EGFR-AKT-mTOR activation mediates epiregulin-induced pleiotropic functions in cultured osteoblasts

Epidermal growth factor (EGF) receptor (EGFR) emerges as an essential molecule for the regulating of osteoblast cellular functions. In the current study, we explored the effect of epiregulin, a new EGFR ligand, on osteoblast functions in vitro, and studied the underlying mechanisms. We found that epiregulin-induced EGFR activation in both primary osteoblasts and osteoblast-like MC3T3-E1 cells. Meanwhile, epiregulin activated AKT-mammalian target of rapamycin (mTOR) and Erk-mitogen-activated protein kinase (MAPK) signalings in cultured osteoblasts, which were blocked by EGFR inhibitor AG1478 or monoclonal antibody against EGFR (anti-EGFR). Further, in primary and MC3T3-E1 osteoblasts, epiregulin promoted cell proliferation and increased alkaline phosphatase activity, while inhibiting dexamethasone (Dex)-induced cell death. Such effects by epiregulin were largely inhibited by AG1478 or anti-EGFR. Notably, AKT-mTOR inhibitors, but not Erk inhibitors, alleviated epiregulin-induced above pleiotropic functions in osteoblasts. Meanwhile, siRNA depletion of Sin1, a key component of mTOR complex 2 (mTORC2), also suppressed epiregulin-exerted effects in MC3T3-E1 cells. Together, these results suggest that epiregulin-induced pleiotropic functions in cultured osteoblasts are mediated through EGFR-AKT-mTOR signalings.     

Effects of Icotinib on Advanced Non-Small Cell Lung Cancer with Different EGFR Phenotypes

Icotinib is the first oral epidermal growth factor receptor (EGFR) tyrosine kinase receptor inhibitor, which has been proven to exert significant inhibitory effects on non-small cell lung cancer in vitro. Clinical evidence has showed that the efficacy of Icotinib on retreating advanced non-small cell lung cancer is comparable to Gefitinib. However, different phenotypes of EGFR can affect the therapeutic outcomes of EGFR tyrosine kinase receptor inhibitor. Therefore, our study focused on efficacy and safety of Icotinib in patients with advanced non-small cell lung cancer of different EGPR phenotypes. Clinical data of patients with advanced non-small cell lung cancer who received Icotinib treatment from August, 2011 to May, 2013 were retrospectively analyzed. Kaplan–Meier analysis was used for survival analysis and comparison. 18 wild-type EGFR and 51 mutant type were found in a total of 69 patients. Objective response rate of patients with mutant type EGFR was 54.9 % and disease control rate was 86.3 %. Objective response rate of wild-type patients was 11.1 % (P = 0.0013 vs mutant type), disease control rate was 50.0 % (P = 0.0017). Median progression-free survival (PFS) of mutant type and wild-type patients were 9.7 and 2.6 months, respectively (P < 0.001). Median PFS of exon 19 mutated mutant patients was 11.3 months, mean PFS of exon 21 L858R mutated mutant patients was 8.7 months (P = 0.3145). Median overall survival (OS) of EGFR mutated patients had not reached. OS time of 13 wild-type patients was 12.9 months (P < 0.001). The common adverse reactions of Icotinib included rash, diarrhea, itching skin with occurrence rates of 24.6 % (17/69), 13.0 % (9/69), and 11.6 % (8/69), respectively. Most adverse reactions were grade I–II. Icotinib has great efficacy in EGFR mutated patients, making it an optimal regimen to treat EGFR mutated patients. Furthermore, most of adverse reactions associated with Icotinib treatment were tolerable.     

