A novel multifunctional nanocomposite C225-conjugated Fe3O4/Ag enhances the sensitivity of nasopharyngeal carcinoma cells to radiotherapy

Radiotherapy is the major treatment for nasopharyngeal carcinoma, a malignant tumor of epithelial origin. In this process, a tracer with high sensitivity is pivotal for diagnostic imaging in radiotherapy. Here, we designed a novel multifunctional magnetic silver nanocomposite, Fe(3)O(4)/Ag conjugated to an epidermal growth factor receptor-specific antibody (C225), which can be potentially used for synchronous cancer therapy and diagnosis via magnetic resonance imaging. Characteristics of Fe(3)O(4)/Ag/C225 were determined by transmission electron microscopy, energy dispersive X-ray spectroscopy, ultraviolet spectra, and dynamic light scattering. The results demonstrated that Fe(3)O(4)/Ag/C225 nanoparticles were spherical and dispersed well in water. The activity of C225 was preserved ～80% in the Fe(3)O(4)/Ag/C225 nanoparticles. Futhermore, we tested the cytotoxicity and radiosensitivity of the nanocomposite for human nasopharyngeal carcinoma cell lines (CNEs) in vitro. MTT analysis revealed that Fe(3)O(4)/Ag/C225 could inhibit the proliferation of CNEs in a dose- and time-dependent manner. The clonogenic assay indicated that Fe(3)O(4)/Ag/C225 combined with X-ray treatment could increase the sensitivity of CNEs to irradiation. In a summary, the novel multifunctional nanocomposite Fe(3)O(4)/Ag/C225 might be a potential radiosensitizer for treating malign tumors in the clinic.     

Tetrandrine enhances radiosensitivity through the CDC25C/CDK1/cyclin B1 pathway in nasopharyngeal carcinoma cells

The increasing resistance of nasopharyngeal carcinoma to irradiation makes the exploration of effective radiosensitizers necessary. Tetrandrine is known to be an antitumor drug, but little is known regarding its radiosensitization effect on nasopharyngeal carcinoma. We investigated the effect of combined treatment of irradiation and maximum non-cytotoxic doses of tetrandrine on the nasopharyngeal carcinoma cell lines CNE1 and CNE2. The maximum non-cytotoxic doses of tetrandrine in CNE1 and CNE2 cells were assessed using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The radiosensitization of cells receiving the maximum non-cytotoxic doses of tetrandrine was assessed by evaluating cell proliferation and DNA damage repair using MTT, clonogenic, comet assays and detection of caspase-3 and phosphorylated histone H2AX (γ-H2AX). The cell cycle was assessed by flow cytometry, and protein expression was detected by western blot analysis. The maximum non-cytotoxic doses of tetrandrine in CNE1 and CNE2 cells were 1.5 μmol/L and 1.8 μmol/L, respectively. When cells were exposed to irradiation and the maximum non-cytotoxic doses of tetrandrine, the survival fraction was decreased. DNA damage and γ-H2AX levels markedly increased. Moreover, tetrandrine abrogated the G2/M phase arrest caused by irradiation. Combined treatment with the maximum non-cytotoxic dose of tetrandrine and irradiation caused suppression of the phosphorylation of CDK1 and CDC25C and increase in the expression of cyclin B1. The study in vivo also showed that the maximum non-cytotoxic dose of tetrandrine could reduce tumor growth in xenograft tumor model. Our results suggest that the maximum non-cytotoxic dose of tetrandrine can enhance the radiosensitivity of CNE1 and CNE2 cells and that the underlying mechanism could be associated with abrogation of radiation-induced G2/M arrest via activation of the CDC25C/CDK1/Cyclin B1 pathway.     

Raf kinase inhibitor protein correlates with sensitivity of nasopharyngeal carcinoma to radiotherapy

