An immunochromatographic assay (ICA) using gold nanoparticles coated with monoclonal antibody (McAb) for the detection of chromium ions (Cr) in water and serum samples was developed, optimized and validated. Gold nanoparticles coated with affinity-purified monoclonal antibodies against isothiocyanobenzyl-EDTA (iEDTA)-chelated Cr3+ were used as the detecting reagent in this completive immunoassay-based one-step test strip. The ICA was investigated to measure chromium speciation (Cr3+ and Cr6+ ions) in water samples. Chromium standard samples of 0-80 ng/mL in water were determined by the test strips. The results showed that the visual lowest detection limit (LDL) of the test strip was 50.0 ng/mL. A portable colorimetric lateral flow reader was used for the quantification of Cr. The results indicated that the linear range of the ICA with colorimetric detection was 5-80 ng/mL. The ICA was also validated for the detection of chromium ions in serum samples. The test trips showed high stability in that they could be stored at 37°C for at least 12 weeks without significant loss of activity. The test strip also showed good selectivity for Cr detection with negligible interference from other heavy metals. Because of its low cost and short testing time (within 5 min), the test strip is especially suitable for on-site large-scale screening of Cr-polluted water samples, biomonitoring of Cr exposure, and many other field applications.
Chromium ions; Gold nanoparticle; Immunochromatography assay; Rapid test; Quantification
Inflammatory myofibroblastic tumor (IMT) is a rare lesion of unclear pathogenesis that shows a wide, highly variable spectrum of clinical behavior. We describe the case of a 17-year-old boy with a large IMT that infiltrated the bladder, ileocecal junction, peritoneum and pelvic retroperitoneal space. The tumor was associated with extensive toughening and thickening of the bladder, and, although it showed a tendency for invasive growth, it affected mainly the bladder and adjacent tissue. To the best of our knowledge, this case report is the first to describe an IMT involving the entire bladder and several adjacent pelviabdominal organs. The bladder wall was tough and could hardly be cut by scalpel. Levels of inflammatory response markers such as C-reactive protein fell after surgery.
Bladder; Inflammatory myofibroblastic tumor
Background and Aims
Brain dysfunction in functional dyspepsia (FD) has been identified by multiple neuroimaging studies. This study aims to investigate the regional gray matter density (GMD) changes in meal-related FD patients and their correlations with clinical variables, and to explore the possible influence of the emotional state on FD patients’s brain structures.
Fifty meal-related FD patients and forty healthy subjects (HS) were included and underwent a structural magnetic resonance imaging scan. Voxel-based morphometry analysis was employed to identify the cerebral structure alterations in meal-related FD patients. Regional GMD changes' correlations with the symptoms and their durations, respectively, have been analyzed.
Compared to the HS, the meal-related FD patients showed a decreased GMD in the bilateral precentral gyrus, medial prefrontal cortex (MPFC), anterior cingulate cortex (ACC) and midcingulate cortex (MCC), left orbitofrontal cortex (OFC) and right insula (p<0.05, FWE Corrected, Cluster size>50). After controlling for anxiety and depression, the meal-related FD patients showed a decreased GMD in the bilateral middle frontal gyrus, left MCC, right precentral gyrus and insula (p<0.05, FWE Corrected, Cluster size>50). Before controlling psychological factors, the GMD decreases in the ACC were negatively associated with the symptom scores of the Nepean Dyspepsia Index (NDI) (r = −0.354, p = 0.048, Bonferroni correction) and the duration of FD (r = −0.398, p = 0.02, Bonferroni correction) respectively.
The regional GMD of meal-related FD patients, especially in the regions of the homeostatic afferent processing network significantly differed from that of the HS, and the psychological factors might be one of the essential factors significantly affecting the regional brain structure of meal-related FD patients.