Mucin 1 (MUC1) signalling contributes to increase the resistance to cell death in human bronchial epithelial cells exposed to nickel acetate

We have previously reported that nickel acetate (Ni²⁺), a well-known human carcinogenic agents, differentially affected apoptosis in two different airway epithelial cell lines derived from the human respiratory tract (A549 and Beas-2B, respectively), suggesting a potential involvement of epidermal growth factor receptor (EGFR)/Neu receptors in mediating this effect. Since ErbBs are closely associated to Mucin 1 (MUC1), a glycoprotein component of airway mucus that is overexpressed in lung tumors, we have investigated the role of this signaling system in the survival response of airway epithelial cells against Ni²⁺-induced cell death. We found that A549 cells exposed to Ni²⁺ do not show any significant increase of MUC1 levels. Conversely, Beas-2B cells exposed to equivalent concentrations of Ni²⁺ showed increased expression of MUC1 levels and this correlated with increased phosphorylation of both EGFR and of the extracellular-regulated kinase 1/2 (ERK1/2) and increase resistance to apoptosis, as indicated by cell viability assessments and DNA damage. Interestingly, suppression of MUC1 by small interfering RNA inhibited the EGFR activation in Beas-2B cells, leading to a significant decrease of survival and enhancement of DNA fragmentation and cleaved Caspase-3 expression. These results strongly suggest a role for MUC1 in Ni²⁺-induced neoplastic transformation, which likely involves the activation of the EGFR-mediated cell survival pathway, highlighting new avenues in the molecular approach to lung cancer prevention.     

The reverse effect of X-ray irradiation on acquired gefitinib resistance in non-small cell lung cancer cell line NCI-H1975 in vitro

The clinical efficacy of gefitinib in the treatment of non-small cell lung cancer (NSCLC) with mutations in exon 18, 19 or 21 of epidermal growth factor receptor (EGFR) is limited by the acquired resistance to the drug. To explore whether X-ray irradiation could reverse the acquired gefitinib resistance in NSCLC cell in vitro. We chose a human NSCLC cell line NCI-H1975 to establish acquired gefitinib-resistant cell line named as NCI-H1975/GR. NCI-H1975/GR was irradiated with X-ray and then named as NCI-H1975/GR/XR. In the three cell lines, subsequently cell growth curves and cell population doubling time were calculated by cell proliferation assay, the changes of cell viability were evaluated by trypan blue dye exclusion method and MTT assay, the cell cycle distribution and apoptosis were investigated by flow cytometry, the expressions of E-cadherin and vimentin used to indicate epithelial-mesenchymal transition (EMT) were determined by western blot analysis, the protein expressions in EGFR/KRAS/BRAF transduction pathway were detected by immunocytochemistry, and the mutations of EGFR, KRAS and BRAF were detected by high resolution melting analysis and direct sequencing. We found that the X-ray irradiation enhanced the growth inhibitory effects of gefitinib on the acquired gefitinib-resistant cell line. Of NCI-H1975/GR/XR following gefitinib treatment, the IC₅₀ decreased significantly, the cell proportion of phase G0/G1 was slightly higher, and the apoptosis cell proportion was significantly higher than those of NCI-H1975/GR. In addition, the reversal of EMT being present in NCI-H1975/GR cells was likely appearing in NCI-H1975/GR/XR cells. These results indicated that the acquired gefitinib resistance could be reversed by X-ray irradiation in NSCLC cell line NCI-H1975 harboring both the L858R and T790M mutation in vitro.     

Quercetin Potentiates the Antitumor Activity of Rituximab in Diffuse Large B-Cell Lymphoma by Inhibiting STAT3 Pathway

STAT3 pathway plays an important role in the growth of diffuse large B-cell lymphoma (DLBCL) cells. Here we investigated the antitumor activity of Quercetin, a flavonoid compound, in combination with rituximab in DLBCL cell lines in vitro. We found that Quercetin synergistically enhanced rituximab-induced growth inhibition and apoptosis in DLBCL cell lines. Moreover, we found Quercetin exerted inhibitory activity against STAT3 pathway and downregulated the expression of survival genes. These results suggest that combining the Quercetin with rituximab may present an attractive and potentially effective way for the treatment of DLBCL.     

