搜索结果: 'methocult media formulations for human hematopoietic cells serum containing'

Both the nonreceptor tyrosine kinase Src and the receptors for transforming growth factor (TGF)-β (TβRI,TβRII) play major roles during tumorigenesis by regulating cell growth,migration/invasion and metastasis. The common Src family kinase inhibitors PP2 and PP1 effectively block Src activity in vitro and in vivo,however,they may exert non-specific effects on other kinases. In this study,we have evaluated PP2 and PP1 for their ability to inhibit TGFβ1-mediated responses in the TGF-β-responsive pancreatic adenocarcinoma cell line Panc1. We show that PP2 and PP1 but not the more specific Src inhibitor SU6656 effectively relieved TGF-b1-induced growth arrest and p21(WAF1) induction,while basal growth was enhanced by PP2 and PP1,and suppressed by SU6656. PP2 and PP1 but not SU6656 also suppressed TGF-β1-induced epithelial-to-mesenchymal transition (EMT) as evidenced by their ability to inhibit downregulation of the epithelial marker E-cadherin,and upregulation of the EMT-associated transcription factor Slug. Likewise,PP2 and PP1 but not SU6656 effectively blocked TGF-β1-induced activation of Smad2 and p38 MAPK and partially suppressed Smad activation and transcriptional activity on TGF-β/Smad-responsive reporters of a kinase-active TβRI mutant ectopically expressed in Panc1 cells. Interestingly,PP2 and PP1 strongly inhibited recombinant TβRI in an in vitro kinase assay,with PP1 being more potent and PP2 being nearly as potent as the established TβRI inhibitor SB431542. PP2 but not PP1 also weakly inhibited the TβRII kinase. Together,these data provide evidence that PP2 and PP1 are powerful inhibitors of TβR function that can block TGF-β/Smad signaling in a Src-unrelated fashion. Both agents may be useful as dual TGF-β/Src inhibitors in experimental therapeutics of late stage metastatic disease. View Publication

Glioblastoma multiforme (GBM) is the most common and aggressive brain cancer,and despite treatment advances,patient prognosis remains poor. During routine animal studies,we serendipitously observed that fenbendazole,a benzimidazole antihelminthic used to treat pinworm infection,inhibited brain tumor engraftment. Subsequent in vitro and in vivo experiments with benzimidazoles identified mebendazole as the more promising drug for GBM therapy. In GBM cell lines,mebendazole displayed cytotoxicity,with half-maximal inhibitory concentrations ranging from 0.1 to 0.3 µM. Mebendazole disrupted microtubule formation in GBM cells,and in vitro activity was correlated with reduced tubulin polymerization. Subsequently,we showed that mebendazole significantly extended mean survival up to 63% in syngeneic and xenograft orthotopic mouse glioma models. Mebendazole has been approved by the US Food and Drug Administration for parasitic infections,has a long track-record of safe human use,and was effective in our animal models with doses documented as safe in humans. Our findings indicate that mebendazole is a possible novel anti-brain tumor therapeutic that could be further tested in clinical trials. View Publication

Although inactivation of the PTEN gene has been implicated in the development of resistance to the HER2 targeting antibody trastuzumab,the mechanisms mediating this resistance remain elusive. We generated trastuzumab resistant cells by knocking down PTEN expression in HER2 overexpressing breast cancer cell lines and demonstrate that development of trastuzumab resistance in these cells is mediated by activation of an IL6 inflammatory feedback loop leading to expansion of the cancer stem cell (CSC) population. Long term trastuzumab treatment generates highly enriched CSCs which display an EMT phenotype secreting over 100-fold more IL6 than parental cells. An IL6 receptor antibody interrupted this inflammatory feedback loop reducing the cancer stem cell population resulting in decreased tumor growth and metastasis in mouse xenographs. These studies demonstrate that trastuzumab resistance may be mediated by an IL6 inflammatory loop and suggest that blocking this loop may provide alternative strategy to overcome trastuzumab resistance. View Publication