Slow axonal transport conveys perikaryally-synthesized cytosolic proteins in a rate-class called Slow Component-b (SCb). One such protein – α-synuclein – is largely conveyed in SCb, and is also a key player in a group of neurodegenerative diseases called synucleinopathies. Axonal transport defects of α-synuclein have been hypothesized to play a role in synucleinopathies, but mechanisms moving α-synuclein in slow axonal transport are unclear. Here we use a recently developed model-system in our laboratory to visualize the slow transport of α-synuclein, comparing it to another SCb protein synapsin. Despite differences inbiological properties and overallsolubility in axons, the anterograde transport of both SCb proteins was strikingly similar, suggesting commonalities in slow axonal transport mechanisms of seemingly diverse cytosolic cargoes. The data support a model where SCb proteins dynamically organize into ‘transport-competent’ complexes that are conveyed via transient associations with other persistently-moving cargoes (“mobile-units”). The identity of the latter is yet unknown. Visualizing normal α-synuclein transport may also open the door to studies of α-synuclein transport in pathologic states.
Cytosolic synaptic proteins; synapsin; α-synuclein; slow axonal transport; transport packets; cargo complexes; diffusion; slow component – b; SCb
The present studies were designed to determine whether the multi-kinase inhibitor sorafenib (Nexavar) interacted with histone deacetylase inhibitors to kill glioblastoma and medulloblastoma cells. In a dose-dependent fashion sorafenib lethality was enhanced in multiple genetically disparate primary human glioblastoma isolates by the HDAC inhibitor sodium valproate (Depakote). Drug exposure reduced phosphorylation of p70 S6K and of mTOR. Similar data to that with valproate were also obtained using the HDAC inhibitor vorinostat (Zolinza). Sorafenib and valproate also interacted to kill medulloblastoma and PNET cell lines. Treatment with sorafenib and HDAC inhibitors radio-sensitized both GBM and medulloblastoma cell lines. Knock down of death receptor (CD95) expression protected GBM cells from the drug combination, as did overexpression of c-FLIP-s, BCL-XL and dominant negative caspase 9. Knock down of PDGFRα recapitulated the effect of sorafenib in combination with HDAC inhibitors. Collectively, our data demonstrate that the combination of sorafenib and HDAC inhibitors kills through activation of the extrinsic pathway, and could represent a useful approach to treat CNS-derived tumors.
HDAC inhibitor; Sorafenib; apoptosis; glioma
Chemotherapy is one of the three most common treatment modalities for cancer. However, its efficacy is limited by multidrug resistant cancer cells. Drug metabolizing enzymes (DMEs) and efflux transporters promote the metabolism, elimination, and detoxification of chemotherapeutic agents. Consequently, elevated levels of DMEs and efflux transporters reduce the therapeutic effectiveness of chemotheraputics and, often, lead to treatment failure. Nuclear receptors, especially pregnane X receptor (PXR, NR1I2) and constitutive androstane activated receptor (CAR, NR1I3), are increasingly recognized for their role in xenobiotic metabolism and clearance as well as their role in the development of multidrug resistance (MDR) during chemotherapy. Promiscuous xenobiotic receptors, including PXR and CAR, govern the inducible expressions of a broad spectrum of target genes that encode phase I DMEs, phase II DMEs, and efflux transporters. Recent studies conducted by a number of groups, including ours, have revealed that PXR and CAR play pivotal roles in the development of MDR in various human carcinomas, including prostate, colon, ovarian, and esophageal squamous cell carcinomas. Accordingly, PXR/CAR expression levels and/or activation statuses may predict prognosis and identify the risk of drug resistance in patients subjected to chemotherapy. Further, PXR/CAR antagonists, when used in combination with existing chemotherapeutics that activate PXR/CAR, are feasible and promising options that could be utilized to overcome or, at least, attenuate MDR in cancer cells.
The present studies were initiated to determine whether inhibitors of MEK1/2 or SRC signaling, respectively, enhance CHK1 inhibitor lethality in primary human glioblastoma cells. Multiple MEK1/2 inhibitors (CI-1040 (PD184352); AZD6244 (ARRY-142886)) interacted with multiple CHK1 inhibitors (UCN-01, AZD7762) to kill multiple primary human glioma cell isolates that have a diverse set of genetic alterations typically found in the disease. Inhibition of SRC family proteins also enhanced CHK1 inhibitor lethality. Combined treatment of glioma cells with (MEK1/2 + CHK1) inhibitors enhanced radiosensitivity. Combined (MEK1/2 + CHK1) inhibitor treatment led to dephosphorylation of ERK1/2 and S6 ribosomal protein, whereas the phosphorylation of JNK and p38 was increased. MEK1/2 + CHK1 inhibitor-stimulated cell death was associated with the cleavage of pro-caspases 3 and 7 as well as the caspase substrate (PARP). We also observed activation of pro-apoptotic BCL-2 effector proteins BAK and BAX and reduced levels of pro-survival BCL-2 family protein BCL-XL. Overexpression of BCL-XL alleviated but did not completely abolish MEK1/2 + CHK1 inhibitor cytotoxicity in GBM cells. These findings argue that multiple inhibitors of the SRC-MEK pathway have the potential to interact with multiple CHK1 inhibitors to kill glioma cells.
