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.
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
Traditionally, ecologists use lattice (regional summary) count data to simulate tree species distributions to explore species coexistence. However, no previous study has explicitly compared the difference between using lattice count and basal area data and analyzed species distributions at both individual species and community levels while simultaneously considering the combined scenarios of life stage and scale. In this study, we hypothesized that basal area data are more closely related to environmental variables than are count data because of strong environmental filtering effects. We also address the contribution of niche and the neutral (i.e., solely dependent on distance) factors to species distributions. Specifically, we separately modeled count data and basal area data while considering life stage and scale effects at the two levels with simultaneous autoregressive models and variation partitioning. A principal coordinates of neighbor matrix (PCNM) was used to model neutral spatial effects at the community level. The explained variations of species distribution data did not differ significantly between the two types of data at either the individual species level or the community level, indicating that the two types of data can be used nearly identically to model species distributions. Neutral spatial effects represented by spatial autoregressive parameters and the PCNM eigenfunctions drove species distributions on multiple scales, different life stages and individual species and community levels in this plot. We concluded that strong neutral spatial effects are the principal mechanisms underlying the species distributions and thus shape biodiversity spatial patterns.
Proteins vital to presynaptic function are synthesized in the neuronal perikarya and delivered into synapses via two modes of axonal transport. While membrane-anchoring proteins are conveyed in fast axonal transport via motor-driven vesicles, cytosolic proteins travel in slow axonal transport; via mechanisms that are poorly understood. We found that in cultured axons, populations of cytosolic proteins tagged to photoactivable-GFP (PA-GFP) move with a slow motor-dependent anterograde bias; distinct from vesicular-trafficking or diffusion of untagged PA-GFP. The overall bias is likely generated by an intricate particle-kinetics involving transient assembly and short-range vectorial spurts. In-vivo biochemical studies reveal that cytosolic proteins are organized into higher-order structures within axon-enriched fractions that are largely segregated from vesicles. Data-driven biophysical modeling best predicts a scenario where soluble molecules dynamically assemble into mobile supra-molecular structures. We propose a model where cytosolic proteins are transported by dynamically assembling into multi-protein complexes that are directly/indirectly conveyed by motors.
Cytosolic synaptic proteins; synapsin; CamKII; slow axonal transport; transport packets; cargo complexes; diffusion
The mammalian HIRA/UBN1/ASF1a complex is a histone chaperone complex that is conserved from yeast (Saccharomyces cerevisiae) to humans. This complex preferentially deposits the histone variant H3.3 into chromatin in a DNA replication-independent manner and is implicated in diverse chromatin regulatory events from gene activation to heterochromatinization. In yeast, the orthologous complex consists of three Hir proteins (Hir1p, Hir2p, and Hir3p), Hpc2p, and Asf1p. Yeast Hir3p has weak homology to CABIN1, a fourth member of the human complex, suggesting that Hir3p and CABIN1 may be orthologs. Here we show that HIRA and CABIN1 interact at ectopic and endogenous levels of expression in cells, and we isolate the quaternary HIRA/UBN1/CABIN1/ASF1a (HUCA) complex, assembled from recombinant proteins. Mutational analyses support the view that HIRA acts as a scaffold to bring together UBN1, ASF1a, and CABIN1 into a quaternary complex. We show that, like HIRA, UBN1, and ASF1a, CABIN1 is involved in heterochromatinization of the genome of senescent human cells. Moreover, in proliferating cells, HIRA and CABIN1 regulate overlapping sets of genes, and these genes are enriched in the histone variant H3.3. In sum, these data demonstrate that CABIN1 is a functional member of the human HUCA complex and so is the likely ortholog of yeast Hir3p.