PURPOSE: This first-in-human dose-escalation trial evaluated the safety,tolerability,maximal-tolerated dose (MTD),dose-limiting toxicities (DLT),pharmacokinetics,pharmacodynamics,and preliminary clinical activity of pictilisib (GDC-0941),an oral,potent,and selective inhibitor of the class I phosphatidylinositol-3-kinases (PI3K). PATIENTS AND METHODS: Sixty patients with solid tumors received pictilisib at 14 dose levels from 15 to 450 mg once-daily,initially on days 1 to 21 every 28 days and later,using continuous dosing for selected dose levels. Pharmacodynamic studies incorporated (18)F-FDG-PET,and assessment of phosphorylated AKT and S6 ribosomal protein in platelet-rich plasma (PRP) and tumor tissue. RESULTS: Pictilisib was well tolerated. The most common toxicities were grade 1-2 nausea,rash,and fatigue,whereas the DLT was grade 3 maculopapular rash (450 mg,2 of 3 patients; 330 mg,1 of 7 patients). The pharmacokinetic profile was dose-proportional and supported once-daily dosing. Levels of phosphorylated serine-473 AKT were suppressed textgreater90% in PRP at 3 hours after dose at the MTD and in tumor at pictilisib doses associated with AUC textgreater20 htextperiodcenteredμmol/L. Significant increase in plasma insulin and glucose levels,and textgreater25% decrease in (18)F-FDG uptake by PET in 7 of 32 evaluable patients confirmed target modulation. A patient with V600E BRAF-mutant melanoma and another with platinum-refractory epithelial ovarian cancer exhibiting PTEN loss and PIK3CA amplification demonstrated partial response by RECIST and GCIG-CA125 criteria,respectively. CONCLUSION: Pictilisib was safely administered with a dose-proportional pharmacokinetic profile,on-target pharmacodynamic activity at dose levels ≥100 mg and signs of antitumor activity. The recommended phase II dose was continuous dosing at 330 mg once-daily. View Publication

OBJECTIVES: Hematopoietic stroma promotes resistance to immune control by APO2L/TRAIL in multiple myeloma (MM) cells in part by increasing synthesis of the anti-apoptotic protein c-FLIP. Here,we tested whether bortezomib can reverse the APO2L/TRAIL environmental mediated-immune resistance (EM-IR). MATERIAL AND METHODS: MM cell lines (RPMI 8226 and U266) and CD138+ patient's MM cells were directly adhered to HS5 stroma exposed to HS5 or bone marrow stroma of patients with MM released soluble factors in a transwell system. Cells were treated with either APO2L/TRAIL (10 ng/mL),bortezomib (10 nm) or both. RESULTS: Pretreatment with bortezomib effectively overcomes APO2L/TRAIL apoptosis resistance in myeloma cell lines and in CD138+ cells while directly adhered or in transwell assay. Bortezomib was not cytotoxic to HS5 stroma cells and only altered monocyte chemotactic protein-2-3 and IL-10 levels in the stroma-myeloma milieu. Factors released by HS5 stroma increased expression of c-FLIP,induced STAT-3 and ERK phosphorylation and reduced DR4 receptor expression in MM cells. HS5 stroma-released factor(s) induced NF-kappaB activation after 20 h exposure in association with an enhanced c-FLIP transcription. Bortezomib effectively reduced c-FLIP protein expression without affecting other proteins. Bortezomib also increased DR4 and DR5 expression in the presence of stroma. CONCLUSIONS: These findings provide the rationale to combine bortezomib and APO2L/TRAIL to disrupt the influence of the stroma microenvironment on MM cells. View Publication

Creation of fusion genes by balanced chromosomal translocations is one of the hallmarks of acute myeloid leukemia (AML) and is considered one of the key leukemogenic events in this disease. In t(12;13)(p13;q12) AML,ectopic expression of the homeobox gene CDX2 was detected in addition to expression of the ETV6-CDX2 fusion gene,generated by the chromosomal translocation. Here we show in a murine model of t(12;13)(p13;q12) AML that myeloid leukemogenesis is induced by the ectopic expression of CDX2 and not by the ETV6-CDX2 chimeric gene. Mice transplanted with bone marrow cells retrovirally engineered to express Cdx2 rapidly succumbed to fatal and transplantable AML. The transforming capacity of Cdx2 depended on an intact homeodomain and the N-terminal transactivation domain. Transplantation of bone marrow cells expressing ETV6-CDX2 failed to induce leukemia. Furthermore,coexpression of ETV6-CDX2 and Cdx2 in bone marrow cells did not accelerate the course of disease in transplanted mice compared to Cdx2 alone. These data demonstrate that activation of a protooncogene by a balanced chromosomal translocation can be the pivotal leukemogenic event in AML,characterized by the expression of a leukemia-specific fusion gene. Furthermore,these findings link protooncogene activation to myeloid leukemogenesis,an oncogenic mechanism so far associated mainly with lymphoid leukemias and lymphomas. View Publication

