The human HDAC (histone deacetylase) family, a well-validated anticancer target, plays a key role in the control of gene expression through regulation of transcription. While HDACs can be subdivided ...into three main classes, the class I, class II and class III HDACs (sirtuins), it is presently unclear whether inhibiting multiple HDACs using pan-HDAC inhibitors, or targeting specific isoforms that show aberrant levels in tumours, will prove more effective as an anticancer strategy in the clinic. To address the above issues, we have tested a number of clinically relevant HDACis (HDAC inhibitors) against a panel of rhHDAC (recombinant human HDAC) isoforms. Eight rhHDACs were expressed using a baculoviral system, and a Fluor de Lystrade mark (Biomol International) HDAC assay was optimized for each purified isoform. The potency and selectivity of ten HDACs on class I isoforms (rhHDAC1, rhHDAC2, rhHDAC3 and rhHDAC8) and class II HDAC isoforms (rhHDAC4, rhHDAC6, rhHDAC7 and rhHDAC9) was determined. MS-275 was HDAC1-selective, MGCD0103 was HDAC1- and HDAC2-selective, apicidin was HDAC2- and HDAC3-selective and valproic acid was a specific inhibitor of class I HDACs. The hydroxamic acid-derived compounds (trichostatin A, NVP-LAQ824, panobinostat, ITF2357, vorinostat and belinostat) were potent pan-HDAC inhibitors. The growth-inhibitory effect of the HDACis on HeLa cells showed that both pan-HDAC and class-I-specific inhibitors inhibited cell growth. The results also showed that both pan-HDAC and class-I-specific inhibitor treatment resulted in increased acetylation of histones, but only pan-HDAC inhibitor treatment resulted in increased tubulin acetylation, which is in agreement with their activity towards the HDAC6 isoform.
Histone deacetylase inhibitors represent a promising new class of anticancer agents. In the current investigation, we examined
the activity of PXD101, a potent histone deacetylase inhibitor, used ...alone or in combination with clinically relevant chemotherapeutics
(docetaxel, paclitaxel, and carboplatin), in preclinical in vitro and in vivo models of ovarian cancer. In vitro activity was examined in ovarian cancer and multidrug-resistant cell lines grown in monolayer culture, and in primary clinical
ovarian cancer specimens grown in three-dimensional organoid culture. PXD101 was found to inhibit in vitro cancer cell growth at sub- to low micromolar IC 50 potency, exhibited synergistic activity when used in combination with relevant chemotherapeutics, and effectively inhibited
the growth of multidrug-resistant cells. In vivo , PXD101 displayed single-agent antitumor activity on human A2780 ovarian cancer s.c. xenografts which was enhanced via combination
therapy with carboplatin. In support of these findings, PXD101 was shown to increase the acetylation of α-tubulin induced
by docetaxel and the phosphorylation of H2AX induced by carboplatin. Taken together, these results support the clinical evaluation
of PXD101 used alone or in combination therapy for the treatment of ovarian cancer. Mol Cancer Ther 2006;5(8):2086–95
Preclinical data show that belinostat (Bel) is synergistic with carboplatin and paclitaxel in ovarian cancer. To further evaluate the clinical activity of belinostat, carboplatin, and paclitaxel ...(BelCaP), a phase 1b/2 study was performed, with an exploratory phase 2 expansion planned specifically for women with recurrent epithelial ovarian cancer (EOC).
Thirty-five women were treated on the phase 2 expansion cohort. BelCap was given as follows: belinostat, 1000 mg/m² daily for 5 days with carboplatin, AUC 5; and paclitaxel, 175 mg/m² given on day 3 of a 21-day cycle. The primary end point was overall response rate (ORR), using a Simon 2 stage design.
The median age was 60 years (range, 39-80 years), and patients had received a median of 3 prior regimens (range, 1-4). Fifty-four percent had received more than two prior platinum-based combinations, sixteen patients (46%) had primary platinum-resistant disease, whereas 19 patients (54%) recurred within 6 months of their most recent platinum treatment. The median number of cycles of BelCaP administered was 6 (range, 1-23). Three patients had a complete response, and 12 had a partial response, for an ORR of 43% (95% confidence interval, 26%-61%). When stratified by primary platinum status, the ORR was 44% among resistant patients and 63% among sensitive patients. The most common drug-related adverse events related to BelCaP were nausea (83%), fatigue (74%), vomiting (63%), alopecia (57%), and diarrhea (37%). With a median follow-up of 4 months (range, 0-23.3 months), 6-month progression-free survival is 48% (95% confidence interval, 31%-66%). Median overall survival was not reached during study follow-up.
Belinostat, carboplatin, and paclitaxel combined was reasonably well tolerated and demonstrated clinical benefit in heavily-pretreated patients with EOC. The addition of belinostat to this platinum-based regimen represents a novel approach to EOC therapy and warrants further exploration.