Apoptosis; CHK 1 inhibitor; glioma; MEK1/2 inhibitors
The mammalian HIRA/UBN1/CABIN1/ASF1a (HUCA) histone chaperone complex deposits the histone H3 variant H3.3 into chromatin, and is linked to gene activation, repression and chromatin assembly in diverse cell contexts. We recently reported that a short N-terminal fragment of UBN1 containing amino acids 1–175 is necessary and sufficient for interaction with the WD repeats of HIRA, and attributed this interaction to a region from residues 120–175 that is highly conserved in a yeast ortholog Hpc2 and so termed the HRD for Hpc2-Related Domain. In this report, through a more comprehensive and refined biochemical and mutational analysis, we identify a smaller and more moderately conserved region within residues 41–77 of UBN1, that we term the NHRD, that is essential for interaction with the HIRA WD repeats; we further demonstrate that the HRD is dispensable for this interaction. We employ analytical ultracentrifugation studies to demonstrate that the NHRD of UBN1 and the WD repeats of HIRA form a tight 1:1 complex with a dissociation constant in the nanomolar range. Mutagenesis experiments identify several key residues in the NHRD that are required for interaction with the HIRA WD repeat domain, stability of the HUCA complex in vitro and in vivo and changes in chromatin organization in primary human cells. Together, these studies implicate the NHRD domain of UBN1 as being an essential region for HIRA interaction and chromatin organization by the HUCA complex.
HIRA; UBN1; Histone Deposition; Chromatin Regulation
The downstream targets of amyloid β (Aβ)-oligomers remain elusive. One hypothesis is that Aβ-oligomers interrupt axonal transport. Although previous studies have demonstrated Aβ-induced transport blockade, early effects of low-n soluble Aβ-oligomers on axonal transport remain unclear. Furthermore, the cargo selectivity for such deficits (if any) or the specific effects of Aβ on the motility kinetics of transported cargoes are also unknown. Toward this, we visualized axonal transport of vesicles in cultured hippocampal neurons treated with picomolar (pm) levels of cell-derived soluble Aβ-oligomers. We examined select cargoes thought to move as distinct organelles and established imaging parameters that allow organelle tracking with consistency and high fidelity – analyzing all data in a blinded fashion. Aβ-oligomers induced early and selective diminutions in velocities of synaptic cargoes but had no effect on mitochondrial motility, contrary to previous reports. These changes were N-methyl d-aspartate receptor/glycogen synthase kinase-3β dependent and reversible upon washout of the oligomers. Cluster-mode analyses reveal selective attenuations in faster-moving synaptic vesicles, suggesting possible decreases in cargo/motor associations, and biochemical experiments implicate tau phosphorylation in the process. Collectively, the data provide a biological basis for Aβ-induced axonal transport deficits.
amyloid β-oligomers; axonal transport; cargo-motor regulation; molecular motors; synaptic loss; transport packets
We describe a strategy for analyzing axonal transport of cytosolic proteins (cps) using photoactivatable GFp—paGFp—with modifications of standard imaging components that can be retroactively fitted to a conventional epifluorescence microscope. the photoactivation and visualization are nearly simultaneous, allowing studies of proteins with rapidly mobile fractions. cultured hippocampal neurons are transfected with paGFp-tagged constructs, a discrete protein population within axons is photoactivated, and then the activated population is tracked by live imaging. We show the utility of this method in analyzing axonal transport of cps that have inherent diffusible pools and distinguish this transport modality from passive diffusion and vesicle transport. the analytical tools used to quantify the motion are also described. aside from the time needed for preparation of neuronal cultures/transfection, the experiment takes 2–3 h, during which time several axons can be imaged and analyzed. these methods should be easy to adopt by most laboratories and may also be useful for monitoring cp movement in other cell types.