Identification of nucleolus-localized PTEN and its function in regulating ribosome biogenesis

The tumor suppressor PTEN is a lipid phosphatase that is found mutated in different types of human cancers. PTEN suppresses cell proliferation by inhibiting the PI3K-Akt signaling pathway at the cell membrane. However, PTEN is also demonstrated to localize in the cell nucleus where it exhibits tumor suppressive activity via a different, unknown mechanism. In this study we report that PTEN also localizes to the nucleolus and that nucleolar PTEN plays an important role in regulating nucleolar homeostasis and maintaining nucleolar morphology. Overexpression of nuclear PTEN in PTEN null cells inhibits Akt phosphorylation and reduces cell size. Knockdown of PTEN in PTEN positive cells leads to nucleolar morphologic changes and an increase in the proportion of cells with a greater number of nucleoli. In addition, knockdown of PTEN in PTEN positive cells increased ribosome biogenesis. These findings expand current understanding of function and relevance of nuclear localized PTEN and provide a foundation for the development of novel therapies targeting PTEN.     

A Systems Biology Approach to Study the Biology Characteristics of Esophageal Squamous Cell Carcinoma by Integrating microRNA and Messenger RNA Expression Profiling

Esophageal squamous cell carcinoma (ESCC) is one of the most malignant tumors. The aim of this study was to investigate the biology characteristics of ESCC by analyzing microRNA and mRNA expression profile. We used BRB-array tools to analyze the deregulated microRNA and mRNA between esophageal squamous cell carcinomas and paired normal adjacent tissues. We used miRTrail and protein–protein interaction methods to explore the related pathways and networks of deregulated microRNA and mRNA. By combining the results of pathways and networks, we found that the deregulated microRNA and their deregulated target mRNA are enriched in the following pathways: DNA replication, cell cycle, ECM-receptor interaction, focal adhesion, mismatch repair, and pathways in cancer. The results showed that many deregulated microRNAs and mRNAs may play a vital role in the pathogenesis of ESCC, and the systems biology approach is very helpful to explore molecular mechanism of ESCC.     

Circulating MicroRNA Biomarkers for Glioma and Predicting Response to Therapy

The need for glioma biomarkers with improved sensitivity and specificity has sparked research into short non-coding RNA known as microRNA (miRNA). Altered miRNA biogenesis and expression in glioma plays a vital role in important signaling pathways associated with a range of tumor characteristics including gliomagenesis, invasion, and malignancy. This review will discuss current research into the role of miRNA in glioma and altered miRNA expression in biofluids as candidate biomarkers with a particular focus on glioblastoma, the most malignant form of glioma. The isolation and characterization of miRNA using cellular and molecular biology techniques from the circulation of glioma patients could potentially be used for improved diagnosis, prognosis, and treatment decisions. We aim to highlight the links between research into miRNA function, their use as biomarkers, and how these biomarkers can be used to predict response to therapy. Furthermore, increased understanding of miRNA in glioma biology through biomarker research has led to the development of miRNA therapeutics which could restore normal miRNA expression and function and improve the prognosis of glioma patients. A panel of important miRNA biomarkers for glioma in various biofluids discovered to date has been summarized here. There is still a need, however, to standardize techniques for biomarker characterization to bring us closer to clinically relevant miRNA-based diagnostic and therapeutic signatures. A clinically validated biomarker panel has potential to improve time to diagnosis, predicting response to treatment and ultimately the prognosis of glioma patients.     

Primary alveolar macrophages exposed to diesel particulate matter increase RAGE expression and activate RAGE signaling