Menin is the product of the tumor suppressor gene Men1 that is mutated in the inherited tumor syndrome multiple endocrine neoplasia type 1 (MEN1). Menin has been shown to interact with SET-1 domain-containing histone 3 lysine 4 (H3K4) methyltransferases including mixed lineage leukemia proteins to regulate homeobox (Hox) gene expression in vitro. Using conditional Men1 knockout mice,we have investigated the requirement for menin in hematopoiesis and myeloid transformation. Men1 excision causes reduction of Hoxa9 expression,colony formation by hematopoietic progenitors,and the peripheral white blood cell count. Menin directly activates Hoxa9 expression,at least in part,by binding to the Hoxa9 locus,facilitating methylation of H3K4,and recruiting the methylated H3K4 binding protein chd1 to the locus. Consistent with signaling downstream of menin,ectopic expression of both Hoxa9 and Meis1 rescues colony formation defects in Men1-excised bone marrow. Moreover,Men1 excision also suppresses proliferation of leukemogenic mixed lineage leukemia-AF9 fusion-protein-transformed myeloid cells and Hoxa9 expression. These studies uncover an important role for menin in both normal hematopoiesis and myeloid transformation and provide a mechanistic understanding of menin's function in these processes that may be used for therapy. View Publication

Combined deletion of chromosomes 1p and 19q is associated with improved prognosis and responsiveness to therapy in patients with anaplastic oligodendroglioma. The deletions usually involve whole chromosome arms,suggesting a t(1;19)(q10;p10). Using stem cell medium,we cultured a few tumors. Paraffin-embedded tissue was obtained from 21 Mayo Clinic patients and 98 patients enrolled in 2 North Central Cancer Treatment Group (NCCTG) low-grade glioma trials. Interphase fusion of CEP1 and 19p12 probes detected the t(1;19). 1p/19q deletions were evaluated by fluorescence in situ hybridization. Upon culture,one oligodendroglioma contained an unbalanced 45,XX,t(1;19)(q10;p10). CEP1/19p12 fusion was observed in all metaphases and 74% of interphase nuclei. Among Mayo Clinic oligodendrogliomas,the prevalence of fusion was 81%. Among NCCTG patients,CEP1/19p12 fusion prevalence was 55%,47%,and 0% among the oligodendrogliomas,mixed oligoastrocytomas,and astrocytomas,respectively. Ninety-one percent of NCCTG gliomas with 1p/19q deletion and 12% without 1p/19q deletion had CEP1/19p12 fusion (P textless 0.001,chi(2) test). The median overall survival (OS) for all patients was 8.1 years without fusion and 11.9 years with fusion (P = 0.003). The median OS for patients with low-grade oligodendroglioma was 9.1 years without fusion and 13.0 years with fusion (P = 0.01). Similar significant median OS differences were observed for patients with combined 1p/19q deletions. The absence of alterations was associated with a significantly shorter OS for patients who received higher doses of radiotherapy. Our results strongly suggest that a t(1;19)(q10;p10) mediates the combined 1p/19q deletion in human gliomas. Like combined 1p/19q deletion,the 1;19 translocation is associated with superior OS and progression-free survival in low-grade glioma patients. View Publication

The present study reports for the first time a dual antiglioma effect of the well-known antidiabetic drug metformin. In low-density cultures of the C6 rat glioma cell line,metformin blocked the cell cycle progression in G(0)/G(1) phase without inducing significant cell death. In confluent C6 cultures,on the other hand,metformin caused massive induction of caspase-dependent apoptosis associated with c-Jun N-terminal kinase (JNK) activation,mitochondrial depolarization and oxidative stress. Metformin-triggered apoptosis was completely prevented by agents that block mitochondrial permeability transition (cyclosporin A) and oxygen radical production (N-acetylcisteine),while the inhibitors of JNK activation (SP600125) or glycolysis (sodium fluoride,iodoacetate) provided partial protection. The antiglioma effect of metformin was reduced by compound C,an inhibitor of AMP-activated protein kinase (AMPK),and was mimicked by the AMPK agonist AICAR. Similar effects were observed in the human glioma cell line U251,while rat primary astrocytes were completely resistant to the antiproliferative and proapoptotic action of metformin. View Publication