PURPOSE: Advanced melanoma is a highly drug-refractory neoplasm representing a significant unmet medical need. We sought to
identify melanoma-associated cell surface molecules and to develop as well ...as preclinically test immunotherapeutic reagents
designed to exploit such targets. EXPERIMENTAL DESIGN AND RESULTS: By transcript profiling, we identified glycoprotein NMB
(GPNMB) as a gene that is expressed by most metastatic melanoma samples examined. GPNMB is predicted to be a transmembrane
protein, thus making it a potential immunotherapeutic target in the treatment of this disease. A fully human monoclonal antibody,
designated CR011, was generated to the extracellular domain of GPNMB and characterized for growth-inhibitory activity against
melanoma. The CR011 monoclonal antibody showed surface staining of most melanoma cell lines by flow cytometry and reacted
with a majority of metastatic melanoma specimens by immunohistochemistry. CR011 alone did not inhibit the growth of melanoma
cells. However, when linked to the cytotoxic agent monomethylauristatin E (MMAE) to generate the CR011-vcMMAE antibody-drug
conjugate, this reagent now potently and specifically inhibited the growth of GPNMB-positive melanoma cells in vitro. Ectopic
overexpression and small interfering RNA transfection studies showed that GPNMB expression is both necessary and sufficient
for sensitivity to low concentrations of CR011-vcMMAE. In a melanoma xenograft model, CR011-vcMMAE induced significant dose-proportional
antitumor effects, including complete regressions, at doses as low as 1.25 mg/kg. CONCLUSION: These preclinical results support
the continued evaluation of CR011-vcMMAE for the treatment of melanoma.
Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer, fibrotic diseases and arteriosclerosis. The PDGF family ...currently consists of at least three gene products, PDGF-A, PDGF-B and PDGF-C, which selectively signal through two PDGF receptors (PDGFRs) to regulate diverse cellular functions. After two decades of searching, PDGF-A and B were the only ligands identified for PDGFRs. Recently, however, database mining has resulted in the discovery of a third member of the PDGF family, PDGF-C, a functional analogue of PDGF-A that requires proteolytic activation. PDGF-A and PDGF-C selectively activate PDGFR-α, whereas PDGF-B activates both PDGFR-α and PDGFR-β. Here we identify and characterize a new member of the PDGF family, PDGF D, which also requires proteolytic activation. Recombinant, purified PDGF-D induces DNA synthesis and growth in cells expressing PDGFRs. In cells expressing individual PDGFRs, PDGF-D binds to and activates PDGFR-β but not PDGFR-α. However, in cells expressing both PDGFRs, PDGF-D activates both receptors. This indicates that PDGFR-α activation may result from PDGFR-α/β heterodimerization.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
To investigate the pharmacological properties of the CR011-vcMMAE fully human antibody-drug conjugate (ADC), such as dose titrations, quantitation of the time (days) to complete regression, ...pharmacokinetics, and schedule dependency. Our prior study characterized a fully human antibody to GPNMB covalently linked to monomethylauristatin E, CR011-vcMMAE, and further demonstrated cell surface staining of melanoma lines susceptible to the immunoconjugate's cytotoxicity (Clin Cancer Res 2005; 12(4): 1373-1382).
The human SK-MEL-2 and SK-MEL-5 melanoma xenografts were used in athymic mice to assess anti-tumor efficacy. After s.c. implantation, tumors became established (60-100 mg), and treatment commenced by i.v. injection of the immunoconjugate or vinblastine or paclitaxel. Short-term anti-tumor effects (inhibition of tumor growth) and long-term effects (complete regression) were observed.
CR011-vcMMAE induced regression of established human SK-MEL-2 and SK-MEL-5 xenografts at doses from 1.25 to 80 mg/kg treatment when administered intravenously every 4 days (4 treatments); strikingly, regressions were not associated with re-growth during the observation period (200 days). The disappearance rate of implants was dose dependent (minimum time, 18.5 days). Detectable serum CR011-vcMMAE >or=1 microg/mL (approximately 0.01 microM) was observed for >30 days post-dose; CR011-vcMMAE showed an elimination half-life of 10.3 days. A low volume of distribution suggested that CR011-vcMMAE was confined to blood and interstitial fluid. CR011-vcMMAE could be delivered by either a single bolus dose or by intermittent dosing (i.e., every 1, 2, 4, 8, or 16 days) with no discernible differences in the proportion of tumor-free survivors, indicating a lack of schedule dependency. The antibody-drug conjugate produced complete regressions, but the equivalent doses of free monomethylauristatin E or unconjugated antibody did not show anti-tumor effects. In addition, decreases in plasma tumor-derived human interleukin-8 coincided with tumor nodule disappearance.
Short-term anti-tumor effects and long-term effects (complete regression) were observed with CR011-vcMMAE, but not with the reference agents. These results suggest that CR011-vcMMAE may provide therapeutic benefit in malignant melanoma.
