional assay. However, among these isoxazole derivatives, the cis-isomer 14b was identified as a potent inducer of apoptosis, and its activity was found to be 6.5 and 4 times superior than that of 13-cis- and 9-cis-retinoic acids, respectively. On the other hand, compound 13b, which has the trans stereochemistry at the double bond, was found not to be active in the apoptotic assay, but it was endowed with appreciable differentiating activity. Therefore, it seems that the different stereochemistry of the double bond may be associated with a different biological activity: potent apoptotic activity for the cis-isomer but differentiating activity for the trans structure. This biological behavior was found, at least in part, for the 9-cis- and 13-cis-retinoic acids with respect to the all-trans-retinoic acid. Thus, structure 14b could offer an interesting model for the design of new compounds endowed with 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 microg/ml, and VP16, at concentrations ranging from 0.05 - 1 microg/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.

Selective induction of apoptosis in multidrug resistant HL60R cells by the thiazolobenzoimidazole derivative 1-(2,6-difluorophenyl)-1H,3H-thiazolo [3,4-a] benzimidazole (TBZ).

We investigated the antitumour effects of 1-(2,6-difluorophenyl)-1H,3H-thiazolo [3,4-a]benzimidazole (TBZ) a new anti-HIV-1 agent, on human promyelocytic HL60 leukaemia, both a parental and a multidrug resistant form (HL60R). HL60R overexpresses P-glycoprotein and, like HL60, lacks p53 protein expression. HL60 and HL60R show similar levels of Bcl-2 protein. In contrast to the conventional chemotherapeutic agents daunorubicin, etoposide and mitoxantrone, TBZ caused equal or even greater cytotoxicity in HL60R than in HL60, and this result was associated with a more marked induction of apoptosis in the drug resistant cells. The antitumour activity of TBZ occurred in the range of concentrations higher than those required to exert antiviral activity. TBZ seems to act in the presence of P-glycoprotein and Bcl-2 and in the absence of p53 and is able to circumvent the mechanisms of drug resistance and anti-apoptosis present in HL60R cells.

Modulation of nitric oxide production by tetracyclines and chemically modified tetracyclines.

Chemically modified tetracyclines (CMTs) dose-dependently decreased inducible nitric oxide synthase (iNOS) and, consequently, nitric oxide (NO) formation by the lipopolysaccharide (LPS)-stimulated J774 line. The inhibitory effect was due to a specific reduction in the iNOS protein content in the cells, as attested by Western blot analysis and by the inhibition of iNOS mRNA accumulation. Furthermore, CMTs cause a dose-dependent increase in cell death in the J774 line mediated by the NO-independent apoptotic mechanism.

Effects of highly purified anthraquinoid compounds from Aloe vera on sensitive and multidrug resistant leukemia cells.

Folk medicine has attributed antitumor properties to preparations from Aloe vera. We have studied the effects of five purified compounds from the plant on human K562 leukemia and on its multidrug resistant (MDR) variant, K562/R. The glycosides aloin A and B, aloesin and aloeresin were devoid of antitumor activity up to 200 mu M concentrations. Only the aglycone aloe emodin produced reproducible antitumor effects, which, interestingly, were more pronounced in the MDR, P-glycoprotein overexpressing, cell line. Its IC50 was in fact 29 mu M in K562 and 10.5 mu M in K562/R. Aloe emodine caused mainly cytostasis and accumulation of the cells in the S and G(2)-M phases of the cell cycle during the first 48 h of treatment. Thereafter, massive cell death ensued. Research on the antitumor activity of compounds extracted from Aloe vera probably deserves continuation.