The aim of this study was to investigate the effect of the μ-opioid receptor gene (OPRM1) A118G polymorphism on the requirement for post-operative fentanyl analgesia in patients undergoing radical gastrectomy. One hundred and twenty-eight gastric cancer patients scheduled to undergo radical gastrectomy under general anesthesia were enrolled in the study. Post-operative, patient-controlled intravenous analgesia of fentanyl was provided for satisfactory analgesia until 48 h after surgery. OPRM1 A118G was screened by DNA sequence analysis of polymerase chain reaction (PCR)-amplified DNA. Differences in fentanyl consumption and adverse effects were compared among the different genotypes at 24 and 48 h after surgery. The ranges of fentanyl dose in the 128 patients at 24 and 48 h after surgery were 5.4–17.3 μg/kg and 12.4–29.9 μg/kg, respectively. Among these patients, there were 54 wild-type homozygotes (AA), 53 heterozygotes (AG) and 21 mutant homozygotes (GG). The frequency of the G allele was 0.371 in the OPRM1 polymorphism. There were no significant differences in fentanyl dose or adverse effects, including nausea, vomiting and dizziness, for the OPRM1 A118G polymorphism (P>0.05). The OPRM1 A118G polymorphism does not play a significant role in post-operative fentanyl analgesic dose or post-operative nausea, vomiting and dizziness in patients undergoing radical gastrectomy.
fentanyl; analgesia; mu opioid receptors; single nucleotide polymorphism
Severe aplastic anemia, which is characterized by immune-mediated bone marrow hypoplasia and pancytopenia, can be treated effectively with immunosuppressive therapy or allogeneic transplantation. One third of patients have disease that is refractory to immunosuppression, with persistent, severe cytopenia and a profound deficit in hematopoietic stem cells and progenitor cells. Thrombopoietin may increase the number of hematopoietic stem cells and progenitor cells.
We conducted a phase 2 study involving patients with aplastic anemia that was refractory to immunosuppression to determine whether the oral thrombopoietin mimetic eltrombopag (Promacta) can improve blood counts. Twenty-five patients received eltrombopag at a dose of 50 mg, which could be increased, as needed, to a maximum dose of 150 mg daily, for a total of 12 weeks. Primary end points were clinically significant changes in blood counts or transfusion independence. Patients with a response continued to receive eltrombopag.
Eleven of 25 patients (44%) had a hematologic response in at least one lineage at 12 weeks, with minimal toxic effects. Nine patients no longer needed platelet transfusions (median increase in platelet count, 44,000 per cubic millimeter). Six patients had improved hemoglobin levels (median increase, 4.4 g per deciliter); 3 of them were previously dependent on red-cell transfusions and no longer needed transfusions. Nine patients had increased neutrophil counts (median increase, 1350 per cubic millimeter). Serial bone marrow biopsies showed normalization of trilineage hematopoiesis in patients who had a response, without increased fibrosis. Monitoring of immune function revealed no consistent changes.
Treatment with eltrombopag was associated with multilineage clinical responses in some patients with refractory severe aplastic anemia. (Funded by the National Heart, Lung, and Blood Institute; ClinicalTrials.gov number, NCT00922883.)
Toll-like receptors are potent activators of the innate immune system and generate signals leading to the initiation of the adaptive immune response that can be utilized for therapeutic purposes. We tested the hypothesis that combined treatment with a toll-like receptor agonist and an anti-tumor monoclonal antibody is effective and induces host-protective anti-tumor immunity. C57BL/6 human mutated HER2 (hmHER2) transgenic mice that constitutively express kinase-deficient human HER2 under control of the CMV promoter were established. These mice demonstrate immunological tolerance to D5-HER2, a syngeneic human HER2-expressing melanoma cell line. This human HER2 tolerant model offers the potential to serve as a preclinical model to test both antibody therapy and the immunization potential of human HER2 targeted therapeutics. Here we show that E6020, a toll like receptor-4 (TLR4) agonist effectively boosted the antitumor efficacy of the monoclonal antibody trastuzumab in immunodeficient C57BL/6 SCID mice as well as in C57BL/6 hmHER2 transgenic mice. E6020 and trastuzumab co-treatment resulted in significantly greater inhibition of tumor growth than was observed with either agent individually. Furthermore, mice treated with the combination of trastuzumab and the TLR4 agonist were protected against re-challenge with human HER2 transfected tumor cells in hmHER2 transgenic mouse strains. These findings suggest that combined treatment with trastuzumab and a TLR4 agonist not only promotes direct anti-tumor effects but also induces a host-protective human HER2-directed adaptive immune response indicative of a memory response. These data provide an immunological rationale for testing TLR4 agonists in combination with antibody therapy in patients with cancer.