Receptors for advanced glycation end-products (RAGE) are members of the immunoglobulin superfamily of cell-surface receptors implicated in mechanisms of pulmonary inflammation. In the current study, we test the hypothesis that RAGE mediates inflammation in primary alveolar macrophages (AMs) exposed to diesel particulate matter (DPM). Quantitative RT-PCR and immunoblotting revealed that RAGE was up-regulated in Raw264.7 cells, an immortalized murine macrophage cell line and primary AMs exposed to DPM for 2 h. Because DPM increased RAGE expression, we exposed Raw264.7 cells and primary AMs isolated from RAGE null and wild-type (WT) mice to DPM prior to the assessment of inflammatory signaling intermediates. DPM led to the activation of Rat sarcoma GTPase (Ras), p38 MAPK and NF-κB in WT AMs and, when compared to WT AMs, these intermediates were diminished in DPM-exposed AMs isolated from RAGE null mice. Furthermore, cytokines implicated in inflammation, including IL-4, IL-12, IL-13 and TNFα, were all significantly decreased in DPM-exposed RAGE null AMs compared to similarly exposed WT AMs. These results demonstrate that diesel-induced inflammatory responses by primary AMs are mediated, at least in part, via RAGE signaling mechanisms. Further work may show that RAGE signaling in both alveolar epithelial cells and resident macrophages is a potential target in the treatment of inflammatory lung diseases exacerbated by environmental pollution.     

Progression of O6-methylguanine-DNA methyltransferase and temozolomide resistance in cancer research

Temozolomide (TMZ) is an alkylating agent that is widely used in chemotherapy for cancer. A key mechanism of resistance to TMZ is the overexpression of O⁶-methylguanine-DNA methyltransferase (MGMT). MGMT specifically repairs the DNA O⁶-methylation damage induced by TMZ and irreversibly inactivates TMZ. Regulation of MGMT expression and research regarding the mechanism of TMZ resistance will help rationalize the clinical use of TMZ. In this review, we provide an overview of recent advances in the field, with particular emphasis on MGMT structure, function, expression regulation, and the association between MGMT and resistance to TMZ.     

Preparation of cinnamoyl-CoA and analysis of the enzyme activity of recombinant CCR protein from Populus tomentosa

Cinnamoyl-CoA reductase (CCR, EC 1.2.1.44), which catalyzes the reduction of cinnamoyl-CoA esters to their respective cinnamaldehydes, is considered as a key enzyme in lignin formation. The substrates of CCR, cinnamoyl-CoA esters, are products of 4-Coumarate-CoA ligase (4CL, EC 6.2.1.12), which is an enzyme upstream of CCR. The PtCCR and Pt4CL were isolated from Populus tomentosa and expressed in E. coli. Results showed that 4CL can catalyze the conversion of hydroxycinnamic acids to cinnamoyl-CoA esters, with high efficiency. The purification of esters using SPE cartridges suggested that 40 % methanol with 0.1 M of acetic acid was the optimal elution buffer for cinnamoyl-CoA esters. The optimization of prokaryotic expression demonstrated that the best expression conditions for recombinant PtCCR was 6 h of 0.4 mM IPTG induction at 37 °C. PtCCR enzyme assay illustrated that the recombinant protein can catalyze the reduction of cinnamoyl-CoA esters. Kinetics analysis showed that feruloyl-CoA has higher affinity to PtCCR with faster reaction speed (Vmax), indicating that feruloyl-CoA was the most favorable substrate for PtCCR catalysis. The recombinant protein was expressed in E. coli, purified through affinity column chromatography, and characterized by SDS-PAGE. SPE cartridges were used to purify the ester products of the Pt4CL reaction. HPLC-MS was used to analyze the structure of esters and evaluate their purity or quantity. Furthermore, the enzyme activity of recombinant CCR to feruloyl-CoA at different pHs indicated that compartmentalization may be an important factor in lignin monomer formation.     

GABA Pharmacology: The Search for Analgesics

Decades of research have been devoted to defining the role of GABAergic transmission in nociceptive processing. Much of this work was performed using rigid, orthosteric GABA analogs created by Povl Krogsgaard-Larsen and his associates. A relationship between GABA and pain is suggested by the anatomical distribution of GABA receptors and the ability of some GABA agonists to alter nociceptive responsiveness. Outlined in this report are data supporting this proposition, with particular emphasis on the anatomical localization and function of GABA-containing neurons and the molecular and pharmacological properties of GABA_A and GABA_B receptor subtypes. Reference is made to changes in overall GABAergic tone, GABA receptor expression and activity as a function of the duration and intensity of a painful stimulus or exposure to GABAergic agents. Evidence is presented that the plasticity of this receptor system may be responsible for the variability in the antinociceptive effectiveness of compounds that influence GABA transmission. These findings demonstrate that at least some types of persistent pain are associated with a regionally selective decline in GABAergic tone, highlighting the need for agents that enhance GABA activity in the affected regions without compromising GABA function over the long-term. As subtype selective positive allosteric modulators may accomplish these goals, such compounds might represent a new class of analgesic drugs.     