Raf kinase inhibitory protein (RKIP) is a metastasis suppressor whose expression is reduced in nasopharyngeal carcinoma (NPC) tissues and is absent in NPC metastases. To investigate the effect of RKIP on radiosensitivity of NPC, high metastatic 5‐8F with low RKIP expression and non‐metastatic 6‐10B with high RKIP expression were stably transfected with plasmids that expressed sense and antisense RKIP cDNA. Overexpression of RKIP sensitized 5‐8F cells to radiation‐induced cell death, G₂‐M cell cycle arrest and apoptosis. In contrast, downexpression of RKIP in 6‐10B cells protected cells from radiation‐induced cell death, G₂‐M cell cycle arrest and apoptosis. In addition, RKIP expression altered the radiosensitivity of NPC cells through MEK and ERK phosphorylation changes of Raf‐1/MEK/ERK signaling pathway. We further investigated the RKIP expression in NPC patients and its association with patients' survival after radiotherapy. Downexpression of RKIP was significantly correlated with advanced clinical stage, lymph node metastasis and radioresistance. Furthermore, survival curves showed that patients with RKIP downexpression had a poor prognosis and induced relapse. Multivariate analysis confirmed that RKIP expression was an independent prognostic indicator. The data suggested that RKIP was a potential biomarker for the radiosensitivity and prognosis of NPC, and its dysregulation might play an important role in the radioresistance of NPC. J. Cell. Biochem. 110: 975–984, 2010. © 2010 Wiley‐Liss, Inc.     

Knockdown of NFBD1/MDC1 enhances chemosensitivity to cisplatin or 5-fluorouracil in nasopharyngeal carcinoma CNE1 cells

Nasopharyngeal carcinoma (NPC) is a rare but highly invasive cancer that is prevalent among people of southern Chinese ancestry in southern China and Southeast Asia. Radiotherapy and cisplatin (CDDP)-based chemotherapy are the main treatment options. Unfortunately, disease response to concurrent chemoradiotherapy varies among patients with NPC, and many cases are resistant to CDDP and radiotherapy. NFBD1 functions in cell cycle checkpoint activation and DNA repair following DNA damage. In this study, we identified the NFBD1 as a tractable molecular target to chemosensitize NPC cells. NFBD1 expression in NPC CNE1 cell lines was depleted using lentivirus-mediated short hairpin RNA, and the elevated sensitivity of these NFBD1-inhibited NPC cells to therapeutic reagent CDDP and 5-fluorouracil (5-FU) was evaluated using MTS assays. Flow cytometry analysis also showed that NFBD1 knockdown led to an obvious induction of apoptosis in CDDP- or 5-FU-treated CNE1 cells. Furthermore, we implicated the involvement of NFBD1 in Rad51 and DNA-PKcs foci formation following CDDP or 5-FU chemotherapy. In conclusion, NFBD1 knockdown improves the chemosensitivity of NPC cells by inhibiting cell growth and promoting apoptosis through the impairment of DNA damage repair, suggesting NFBD1 as a novel therapeutic target for NPC.

     

Downregulation of BMI-1 enhances 5-fluorouracil-induced apoptosis in nasopharyngeal carcinoma cells

5-Fluorouracil (5-FU) is an important chemotherapeutic agent for nasopharyngeal carcinoma (NPC). However, drug resistance may occur after several cycles of 5-FU-based chemotherapy. The oncogene B-cell-specific Moloney murine leukemia virus insertion site 1 (BMI-1) has been shown to be involved in the protection of cancer cells from apoptosis. In this study, 5-FU treatment could increase the percentage of apoptotic NPC cells among BMI-1/RNAi-transfected cells than that among cells transfected with the empty vector. The 50% inhibitory concentration (IC₅₀) values of 5-FU were significantly decreased to a greater extent in the cells transfected with BMI-1/RNAi. Most importantly, the expression of phospho-AKT and the anti-apoptotic protein BCL-2 were downregulated in the cells in which BMI-1 expression was inhibited, whereas the apoptosis-inducer BAX was observed to be upregulated. Abrogation of AKT pathway by a PI3K inhibitor could not further increase the sensitivity to 5-FU in the cells with reduced BMI-1 expression. Taken together, BMI-1 depletion enhanced the chemosensitivity of NPC cells by inducing apoptosis; which is associated with inhibition of the PI3K/AKT pathway.     

The Rac/Cdc42 guanine nucleotide exchange factor beta1Pix enhances mastoparan-activated Gi-dependent pathway in mast cells

Carbachol stimulates granule exocytosis, phospholipase C (PLC), and phospholipase D (PLD) in RBL-2H3hm1 mast cells by a mechanism that involves Galphaq. However, mastoparan stimulates the same responses through Gi protein. Both Gi and Galphaq pathways are suppressed by Clostridium difficile toxin B, suggesting that Rac and Cdc42 small GTPases are also involved. Over-expression of beta1Pix, a guanine nucleotide exchange factor for Rac and Cdc42, enhances mastoparan-but not carbachol-induced hexosaminidase secretion and PLC and PLD activation. Furthermore, cells expressing beta1Pix exhibit elevated levels of mastoparan-stimulated IP3 production. Taken together, these findings implicate beta1Pix in regulating hexoasaminidase secretion and IP3 production in early stage upon mastoparan stimulation.     