toll like receptor 4 agonist; HER2; antibody therapy; transgenic mouse; antitumor immunity
The present study was designed to investigate the pattern of time-dependent expression of peroxisome proliferator-activated receptors (PPARα, β, and γ) after global cerebral ischemia and reperfusion (I/R) damage in the rat hippocampus. Male Sprague Dawley (SD) rats were subjected to global cerebral I/R. The rat hippocampi were isolated to detect the expression of PPARs mRNA and protein levels at 30 min–30 d after I/R by RT-PCR and Western blot analysis, respectively. The expression levels of PPARs mRNA and protein in the rat hippocampus significantly increased and peaked at 24 h for PPARα and γ (at 48 h for PPARβ) after I/R, then gradually decreased, and finally approached control levels on d 30. The present results suggest that global cerebral I/R can cause obvious increases of hippocampal PPARs mRNA and protein expression within 15 d after I/R. These findings may help to guide the experimental and clinical therapeutic use of PPARs agonists against brain injury.
Isocitrate dehydrogenase 1 (IDH1) gene aberrations have recently been reported in acute myeloid leukemia (AML). To evaluate the prognostic significance of IDH1 mutations in AML, we performed a meta-analysis. Fifteen studies covering a total of 8121 subjects were included in this analysis. The frequency of IDH1 R132 mutations were 4.4–9.3% for AML patients and 10.9–16.0% for cytogenetically normal (CN)-AML patients. The IDH1 mutations were associated with NPM1 mutations in 6 studies and normal cytogenetics in 5 studies. AML patients with IDH1 mutations had inferior overall survival compared to patients without the mutations (hazard ratio 1.17, 95% CI: 1.02–1.36). Additionally, in CN-AML patients, IDH1 mutations were associated with a lower complete remission rate (risk ratio 1.30, 95% CI: 1.04–1.63). Although the available literature is limited to observational studies, these results may justify the risk-adapted therapeutic strategies for AML according to the IDH1 status.
Acute myeloid leukemia; IDH1; mutation; prognosis; meta-analysis
The theory of acupoint specificity is the basis for elucidating the actions of acupoints as employed in clinical practice. Acupoint specificity has become a focus of attention in international research efforts by scholars in the areas of acupuncture and moxibustion. In 2006, the Chinese Ministry of Science approved and initiated the National Basic Research Program (973 Program), one area of which was entitled Basic Research on Acupoint Specificity Based on Clinical Efficacy. Using such approaches as data mining, evidence-based medicine, clinical epidemiology, neuroimaging, molecular biology, neurophysiology, and metabolomics, fruitful research has been conducted in the form of literature research, clinical assessments, and biological studies. Acupoint specificity has been proved to exist, and it features meridian-propagated, relative, persistent, and conditional effects. Preliminarily investigations have been made into the biological basis for acupoint specificity.