Transarterial chemoembolization combined with sorafenib for unresectable hepatocellular carcinoma: a systematic review and meta-analysis

Sorafenib in combination with Transarterial chemoembolization (TACE) is increasingly used in patients with unresectable hepatocellular carcinoma (HCC), but the current evidence is still controversial. The aim of this systematic review was to evaluate the effectiveness and safety of TACE plus sorafenib versus TACE alone for unresectable HCC. We searched PubMed, EMBASE and the Cochrane Library for clinical trials comparing TACE plus sorafenib with TACE alone for unresectable HCC. The study outcomes included overall survival (OS), time to progression (TTP), objective response and adverse events (AEs). Six studies including 1,181 patients were included. Meta-analysis of all studies suggested that the combination therapy group had significant longer OS than TACE group [hazard ratio (HR) = 0.64, 95 % confidence interval (CI) = 0.43–0.97], but the pooled HR of randomized controlled trials (RCTs) failed to achieve statistical significance. For TTP, meta-analysis in both RCTs subgroup and retrospective studies subgroup suggested that combination therapy was superior to TACE group. The combination therapy was also associated with better response to treatment (risk ratio = 1.45, 95 % CI = 1.04–2.02) when both RCTs and retrospective studies were pooled. However, the sorafenib associated AEs were more frequent in the combination therapy group. In conclusion, the combination of TACE and sorafenib is likely to improve OS, TTP and response to treatment when compared with TACE monotherapy. The combination group is also associated with more sorafenib-related AEs.     

Implementation of Transportation Distance for Analyzing FLIM and FRET Experiments

Analysis of fluorescence lifetime imaging microscopy (FLIM) and Förster resonance energy transfer (FRET) experiments in living cells is usually based on mean lifetimes computations. However, these mean lifetimes can induce misinterpretations. We propose in this work the implementation of the transportation distance for FLIM and FRET experiments in vivo. This non-fitting indicator, which is easy to compute, reflects the similarity between two distributions and can be used for pixels clustering to improve the estimation of the FRET parameters. We study the robustness and the discriminating power of this transportation distance, both theoretically and numerically. In addition, a comparison study with the largely used mean lifetime differences is performed. We finally demonstrate practically the benefits of the transportation distance over the usual mean lifetime differences for both FLIM and FRET experiments in living cells.     

Suppression of PI3K/Akt signaling by synthetic bichalcone analog TSWU-CD4 induces ER stress- and Bax/Bak-mediated apoptosis of cancer cells

Suppression of the activity of pro-apoptotic Bcl-2-family proteins frequently confers chemoresistance to many human cancer cells. Using subcellular fractionation, the ER calcium (Ca⁺⁺) channel inhibitor dantrolene and small interfering RNA (siRNA) against Bax or Bak, we show that the new synthetic bichalcone analog TSWU-CD4 induces apoptosis in human cancer cells by releasing endoplasmic reticulum (ER)-stored Ca⁺⁺ through ER/mitochondrial oligomerization of Bax/Bak. Blockade of the protein kinase RNA-like ER kinase or the unfolded protein response regulator glucose-regulated protein 78 expression by siRNA not only suppressed oligomeric Bax/Bak-mediated pro-caspase-12 cleavage and apoptosis but also resulted in an inhibition of Bcl-2 downregulation induced by TSWU-CD4. Induction of the ER oligomerization of Bax/Bak and apoptosis by TSWU-CD4 were suppressed by Bcl-2 overexpression. Inhibition of lipid raft-associated phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) signaling by TSWU-CD4 induced ER stress- and oligomeric Bax/Bak-mediated apoptosis, which were substantially reversed by overexpression of the wt PI3K p85α subunit. Taken together, these results suggest that suppression of lipid raft-associated PI3K/Akt signaling is required for the ER stress-mediated apoptotic activity of Bax/Bak, which is responsible for the ability of TSWU-CD4-treated cancer cells to exit the ER-mitochondrial apoptotic cell death pathway.     