Hippo pathway contributes to cisplatin resistant-induced EMT in nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) is a kind of head-neck malignant tumor derived from the nasopharyngeal epithelium and is mainly prevalent in Southern China and Southeast Asia countries. Cisplatin (DDP) provides the first-line therapeutic administration in NPC patients. However, chemoresistance has been a main barrier and caused bad treatment outcome in NPC therapy. To understand the molecular mechanism of acquired resistance to DDP, multiple methods were performed to examine the morphocytology and molecular changes in DDP-resistant NPC cells. We found that drug resistance cells displayed epithelial-mesenchymal transition (EMT) characteristics. DDP-resistant NPC cells exhibited enhanced migration and invasion potential. Moreover, overexpression of TAZ, one key gene in Hippo pathway, is closely associated with the DDP resistance of NPC cells and its EMT properties. Depletion of TAZ in DDP-resistant cells reversed EMT phenotypes to MET characteristics and restored chemosensitivity of DDP-resistant cells to DDP treatment. These results suggest that inactivation of TAZ could be a promising approach for the treatment of NPC patients.     

A novel pathway spatiotemporally activates Rac1 and redox signaling in response to fluid shear stress

Hemodynamic forces regulate embryonic organ development, hematopoiesis, vascular remodeling, and atherogenesis. The mechanosensory stimulus of blood flow initiates a complex network of intracellular pathways, including activation of Rac1 GTPase, establishment of endothelial cell (EC) polarity, and redox signaling. The activity of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase can be modulated by the GTP/GDP state of Rac1; however, the molecular mechanisms of Rac1 activation by flow are poorly understood. Here, we identify a novel polarity complex that directs localized Rac1 activation required for downstream reactive oxygen species (ROS) production. Vav2 is required for Rac1 GTP loading, whereas, surprisingly, Tiam1 functions as an adaptor in a VE-cadherin–p67phox–Par3 polarity complex that directs localized activation of Rac1. Furthermore, loss of Tiam1 led to the disruption of redox signaling both in vitro and in vivo. Our results describe a novel molecular cascade that regulates redox signaling by the coordinated regulation of Rac1 and by linking components of the polarity complex to the NADPH oxidase.     

Activation of NADPH oxidase 1 in tumour colon epithelial cells

In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22(phox), NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium) or NADP(+). The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1(Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O(2)(-)-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N-Rac1(Q61L)] between truncated NOXA1(1-211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox1 activity, but NOXO1N(1-292) [C-terminal truncated NOXO1(1-292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox1 activation and their interactions might be responsible for regulating the O(2)(-)-producing activity in Caco-2 cells.     

Macrophage/microglia-specific protein Iba1 enhances membrane ruffling and Rac activation via phospholipase C-gamma -dependent pathway

Iba1 is a macrophage/microglia-specific calcium-binding protein that is involved in RacGTPase-dependent membrane ruffling and phagocytosis. In this study, we introduced Iba1 into Swiss 3T3 fibroblasts and demonstrated the enhancement of platelet-derived growth factor (PDGF)-induced membrane ruffling and chemotaxis. Wortmannin treatment did not completely suppressed this enhanced membrane ruffling in Iba1-expressing cells, whereas it did in Iba1-nonexpressing cells, suggesting that the enhancement is mediated through a phosphatidylinositol 3-kinase (PI3K)-independent signaling pathway. Porcine aorta endothelial cells transfected with expression constructs of Iba1 and PDGF receptor add-back mutants were used to analyze the signaling pathway responsible for the Iba1-induced enhancement of membrane ruffling. In the absence of Iba1 expression, PDGF did not induced membrane ruffling in cells expressing the Tyr-1021 receptor mutant, which is capable of activating phospholipase C-gamma (PLC-gamma) but not PI3K. In contrast, in the presence of Iba1 expression, membrane ruffling was formed in cells expressing the Tyr-1021 mutant. In addition, Rac was shown to be activated during membrane ruffling in cells expressing Iba1 and the Tyr-1021 mutant. Furthermore, dominant negative forms of PLC-gamma completely suppressed PDGF-induced Iba1-dependent membrane ruffling and Rac activation. These results indicate the existence of a novel signaling pathway where PLC-gamma activates Rac in a manner dependent on Iba1.     