PDGF-B is of central importance in mesangioproliferative diseases. PDGF-D, a new PDGF isoform, like PDGF-B, signals through the PDGF betabeta-receptor. The present study first determined that PDGF-D ...is mitogenic for rat mesangial cells and is not inhibited by a PDGF-B antagonist. Low levels of PDGF-D mRNA were detected in normal rat glomeruli. After induction of mesangioproliferative nephritis in rats by anti-Thy 1.1 mAb, glomerular PDGF-D mRNA and protein expression increased significantly from days 4 to 9 in comparison with nonnephritic rats. Peak expression of PDGF-D mRNA occurred 2 d later than peak PDGF-B mRNA expression. In addition, PDGF-D serum levels increased significantly in the nephritic animals on day 7. For investigating the functional role of PDGF-D, neutralizing fully human mAb were generated using the XenoMouse technology. Rats with anti-Thy 1.1-induced nephritis were treated on days 3 and 5 with different amounts of a fully human PDGF-DD-specific neutralizing mAb (CR002), equal amounts of irrelevant control mAb, or PBS by intraperitoneal injection. Specific antagonism of PDGF-D led to a dose-dependent (up to 67%) reduction of glomerular cell proliferation. As judged by double immunostaining for 5-bromo-2'-deoxyuridine and alpha-smooth muscle actin, glomerular mesangial cell proliferation was reduced by up to 57%. Reduction of glomerular cell proliferation in the rats that received CR002 was not associated with reduced glomerular expression of PDGF-B mRNA. PDGF-D antagonism also led to reduced glomerular infiltration of monocytes/macrophages (day 5) and reduced accumulation of fibronectin (day 8). In contrast, no effect was noted in normal rats that received an injection of CR002. These data show that PDGF-D is overexpressed in mesangioproliferative states and can act as an auto-, para-, or even endocrine glomerular cell mitogen, indicating that antagonism of PDGF-D may represent a novel therapeutic approach to mesangioproliferative glomerulonephritides.
The Semaphorins are a large family of transmembrane, GPI-anchored and secreted proteins that play an important role in neuronal and endothelial cell guidance. A human gene related to the class VI ...Semaphorin family, Semaphorin 6A-1 (Sema 6A-1) was identified by homology-based genomic mining. Recent implication of Sema 3 family members in tumor angiogenesis and our expression analysis of Sema 6A-1 suggested that class VI Semaphorin might effect tumor neovascularization. The mRNA expression of Sema 6A-1 was elevated in several renal tumor tissue samples relative to adjacent non-tumor tissue samples from the same patient. Sema 6A-1 transcript was also expressed in the majority of renal clear cell carcinoma (RCC) cell lines and to a lesser extent in endothelial cells. To test the role of Sema 6A-1 in tumor angiogenesis, we engineered, expressed and purified the Sema 6A-1 soluble extracellular domain (Sema-ECD). The purified Sema-ECD was screened in a variety of endothelial cell-based assay both in vitro and in vivo. In vitro, Sema-ECD blocked VEGF-mediated endothelial cell migration. These effects were explained in part by our observation in endothelial cells that Sema-ECD inhibited VEGF-mediated Src, FAK and ERK phosphorylation. In vivo, mouse Matrigel assays demonstrated that the intraperitoneal administration of recombinant Sema-ECD inhibited both bFGF/VEGF and tumor cell line-induced neovascularization. These findings reveal a novel therapeutic utility for Sema 6A-1 (Sema-ECD) as an inhibitor of growth factor as well as tumor-induced angiogenesis.
The p38 mitogen-activated protein kinases (MAPK) are activated by cellular stresses and play an important role in regulating gene expression. We have isolated a cDNA encoding a novel protein kinase ...that has significant homology (57% amino acid identity) to human p38α/CSBP. The novel kinase, p38δ, has a nucleotide sequence encoding a protein of 365 amino acids with a putative TGY dual phosphorylation motif. Dot-blot analysis of p38δ mRNA in 50 human tissues revealed a distribution profile of p38δ that differs from p38α. p38δ is highly expressed in salivary gland, pituitary gland, and adrenal gland, whereas p38α is highly expressed in placenta, cerebellum, bone marrow, thyroid gland, peripheral leukocytes, liver, and spleen. Like p38α, p38δ is activated by cellular stress and proinflammatory cytokines. p38δ phosphorylates ATF-2 and PHAS-I, but not MAPK-activated protein kinase-2 and -3, knownin vivo and in vitro substrates of p38α. We also observed that p38δ was strongly activated by MKK3 and MKK6, while p38α was preferentially activated by MKK6. Other experiments showed that a potent p38α kinase inhibitor AMG 2372 minimally inhibited the kinase activity of p38δ. Taken together, these data indicate that p38δ is a new member of the p38 MAPK family and that p38δ likely has functions distinct from that of p38α.
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