Antibody drugs are widely used in cancer therapy, but conditions to maximize tumor penetration and efficacy have yet to be fully elucidated. In this study, we investigated the impact of antibody binding affinity on tumor targeting and penetration with affinity variants that recognize the same epitope. Specifically, we compared four derivatives of the C6.5 monoclonal antibody (MAb) which recognizes the same HER2 epitope (monovalent KDs ranging from 270nM to 0.56nM). Moderate affinity was associated with the highest tumor accumulation at 24hr and 120hr post i.v. injection, whereas high affinity was found to produce the lowest tumor accumulation. Highest affinity MAb were confined to the perivascular space of tumors with an average penetration of 20.4 +/− 7.5 microns from tumor blood vessels. Conversely, lowest affinity MAb exhibited a broader distribution pattern with an average penetration of 84.8 +/− 12.8 microns. In vitro internalization assays revealed that antibody internalization and catabolism generally increased with affinity, plateauing once the rate of HER2 internalization exceeded the rate of antibody dissociation. Effects of internalization and catabolism on tumor targeting were further examined using antibodies of moderate (C6.5) or high affinity (trastuzumab) labeled with residualizing (111In-labeled) or non-residualizing (125I-labeled) radioisotopes. Significant amounts of antibody of both affinities were degraded by tumors in vivo. Further, moderate to high affinity MAbs targeting the same HER2 epitope with monovalent affinity above 23nM had equal tumor accumulation of residualizing radiolabel over 120hrs. Results indicated equal tumor exposure, suggesting that MAb penetration and retention in tumors reflected affinity-based differences in tumor catabolism. Together, these results suggest that high-density, rapidly internalizing antigens subject high-affinity antibodies to greater internalization and degradation, thereby limiting their penetration of tumors. In contrast, lower affinity antibodies penetrate tumors more effectively when rates of antibody-antigen dissociation are higher than rates of antigen internalization. Together, our findings offer insights into how to optimize the ability of therapeutic antibodies to penetrate tumors.
Yeast Rtt109 promotes nucleosome assembly and genome stability by acetylating K9, K27 and K56 of histone H3 through interaction with either of two distinct histone chaperones, Vps75 or Asf1. We report the crystal structure of an Rtt109-AcCoA/Vps75 complex revealing an elongated Vps75 homodimer bound to two globular Rtt109 molecules to form a symmetrical holoenzyme with a ~12 Å diameter central hole. Vps75 and Rtt109 residues that mediate complex formation in the crystals are also important for Rtt109-Vps75 interaction and H3K9/K27 acetylation both in vitro and in yeast cells. The same Rtt109 residues do not participate in Asf1-mediated Rtt109 acetylation in vitro or H3K56 acetylation in yeast cells, demonstrating that Asf1 and Vps75 dictate Rtt109 substrate specificity through distinct mechanisms. These studies also suggest that Vps75 binding stimulates Rtt109 catalytic activity by appropriately presenting the H3–H4 substrate within the central cavity of the holoenzyme to promote H3K9/K27 acetylation of new histones prior to deposition.
Retinoblastoma gene (Rb1) is required for proper cell cycle exit in the developing mouse inner ear and its deletion in the embryo leads to proliferation of sensory progenitor cells that differentiate into hair cells and supporting cells. In a conditional hair cell Rb1 knockout mouse, Pou4f3-Cre-pRb™/™, pRb™/™ utricular hair cells differentiate and survive into adulthood whereas differentiation and survival of pRb™/™ cochlear hair cells are impaired. To comprehensively survey the pRb pathway in the mammalian inner ear, we performed microarray analysis of pRb™/™ cochlea and utricle. The comparative analysis shows that the core pathway shared between pRb™/™ cochlea and utricle is centered on e2F, the key pathway that mediates pRb function. A majority of differentially expressed genes and enriched pathways are not shared but uniquely associated with pRb™/™ cochlea or utricle. In pRb™/™ cochlea, pathways involved in early inner ear development such as Wnt/β-catenin and Notch were enriched, whereas pathways involved in proliferation and survival are enriched in pRb™/™ utricle. Clustering analysis showed that the pRb™/™ inner ear has characteristics of a younger control inner ear, an indication of delayed differentiation. We created a transgenic mouse model (ER-Cre-pRbflox/flox) in which Rb1 can be acutely deleted postnatally. Acute Rb1 deletion in the adult mouse fails to induce proliferation or cell death in inner ear, strongly indicating that Rb1 loss in these postmitotic tissues can be effectively compensated for, or that pRb-mediated changes in the postmitotic compartment result in events that are functionally irreversible once enacted. This study thus supports the concept that pRb-regulated pathways relevant to hair cell development, encompassing proliferation, differentiation and survival, act predominantly during early development.