Mesothelin, a secreted protein, is overexpressed in some cancers, including pancreatic cancer. Rescent studies have shown that overexpression of mesothelin significantly increased tumor cell proliferation, and downregulation of mesothelin inhibited cell proliferation in pancreatic cancer cells, but its exact function and mechanism remains unclear. The aim of the present study was to evaluate the effects of mesothelin on proliferation and apoptosis in pancreatic cancer cells with different p53 status and to explore its signal pathway. Mesothelin levels were detected by western blot and RT-PCR assay in human pancreatic cancer AsPC-1, HPAC and Capan-2, Capan-1 and MIA PaCa-2 cell lines. Mesothelin was slienced by shRNA in AsPC-1, Capan-2 and Capan-1 cells with rich mesothelin level, and mesothelin was overexpressed in the HPAC and Capan-2 cells with less mesothelin level. We observed that in the AsPC-1 and Capan-1cells with mt-p53, and Capan-2 cells with wt-p53, shRNA mediated sliencing of the mesothelin significantly increased PUMA and Bax expression and caspase-3 activity, and decreased bcl-2 expression, followed by the reduced proliferation and colony forming capability and increased cell apoptosis. When PUMA was slienced by siRNA in the stable mesothelin shRNA transfected cells, proliferative capability was significantly increased, and apoptosis was decreased. However, in the Capan-2 cells with wt-p53, suppression of the mesothelin significantly increased wt-p53 levels. When p53 was blocked by siRNA in the stable mesothelin shRNA transfected Capan-2 cells, PUMA was inhibited, followed by increased proliferative capability and decreased cell apoptosis. In the HPAC and Capan-2 cells with wt-p53 and in the MIA PaCa-2 cells with mt-p53, overexpression of the mesothelin significantly decreased bax levels and increased bcl-2 levels, followed by increased proliferative and colony forming capability. Furthermore, mesothelin-shRNA-transfected cells exhibited a reduced rate of tumor growth under in vivo conditions. However, mesothelin-transfected cells exhibited a increased rate of tumor growth under in vivo conditions. Our data demonstrated that mesothelin promotes proliferation and inhibited apoptosis through p53-dependent pathway in pancreatic cancer cells with wt-p53, and p53-independent pathway in pancreatic cancer cells with mt-p53. Targeting mesothelin by shRNA is the important method for pancreatic cancer therapy.
Pancreatic cancer; Proliferation, apoptosis; Mesothelin; P53
It has been proven that nuclear factor-kappa B (NF-κB) is activated as a well-known transcription factor after subarachnoid hemorrhage (SAH). However, the panoramic view of NF-κB activity after SAH remained obscure. Cultured neurons were signed into control group and six hemoglobin- (Hb-) incubated groups. One-hemorrhage rabbit SAH model was produced, and the rabbits were divided randomly into one control group and five SAH groups. NF-κB activity was detected by electrophoretic mobility shift assay (EMSA) and immunohistochemistry. Real-time polymerase chain reaction (PCR) was performed to assess the downstream genes of NF-κB. NeuN immunofluorescence and lactate dehydrogenase (LDH) quantification were used to estimate the neuron injury. Double drastically elevated NF-κB activity peaks were detected in rabbit brains and cultured neurons. The downstream gene expressions showed an accordant phase peaks. NeuN-positive cells decreased significantly in day 3 and day 10 groups. LDH leakage exhibited a significant increase in Hb-incubated groups, but no significant difference was found between the Hb incubated groups. These results suggested that biphasic increasing of NF-κB activity was induced after SAH, and the early NF-κB activity peak indicated the injury role on neurons; however, the late peak might not be involved in the deteriorated effect on neurons.
The aim of this study was to investigate the molecular mechanism of 8-chloroadenosine 3′,5′-monophosphate (8-Cl-cAMP) in the inhibition of the growth and induction of apoptosis of multiple myeloma (MM) cells. Two MM-derived cell lines, RPMI-8226 and U266, were used. Cell viability, apoptosis induction and mitochondrial transmembrane potential were determined and the expression levels of cell cycle regulatory proteins (Cdk2, cyclin E, p27 and c-myc) and p38 mitogen-activated protein kinase (MAPK) protein were detected. Following treatment with 8-Cl-cAMP, the percentage of apoptotic cells increased in a concentration- and time-dependent manner and the mitochondrial transmembrane potential collapsed to reveal typical apoptotic features. Our data further demonstrated that 8-Cl-cAMP induced progressive phosphorylation of p38 MAPK and that the expression levels of p27 proteins in the MM cells were increased whereas those of c-myc were significantly decreased. Notably, the proapoptotic effect of 8-Cl-cAMP was largely prevented by a p38 MAPK inhibitor. Furthermore, knockdown of p27 was able to decrease the 8-Cl-cAMP-induced apoptosis in the MM cells. These results indicate that 8-Cl-cAMP induced p27-dependent cell cycle arrest and apoptosis in the MM cells, which demonstrates the potential of cAMP-modulating agents for use in the treatment of MM.
8-chloroadenosine 3′,5′-monophosphate; multiple myeloma; apoptosis; p27; cell cycle regulators
To study the hypothesis that gemcitabine treatment augments the chemoresistance to gemcitabine by clusterin (sCLU) upregulation. Clusterin inhibition could augment the chemosensitivity of human pancreatic cancer cells by inhibition of clusterin-dependent pERK1/2 activation.