Relative mRNA expression and immunolocalization for transforming growth factor-beta (TGF-β) and their effect on in vitro development of caprine preantral follicles

This study aimed to evaluate the immunolocalization and messenger RNA (mRNA) expression for transforming growth factor-beta (TGF-β) and its receptors (TGF-βRI and RII), as well as mRNA expression for P450 aromatase and FSH receptor in caprine preantral follicles. The effects of TGF-β, FSH alone, or in association on the in vitro follicular development were also assessed. Immunohistochemical analyses showed the expression of TGF-β and its receptors in oocytes of all follicle stages and granulosa cells of primary and secondary follicles. mRNA for TGF-β receptors and for FSH receptor (FSHR) was present in preantral follicles as well as in oocytes and granulosa cells of antral follicles. Isolated secondary follicles were cultured in α-minimum essential medium (MEM) alone or supplemented with either FSH (100 ng/ml), TGF-β (10 ng/ml), or TGF-β + FSH for 18 d. TGF-β increased significantly oocyte diameter when compared to FSH alone and control. After 18 d of culture, all groups showed a significant reduction in P450 aromatase and FSHR mRNA levels in comparison to fresh control. In contrast, treatment with FSH significantly increased the mRNA expression for TGF-β in comparison to fresh control and other treatments. In conclusion, the findings showed that TGF-β and its receptors are present in caprine ovarian follicles. Furthermore, they showed a positive effect on oocyte growth in vitro.     

Effect of Recombinant hPTEN Gene Expression on PDGF Induced VSMC Proliferation

Human phosphatase and tensin homolog (hPTEN) gene was expressed in vascular smooth muscle cells (VSMCs) to study its effect on VSMC proliferation induced in platelet-derived growth factor (PDGF) conditioned medium. After G418 selection, MTT assay was conducted to examine transfected VSMC proliferation induced in human PDGF conditioned medium. We successfully constructed eukaryotic expression vector pcDNA4/myc-His-PTEN and transferred into VSMC cells. We report that in vitro proliferation of VSMC was inhibited in PTEN transfected VSMCs induced in PDGF conditioned medium. RT-PCR and Western blot results indicated significantly high levels of protein kinase B-PKB and nuclear factor kappa B mRNA and protein, respectively, in PDGF group as compared with the control group.     

Optimization of conditions for expression of recombinant interferon-γ in E.coli

Interferon gamma (IFN-γ) is an important immunoregulatory cytokine that has a central role against viral and bacterial infections. In this study, the cDNA encoding 141 amino acids of mature IFN-γ from mice splenocytes was cloned in a prokaryotic expression vector pQE 30. Optimization of expression conditions resulted in high IFN-γ protein. Western blot showed that recombinant IFN-γ was specifically recognized by its counterpart anti-mouse IFN-γ antibodies. In vitro dose-dependent studies, with A549 and HeLa cell lines, showed that cloned IFN-γ was safe and had no effect on cell proliferation. The protein prediction and analysis using SOPMA program, revealed that IFN-γ had 80 α-helices, 8 β-turns jointed by 9 extended strands and 44 random coils. A total of four major clusters were observed with murine IFN-γ sharing 39 % homology with human IFN-γ. Pair-wise alignment studies with human revealed 26 % identity and 43.3 % similarity. The recovery of bioactive proteins from inclusion bodies (IBs) is a complex process and various protocols have been developed. We report here a simple, robust and inexpensive purification approach for obtaining recombinant IFN-γ protein expressed as IBs in E.coli.