A novel lncRNA promotes myogenesis of bovine skeletal muscle satellite cells via PFN1-RhoA/Rac1

Myogenesis, the process of skeletal muscle formation, is a highly coordinated multistep biological process. Accumulating evidence suggests that long non-coding RNAs (lncRNAs) are emerging as a gatekeeper in myogenesis. Up to now, most studies on muscle development-related lncRNAs are mainly focussed on humans and mice. In this study, a novel muscle highly expressed lncRNA, named lnc23, localized in nucleus, was found differentially expressed in different stages of embryonic development and myogenic differentiation. The knockdown and over-expression experiments showed that lnc23 positively regulated the myogenic differentiation of bovine skeletal muscle satellite cells. Then, TMT 10-plex labelling quantitative proteomics was performed to screen the potentially regulatory proteins of lnc23. Results indicated that lnc23 was involved in the key processes of myogenic differentiation such as cell fusion, further demonstrated that down-regulation of lnc23 may inhibit myogenic differentiation by reducing signal transduction and cell fusion among cells. Furthermore, RNA pulldown/LC-MS and RIP experiment illustrated that PFN1 was a binding protein of lnc23. Further, we also found that lnc23 positively regulated the protein expression of RhoA and Rac1, and PFN1 may negatively regulate myogenic differentiation and the expression of its interacting proteins RhoA and Rac1. Hence, we support that lnc23 may reduce the inhibiting effect of PFN1 on RhoA and Rac1 by binding to PFN1, thereby promoting myogenic differentiation. In short, the novel identified lnc23 promotes myogenesis of bovine skeletal muscle satellite cells via PFN1-RhoA/Rac1.     

Correction to: Inhibition of PAD4 enhances radiosensitivity and inhibits aggressive phenotypes of nasopharyngeal carcinoma cells

Background

ZNF674-AS1, a recently characterized long noncoding RNA, shows prognostic significance in hepatocellular carcinoma and glioma. However, the expression and function of ZNF674-AS1 in non-small cell lung cancer (NSCLC) are unclear.

Methods

In this work, we investigated the expression of ZNF674-AS1 in 83 pairs of NSCLC specimens and adjacent noncancerous lung tissues. The clinical significance of ZNF674-AS1 in NSCLC was analyzed. The role of ZNF674-AS1 in NSCLC growth and cell cycle progression was explored.

Results

Our data show that ZNF674-AS1 expression is decreased in NSCLC compared to normal tissues. ZNF674-AS1 downregulation is significantly correlated with advanced TNM stage and decreased overall survival of NSCLC patients. Overexpression of ZNF674-AS1 inhibits NSCLC cell proliferation, colony formation, and tumorigenesis, which is accompanied by a G0/G1 cell cycle arrest. Conversely, knockdown of ZNF674-AS1 enhances the proliferation and colony formation of NSCLC cells. Biochemically, ZNF674-AS1 overexpression increases the expression of p21 through downregulation of miR-423-3p. Knockdown of p21 or overexpression of miR-423-3p blocks ZNF674-AS1-mediated growth suppression and G0/G1 cell cycle arrest. In addition, ZNF674-AS1 expression is negatively correlated with miR-423-3p in NSCLC specimens.

Conclusions

ZNF674-AS1 suppresses NSCLC growth by downregulating miR-423-3p and inducing p21. This work suggests the therapeutic potential of ZNF674-AS1 in the treatment of NSCLC.

     

Activation of Rac1 and the p38 mitogen-activated protein kinase pathway in response to arsenic trioxide

Arsenic trioxide induces differentiation and apoptosis of malignant cells in vitro and in vivo, but the mechanisms by which such effects occur have not been elucidated. In the present study we provide evidence that arsenic trioxide induces activation of the small G-protein Rac1 and the alpha and beta isoforms of the p38 mitogen-activated protein (MAP) kinase in several leukemia cell lines. Such activation of Rac1 and p38-isoforms results in downstream engagement of the MAP kinase-activated protein kinase-2 and is enhanced by pre-treatment of cells with ascorbic acid. Interestingly, pharmacological inhibition of p38 potentiates arsenic-dependent apoptosis and suppression of growth of leukemia cell lines, suggesting that this signaling cascade negatively regulates induction of antileukemic responses by arsenic trioxide. Consistent with this, overexpression of a dominant-negative p38 mutant (p38betaAGF) enhances the antiproliferative effects of arsenic trioxide on target cells. To further define the relevance of activation of the Rac1/p38 MAP kinase pathway in the induction of arsenic-dependent antileukemic effects, studies were performed using bone marrows from patients with chronic myelogenous leukemia. Arsenic trioxide suppressed the growth of leukemic myeloid (CFU-GM) progenitors from such patients, whereas concomitant pharmacological inhibition of the p38 pathway enhanced its growth-suppressive effects. Altogether, these data provide evidence for a novel function of the p38 MAP kinase pathway, acting as a negative regulator of arsenic trioxide-induced apoptosis and inhibition of malignant cell growth.     