Clusterin was silenced by serial concentration of OGX-011 transfection in pancreatic cancer MIAPaCa-2 and BxPC-3 cell lines, then treated with serial concentration of gemcitabine. After the cells were treated with OGX-011 for 8 h, the cells were then treated with 5 μM ERK inhibitor PD98059 for 18 h or transfected with a wt-pERK-expressing plasmid into these cells for 24 h, after which the cells were treated with 1.0 uM gemcitabine for 24–72 h. Cell proliferation was determined by MTT. Apoptosis was quantified by flow cytometry,.sCLU and pERK1/2 production was analyzed by western blot, and sCLU mRNA was analyzed by RT-PCR. Xenograft of established tumors was used to evaluate primary tumor growth and apoptosis after treatment with gemcitabine alone or in combination with OGX-011. Phosphorylated ERK1/2 and sCLU levels in tumor tissues were measured by TUNEL analysis.
As detected by MTT and FACS assay, a combination of gemcitabine + OGX-011 reflected the chemotherapeutic sensitivity and increased the gemcitabine -induced apoptosis in MIAPaCa-2 and BxPC-3 cells. Western blotting and RT-PCR analysis revealed that the expression of clusterin was higher in gemcitabine -resistant MIAPaCa-2 cells, however, decreased significantly after pretreatment with OGX-011. Furthermore, the OGX-011 or combination of gemcitabine + OGX-011 decreased the gemcitabine -induced activation of pERK1/2. wt-pERK-re-expression decreased OGX-011+ gemcitabine -induced apoptosis. Finally, OGX-011 in combination with gemcitabine substantially decreased the in vivo tumor growth and promoted apoptosis. Taken together, clusterin confers gmcitabine resistance in pancreatic cancer cells.
Knockdown of clusterin by OGX-011 transfection sensitizes pancreatic cancer cells to gemcitabine by inhibition of gemcitabine -induced clusterin-pERK1/2 activation.
Pancreatic cancer; Chemoresistance; Gemcitabine; Gene treatment; Clusterin;ERK1/2
The present studies were initiated to determine in greater molecular detail the regulation of CHK1 inhibitor lethality in transfected and infected breast cancer cells and using genetic models of transformed fibrobalsts. Multiple MEK1/2 inhibitors (PD184352, AZD6244 [ARRY-142886]) interacted with multiple CHK1 inhibitors (UCN-01 [7-hydroxystaurosporine], AZD7762) to kill mammary carcinoma cells and transformed fibroblasts. In transformed cells, CHK1 inhibitor-induced activation of ERK1/2 was dependent upon activation of SRC family non-receptor tyrosine kinases as judged by use of multiple SRC kinase inhibitors (PP 2, Dasatinib; AZD0530), use of SRC/FYN/YES deleted transformed fibroblasts or by expression of dominant negative SRC. Cell killing by SRC family kinase inhibitors and CHK1 inhibitors was abolished in BAX/BAK−/− transformed fibroblasts and suppressed by overexpression of BCL-XL. Treatment of cells with BCL-2/BCL-XL antagonists promoted SRC inhibitor + CHK1 inhibitor-induced lethality in a BAX/BAK-dependent fashion. Treatment of cells with [SRC + CHK1] inhibitors radio-sensitized tumor cells. These findings argue that multiple inhibitors of the SRC-RAS-MEK pathway interact with multiple CHK1 inhibitors to kill transformed cells.
CHK1; SRC; apoptosis; breast cancer; kinase; therapeutics; intrinsic; caspase
To investigate the efficacy and safety of regional intra-arterial chemotherapy (RIAC) versus systemic chemotherapy for stage III/IV pancreatic cancer.
Randomized controlled trials of patients with advanced pancreatic cancer treated by regional intra-arterial or systemic chemotherapy were identified using PubMed, ISI, EMBASE, Cochrane Library, Google, Chinese Scientific Journals Database (VIP), and China National Knowledge Infrastructure (CNKI) electronic databases, for all publications dated between 1960 and December 31, 2010. Data was independently extracted by two reviewers. Odds ratios and relative risks were pooled using either fixed- or random-effects models, depending on I2 statistic and Q test assessments of heterogeneity. Statistical analysis was performed using RevMan 5.0.
