Sensitization of TRAIL-resistant cells by inhibition of heat shock protein 90 with low-dose geldanamycin

Due to its specificity and effectiveness, tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) is being tested for cancer therapy. Inhibition of the function of heat shock protein 90 (HSP90) is under clinical trials for cancer therapy. However, some cancer cells are resistant to TRAIL, and at the dose required for inducing apoptosis, geldanamycin, a drug that inhibits HSP90 function, has shown adverse effects. Therefore, our working plan was to identify a sublethal dose of geldanamycin and combine it with TRAIL to induce apoptosis in TRAIL-resistant prostate cancer cells. Treatment of LNCaP with 250 nmol/L geldanamycin inhibited HSP90 function but did not induce significant apoptosis. However, combination of geldanamycin and TRAIL induced highly significant apoptosis in TRAIL-resistant LNCaP cells. In addition to inducing caspase activity and apoptosis, treatment with geldanamycin and TRAIL decreased inhibitor of kappaB (IkappaB) kinase (IKK) complex proteins, IKKalpha, IKKbeta, and IKKgamma. The loss of IKK affected IkappaBalpha/nuclear factor-kappaB (NF-kappaB) interaction and reduced nuclear transport of NF-kappaB, resulting in reduced NF-kappaB activity. Our data show increase in apoptosis using low, suboptimal dose of geldanamycin when used with TRAIL. These results provide a means to alleviate two problems: resistance to TRAIL and adverse effects of high-dose geldanamycin.     

Structure-activity relationship of neomycin, paromomycin, and neamine-arginine conjugates, targeting HIV-1 gp120-CXCR4 binding step

We have recently designed and synthesized aminoglycoside–arginine conjugates (AACs) as potential anti-HIV-1 agents. AACs exert a number of activities related to Tat antagonism. We here present a new set of AACs, conjugates of neomycin B, paromomycin, and neamine with different number of arginines (1–6), their (a) uptake by human T-cell lines, (b) antiviral activities, (c) competition with monoclonal antibody (mAb) 12G5 binding to CXCR4, (d) competition with stromal cell-derived factor-1 (SDF-1) binding to CXCR4, and (e) competition with HIV-1 coat protein gp120 cell penetration. The appearance of mutations in HIV-1 gp120 gene in AACs resistant HIV-1 isolates, supports that AACs inhibit HIV-1 infectivity via interference of gp120–CXCR4 interaction. Our results point that the most potent AACs is the hexa-arginine–neomycin conjugate, the other multi-arginine–aminoglycoside conjugates are less active, and the mono-arginine conjugates display the lowest activity. Our studies demonstrate that, in addition to the core, the number of arginines attached to a specific aminoglycoside, are also important in the design of potent anti-HIV agents. The AACs play an important role, not only as HIV-1 RNA binders but also as inhibitors of viral entry into human cells.     

Plain roentgenographic estimation of systolic pulmonary artery pressure by the pulmonary arterial angle method.

Chest roentgenograms of 125 patients (115 with pure or predominant mitral stenosis) were studied by the pulmonary arterial angle method to estimate systolic pulmonary artery pressure without prior knowledge of catheterization data. First the angle between the line drawn along the right upper lobe artery and the tangent drawn along the point of junction of superior and lateral borders of the right pulmonary artery was determined. Next the angle between the right middle lobe artery and the descending pulmonary artery is determined. The difference between these angles equals systolic pulmonary artery pressure. Catheter and angle values were identical in 26 patients. There was a 1- to 5-mm difference in 70, a 6- to 10-mm difference in 18, an 11- to 15-mm difference in 8, and a difference greater than 15 mm in 3.     