The present study explored the sensitivity of leukaemic blasts derived from 30 acute myeloid leukaemia (AML) patients to Bortezomib. Bortezomib induced apoptosis of primary AML blasts: 18/30 AMLs were clearly sensitive to the proapoptotic effects of Bortezomib,while the remaining cases were moderately sensitive to this molecule. The addition of tumour necrosis factor-related-apoptosis-inducing ligand,when used alone,did not induce apoptosis of AML blasts and further potentiated the cytotoxic effects of Bortezomib. The majority of AMLs sensitive to Bortezomib showed immunophenotypic features of the M4 and M5 French-American-British classification subtypes and displayed myelomonocytic features. All AMLs with mutated FLT3 were in the Bortezomib-sensitive group. Biochemical studies showed that: (i) Bortezomib activated caspase-8 and caspase-3 and decreased cellular FLICE [Fas-associated death domain (FADD)-like interleukin-1beta-converting enzyme]-inhibitory protein (c-FLIP) levels in AML blasts; (ii) high c-FLIP levels in AML blasts were associated with low Bortezomib sensitivity. Finally,analysis of the effects of Bortezomib on leukaemic cells displaying high aldehyde dehydrogenase activity suggested that this drug induced in vitro killing of leukaemic stem cells. The findings of the present study,further support the development of Bortezomib as an anti-leukaemic drug and provide simple tools to predict the sensitivity of AML cells to this drug. View Publication

The molecular mechanisms by which tumor cells metastasize to bone are likely to involve invasion,cell adhesion to bone,and the release of soluble mediators from tumor cells that stimulate osteoclast-mediated bone resorption. Bisphosphonates (BPs) are powerful inhibitors of the osteoclast activity and are,therefore,used in the treatment of patients with osteolytic metastases. However,an added beneficial effect of BPs may be direct antitumor activity. We previously reported that BPs inhibit breast and prostate carcinoma cell adhesion to bone (Boissier et al.,Cancer Res.,57: 3890-3894,1997). Here,we provided evidence that BP pretreatment of breast and prostate carcinoma cells inhibited tumor cell invasion in a dose-dependent manner. The order of potency for four BPs in inhibiting tumor cell invasion was: zoledronate textgreater ibandronate textgreater NE-10244 (active pyridinium analogue of risedronate) textgreater clodronate. In addition,NE-58051 (the inactive pyridylpropylidene analogue of risedronate) had no inhibitory effect,whereas NE-10790 (a phosphonocarboxylate analogue of risedronate in which one of the phosphonate groups is substituted by a carboxyl group) inhibited tumor cell invasion to an extent similar to that observed with NE-10244,indicating that the inhibitory activity of BPs on tumor cells involved the R2 chain of the molecule. BPs did not induce apoptosis in tumor cells,nor did they inhibit tumor cell migration at concentrations that did inhibit tumor cell invasion. However,although BPs did not interfere with the production of matrix metalloproteinases (MMPs) by tumor cells,they inhibited their proteolytic activity. The inhibitory effect of BPs on MMP activity was completely reversed in the presence of an excess of zinc. In addition,NE-10790 did not inhibit MMP activity,suggesting that phosphonate groups of BPs are responsible for the chelation of zinc and the subsequent inhibition of MMP activity. In conclusion,our results provide evidence for a direct cellular effect of BPs in preventing tumor cell invasion and an inhibitory effect of BPs on the proteolytic activity of MMPs through zinc chelation. These results suggest,therefore,that BPs may be useful agents for the prophylactic treatment of patients with cancers that are known to preferentially metastasize to bone. View Publication

Primary sclerosing cholangitis (PSC),a chronic inflammatory liver disease characterized by progressive bile duct destruction,develops as an extra-intestinal complication of inflammatory bowel disease (IBD) (Chapman,R.W. 1991. Gut. 32:1433-1435). However,the liver and bowel inflammation are rarely concomitant,and PSC can develop in patients whose colons have been removed previously. We hypothesized that PSC is mediated by long-lived memory T cells originally activated in the gut,but able to mediate extra-intestinal inflammation in the absence of active IBD (Grant,A.J.,P.F. Lalor,M. Salmi,S. Jalkanen,and D.H. Adams. 2002. Lancet. 359:150-157). In support of this,we show that liver-infiltrating lymphocytes in PSC include mucosal T cells recruited to the liver by aberrant expression of the gut-specific chemokine CCL25 that activates alpha4beta7 binding to mucosal addressin cell adhesion molecule 1 on the hepatic endothelium. This is the first demonstration in humans that T cells activated in the gut can be recruited to an extra-intestinal site of disease and provides a paradigm to explain the pathogenesis of extra-intestinal complications of IBD. View Publication