Six randomized controlled trials comprised of 298 patients met the standards for inclusion in the meta-analysis, among 492 articles that were identified. Eight patients achieved complete remission (CR) with regional intra-arterial chemotherapy (RIAC), whereas no patients achieved CR with systemic chemotherapy. Compared with systemic chemotherapy, patients receiving RIAC had superior partial remissions (RR = 1.99, 95% CI: 1.50, 2.65; 58.06% with RIAC and 29.37% with systemic treatment), clinical benefits (RR = 2.34, 95% CI: 1.84, 2.97; 78.06% with RAIC and 29.37% with systemic treatment), total complication rates (RR = 0.72, 95% CI: 0.60, 0.87; 49.03% with RIAC and 71.33% with systemic treatment), and hematological side effects (RR = 0.76, 95% CI: 0.63, 0.91; 60.87% with RIAC and 85.71% with systemic treatment). The median survival time with RIAC (5–21 months) was longer than for systemic chemotherapy (2.7–14 months). Similarly, one year survival rates with RIAC (28.6%−41.2%) were higher than with systemic chemotherapy (0%−12.9%.).
Regional intra-arterial chemotherapy is more effective and has fewer complications than systemic chemotherapy for treating advanced pancreatic cancer.
Pemetrexed (ALIMTA) is a folate anti-metabolite that has been approved for the treatment of non-small cell lung cancer, and has been shown to stimulate autophagy. In the present study, we sought to further understand the role of autophagy in the response to pemetrexed and to test if combination therapy could enhance the level of toxicity through altered autophagy in tumor cells. The multi-kinase inhibitor sorafenib (NEXAVAR), used in the treatment of renal and hepatocellular carcinoma, suppresses tumor angiogenesis and promotes autophagy in tumor cells. We found that sorafenib interacted in a greater than additive fashion with pemetrexed to increase autophagy and to kill a diverse array of tumor cell types. Tumor cell types that displayed high levels of cell killing after combination treatment showed elevated levels of AKT, p70 S6K and/or phosphorylated mTOR, in addition to Class III RTKs such as PDGFRβ and VEGFR1, known in vivo targets of sorafenib. In xenograft and in syngeneic animal models of mammary carcinoma and glioblastoma, the combination of sorafenib and pemetrexed suppressed tumor growth without deleterious effects on normal tissues or animal body mass. Taken together, the data suggest that premexetred and sorafenib act synergistically to enhance tumor killing via the promotion of a toxic form of autophagy that leads to activation of the intrinsic apoptosis pathway, and predict that combination treatment represents a future therapeutic option in the treatment of solid tumors.
Drug resistance is a significant barrier to an effective treatment of breast cancer. Human pregnane X receptor (hPXR), an orphan nuclear receptor known for its activation by many important clinical drugs, is a major transcription factor of drug metabolism enzymes (DMEs), such as cytochrome P450 3A4 (CYP3A4), and efflux transporters such as multi-drug resistance gene (MDR1). hPXR has been detected in human breast cancers but its role in responses of cancers toward drugs remains unknown. In this study, hPXR expression was confirmed in breast cancer cell lines and in normal and cancerous human breast specimens. Preactivation of hPXR by SR12813 in MDA-MB-231 cells led to an increased resistance to Taxol at concentrations of 20 and 50 nmol/L. A significant increase in resistance toward tamoxifen was also observed in MCF-7 with hPXR preactivation. Activation of hPXR led to an increased expression of CYP3A4 and MDR1, two possible mediators for hPXR-mediated drug resistance in breast cancers. Furthermore, knockdown of hPXR via small hairpin RNA (shRNA) sensitized MDA-MB-231 and MCF-7 cells to the treatment of Taxol, vinblastine or tamoxifen. The reduction in resistance of hPXR knockdown cells was further confirmed by reduced colony formation under the pressure of cancer treatment drugs. Taken together, our data suggest a potential role of hPXR in breast cancer resistance to drug treatments.
pregnane X receptor; steroid and xenobiotic receptor; breast cancer; chemotherapy; drug resistance; taxol; tamoxifen; vinblastine; drug metabolism enzymes; transcriptional regulation