Rap1 promotes VEGFR2 activation and angiogenesis by a mechanism involving integrin αvβ₃

Vascular endothelial growth factor (VEGF) acting through VEGF receptor 2 (VEGFR2) on endothelial cells (ECs) is a key regulator of angiogenesis, a process essential for wound healing and tumor metastasis. Rap1a and Rap1b, 2 highly homologous small G proteins, are both required for angiogenesis in vivo and for normal EC responses to VEGF. Here we sought to determine the mechanism through which Rap1 promotes VEGF-mediated angiogenesis. Using lineage-restricted Rap1-knockout mice we show that Rap1-deficiency in endothelium leads to defective angiogenesis in vivo, in a dose-dependent manner. Using ECs obtained from Rap1-deficient mice we demonstrate that Rap1b promotes VEGF-VEGFR2 kinase activation and regulates integrin activation. Importantly, the Rap1b-dependent VEGF-VEGFR2 activation is in part mediated via integrin α(v)β(3). Furthermore, in an in vivo model of zebrafish angiogenesis, we demonstrate that Rap1b is essential for the sprouting of intersomitic vessels, a process known to be dependent on VEGF signaling. Using 2 distinct pharmacologic VEGFR2 inhibitors we show that Rap1b and VEGFR2 act additively to control angiogenesis in vivo. We conclude that Rap1b promotes VEGF-mediated angiogenesis by promoting VEGFR2 activation in ECs via integrin α(v)β(3). These results provide a novel insight into the role of Rap1 in VEGF signaling in ECs.     

Hepatitis B virus subgenotype A1 predominates in liver disease patients from Kerala,India

AIM:To molecularly characterize hepatitis B virus(HBV)isolates from Kerala and to relate them to the clinical manifestation of infection.METHODS:Sera and clinical data were collected from91 patients diagnosed with chronic HBV infection and HBV-related hepatocellular carcinoma(HCC).HBV from44 HCC,22 cirrhotic and 25 chronic hepatitis patients were genotyped by sequencing of the complete S region or by restriction fragment length polymorphism assays.The basic core promoter/precore region was sequenced.The complete surface DNA sequences were assembled and aligned manually,and then compared with the sequences of HBV of genotypes(A-J)from GenBank.The evolutionary history was inferred using the Neighbor-Joining method and the evolutionary distances computed using the Kimura 2-parameter method.Bootstrapping was performed using 1000 replicates.The TaqMan BS-1 probe was used to quantify HBV DNA at a lower detection limit of approximately20 IU/mL.Continuous variables were compared using an independent Student’s t test.Theχ2test or Fisher’s exact test was used to compare categorical variables.The differences were considered statistically significant at P<0.05.RESULTS:Irrespective of disease status,the predominant genotype was A(72%);95%belonging to subgenotype A1,followed by genotypes D(27%)and C(1%).HCC patients infected with subgenotype A1were significantly younger than those infected with D.Mutation A1762T/G1764A was significantly associated with HCC in both genotypes A and D.Mutation G1862T was more frequent in subgenotype A1(P<0.0001),and in combination with A1762T/G1764A,it was significantly associated with HBV from HCC patients.Mutation C1766T/T1768A was significantly associated with genotype A(P=0.05)and HCC(P=0.03).The preS2start codon M1T/I mutation was unique to genotype A strains(15.6%)from all disease groups and occurred at a higher frequency in isolates from HCC patients(P=0.076).A higher frequency of preS deletion mutants(33.3%)was observed in genotype A from HCC compared with non-HCC patients,but     

Combination of proteasomal inhibitors lactacystin and MG132 induced synergistic apoptosis in prostate cancer cells

The proteasome inhibitor Velcade (bortezomib/PS-341) has been shown to block the targeted proteolytic degradation of short-lived proteins that are involved in cell maintenance, growth, division, and death, advocating the use of proteasomal inhibitors as therapeutic agents. Although many studies focused on the use of one proteasomal inhibitor for therapy, we hypothesized that the combination of proteasome inhibitors Lactacystin (AG Scientific, Inc., San Diego CA) and MG132 (Biomol International, Plymouth Meeting, PA) may be more effective in inducing apoptosis. Additionally, this regimen would enable the use of sublethal doses of individual drugs, thus reducing adverse effects. Results indicate a significant increase in apoptosis when LNCaP prostate cancer cells were treated with increasing levels of Lactacystin, MG132, or a combination of sublethal doses of these two inhibitors. Furthermore, induction in apoptosis coincided with a significant loss of IKKalpha, IKKbeta, and IKKgamma proteins and NFkappaB activity. In addition to describing effective therapeutic agents, we provide a model system to facilitate the investigation of the mechanism of action of these drugs and their effects on the IKK-NFkappaB axis.