MiR-27a-3p enhances the cisplatin sensitivity in hepatocellular carcinoma cells through inhibiting PI3K/Akt pathway

MicroRNAs (miRNAs) play an important role in drug resistance, and it is reported that miR-27a-3p regulated the sensitivity of cisplatin in breast cancer, lung cancer and ovarian cancer. However, the relationship between miR-27a-3p and chemosensitivity of cisplatin in hepatocellular carcinoma (HCC) was unclear, especially the underlying mechanism was unknown. In the present study, we analyzed miR-27a-3p expression levels in 372 tumor tissues and 49 adjacent tissues in HCC samples from TCGA database, and found that the miR-27a-3p was down-regulated in HCC tissues. The level of miR-27a-3p was associated with metastasis, Child–Pugh grade and race. MiR-27a-3p was regarded as a favorable prognosis indicator for HCC patients. Then, miR-27a-3p was overexpressed in HepG2 cell, and was knocked down in PLC cell. Next, we conducted a series of in vitro assays, including MTT, apoptosis and cell cycle assays to observe the biological changes. Further, inhibitor rate and apoptosis rate were detected with pre- and post-cisplatin treatment in HCC. The results showed that overexpression of miR-27a-3p repressed the cell viability, promoted apoptosis and increased the percentage of cells in G₀/G₁ phase. Importantly, overexpression of miR-27a-3p significantly increased the inhibitor rate and apoptosis rate with cisplatin intervention. Besides, we found that miR-27a-3p added cisplatin sensitivity potentially through regulating PI3K/Akt signaling pathway. Taken together, miR-27a-3p acted as a tumor suppressor gene in HCC cells, and it could be useful for modulating cisplatin sensitivity in chemotherapy.     

Role of Rac1 GTPase in NADPH Oxidase Activation and Cognitive Impairment Following Cerebral Ischemia in the Rat

Background

Recent work by our laboratory and others has implicated NADPH oxidase as having an important role in reactive oxygen species (ROS) generation and neuronal damage following cerebral ischemia, although the mechanisms controlling NADPH oxidase in the brain remain poorly understood. The purpose of the current study was to examine the regulatory and functional role of the Rho GTPase, Rac1 in NADPH oxidase activation, ROS generation and neuronal cell death/cognitive dysfunction following global cerebral ischemia in the male rat.

Methodology/Principal Findings

Our studies revealed that NADPH oxidase activity and superoxide (O₂ ⁻) production in the hippocampal CA1 region increased rapidly after cerebral ischemia to reach a peak at 3 h post-reperfusion, followed by a fall in levels by 24 h post-reperfusion. Administration of a Rac GTPase inhibitor (NSC23766) 15 min before cerebral ischemia significantly attenuated NADPH oxidase activation and O₂ ⁻ production at 3 h after stroke as compared to vehicle-treated controls. NSC23766 also attenuated “in situ” O₂ ⁻ production in the hippocampus after ischemia/reperfusion, as determined by fluorescent oxidized hydroethidine staining. Oxidative stress damage in the hippocampal CA1 after ischemia/reperfusion was also significantly attenuated by NSC23766 treatment, as evidenced by a marked attenuation of immunostaining for the oxidative stress damage markers, 4-HNE, 8-OHdG and H2AX at 24 h in the hippocampal CA1 region following cerebral ischemia. In addition, Morris Water maze testing revealed that Rac GTPase inhibition after ischemic injury significantly improved hippocampal-dependent memory and cognitive spatial abilities at 7–9 d post reperfusion as compared to vehicle-treated animals.

Conclusions/Significance

The results of the study suggest that Rac1 GTPase has a critical role in mediating ischemia/reperfusion injury-induced NADPH oxidase activation, ROS generation and oxidative stress in the hippocampal CA1 region of the rat, and thus contributes significantly to neuronal degeneration and cognitive dysfunction following cerebral ischemia.