hair cells; retinoblastoma; Rb1; proliferation; regeneration; apoptosis; inner ear
CD14 is the pivotal molecule in the diagnosis and therapy of CD14-associated diseases, and is important in bacteremia. The HeLa cell line is regarded as immortal due to its prolific character. The HeLa cell line is derived from human cervical cancer cells and has been widely used in cancer research and gene transfection. In the present study, we established the expression plasmid pcDNA3.1(+)-CD14, and transfected it into the human cervical cancer cell line HeLa to establish a stable cell line (HeLa-CD14) expressing human CD14 antigen on the membrane. After the human CD14 gene was cloned and sequenced through RT-PCR and T-A cloning techniques, the eukaryotic expression vector pcDNA3.1(+)-CD14 was constructed by cleaving with double restriction endonucleases and ligating with T4 ligase. HeLa cells were transfected with the pcDNA3.1(+)-CD14 recombinant plasmid using Superfect transfection reagent. The cells were selected using G418 and the expression of human CD14 on the transfectant was confirmed by RT-PCR and immunohistochemistry. The expression of CD14 mRNA was significantly different between the blank pcDNA3.1(+)-transfected cell group and the pcDNA3.1(+)-CD14-transfected cell group (p<0.01). The fluorescence was significantly stronger on the established stable cell line than on the transiently transfected HeLa cells, and no visible fluorescence was observed in blank pcDNA3.1(+)-transfected cells. In this study, the human CD14 transfectant, stable cell line HeLa-CD14, was successfully established, which may be used to study CD14 and cervical cancer in vitro and in vivo.
CD14; transfectant; HeLa
Short-chain fatty acids are the major by-products of bacterial fermentation of undigested dietary fibers in human large intestine. SCFAs, mostly propionate and butyrate, inhibit proliferation and induce apoptosis in colon cancer cells, but clinical trials had mixed results regarding the anti-tumor activities of SCFAs. Herein we demonstrate that propionate and butyrate induced autophagy in human colon cancer cells to dampen apoptosis whereas inhibition of autophagy potentiated SCFA induced apoptosis. Colon cancer cells, after propionate treatment, exhibited extensive characteristics of autophagic proteolysis: increased LC3-I to LC3-II conversion, acidic vesicular organelle development and reduced p62/SQSTM1 expression. Propionate-induced autophagy was associated with decreased mTOR activity and enhanced AMP kinase activity. The elevated AMPKα phosphorylation was associated with cellular ATP depletion and overproduction of reactive oxygen species due to mitochondrial dysfunction involving the induction of MPT and loss of Δψ. In this context, mitochondria biogenesis was initiated to recover cellular energy homeostasis. Importantly, when autophagy was prevented either pharmacologically (3-MA or chloroquine) or genetically (knockdown of ATG5 or ATG7), the colon cancer cells became sensitized toward propionate induced apoptosis through activation of caspase 7 and its downstream effector caspase-3. The observations indicate that propionate-triggered autophagy serves as an adaptive strategy for retarding mitochondria-mediated apoptotic cell death, whereas application of an autophagy inhibitor (Chloroquine) is expected to enhance the therapeutic efficacy of SCFAs in inducing colon tumor cell apoptosis.
Short-chain fatty acids; autophagy; apoptosis; mitochondrial; colon 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 multikinase 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.
pemetrexed; sorafenib; autophagy; apoptosis; PDGFR; ZMP; AMP; thymidylate synthase
AIM: To summarize clinical, endoscopic, radiologic and pathologic features of special diaphragm-like strictures found in small bowel, with no patient use of non-steroidal anti-inflammatory drugs (NSAIDs).
METHODS: From January 2000 to December 2009, 5 cases (2 men and 3 women, with a mean age of 41.6 years) were diagnosed as having diaphragm-like strictures of small bowel on imaging, operation and pathology. All the patients denied the use of NSAIDs. The clinical, endoscopic, radiologic and pathologic findings in these 5 patients were retrospectively reviewed from the hospital database. Images of capsule endoscopy (CE) and small bowel follow-through (SBFT) obtained in 3 and 3 patients, respectively, and images of double-balloon enteroscopy and computed tomography enterography (CTE) obtained in all 5 patients were available for review.
RESULTS: All patients presented with long-term (2-16 years) symptoms of gastrointestinal bleeding and varying degrees of anemia. There was only one stricture in four cases and three lesions in one case, and all the lesions were located in the middle or distal segment of ileum. Circumferential stricture was shown in the small bowel in three cases in the CE image, but the capsule was retained in the small bowel of 2 patients. Routine abdomen computed tomography scan showed no other abnormal results except gallstones in one patient. The lesions were shown as circumferential strictures accompanied by dilated small bowel loops in the small bowel on the images of CTE (in all 5 cases), SBFT (in 2 cases) and double-balloon enteroscopy (in all cases). On microscopy, a chronic inflammatory infiltrate and circumferential diaphragm were found in all lesions.
CONCLUSION: Diaphragm-like strictures of small bowel might be a special consequence of unclear damaging insults to the intestine, having similar clinical, endoscopic, radiologic and pathologic features.
Small bowel; Gastrointestinal bleeding; Diaphragm; Stricture; Endoscopy; Computed tomography; Enterography