Brefeldin A delivery nanomicelles in hepatocellular carcinoma therapy: Characterization, cytotoxic evaluation in vitro, and antitumor efficiency in vivo.

Hepatocellular carcinoma (HCC) is one of the major cancers with high mortality rate. Traditional drugs used in clinic are usually limited by the drug resistance and side effect and novel agents are still needed. Macrolide brefeldin A (BFA) is a well-known lead compound in cancer chemotherapy, however, with poor solubility and instability. In this study, to overcome these disadvantages, BFA was encapsulated in mixed nanomicelles based on TPGS and F127 copolymers (M-BFA). M-BFA was conferred high solubility, colloidal stability, and capability of sustained release of intact BFA. In vitro, M-BFA markedly inhibited the proliferation, induced G0/G1 phase arrest, and caspase-dependent apoptosis in human liver carcinoma HepG2 cells. Moreover, M-BFA also induced autophagic cell death via Akt/mTOR and ERK pathways. In HepG2 tumor-bearing xenograft mice, indocyanine green (ICG) as a fluorescent probe loaded in M-BFA distributed to the tumor tissue rapidly, prolonged the blood circulation, and improved the tumor accumulation capacity. More importantly, M-BFA (10 mg/kg) dramatically delayed the tumor progression and induced extensive necrosis of the tumor tissues. Taken together, the present work suggests that M-BFA has promising potential in HCC therapy.

Regulating the Golgi apparatus by co-delivery of a COX-2 inhibitor and Brefeldin A for suppression of tumor metastasis.

Finding a cure for breast cancer currently remains a medical challenge in due to the failure of common treatment methods to inhibit invasion and metastasis of cancer cells, which eventually leads to recurrence of breast cancer. Many secreted proteins are overexpressed and play crucial roles in tumorigenesis and development. The Golgi apparatus is a key protein processing and secretion factory in which metastasis-associated proteins are modified, transported and secreted; thus, regulating the Golgi apparatus of tumor cells is a viable strategy to inhibit tumor metastasis. Herein, celecoxib (CLX) and Brefeldin A (BFA) were encapsulated into the biocompatible polymer PLGA-PEG to form nanoparticles that act on the Golgi apparatus to treat metastatic breast cancer; CLX is a specific COX-2 inhibitor which accumulates in the Golgi apparatus, and BFA is a protein transport inhibitor fusing the Golgi apparatus into endoplasmic reticulum. The optimized CLX and BFA co-loaded nanoparticles (CBNPs) possessed good physicochemical properties. CBNPs efficiently damaged the Golgi apparatus within 30 min and showed enhanced cytotoxicity of CLX and BFA toward murine metastatic breast cancer 4T1 cells. The migration and invasion abilities of the cells were dramatically suppressed by the CBNPs. Further, the expression and secretion of metastasis-associated proteins such as matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor (VEGF) were remarkably decreased. Our findings showed that co-delivering CLX and BFA to regulate the Golgi apparatus may be an efficient strategy to inhibit breast cancer growth and suppress tumor cell metastasis.

Combining autophagy-inducing peptides and brefeldin A delivered by perinuclear-localized mesoporous silica nanoparticles: a manipulation strategy for ER-phagy.

Autophagic degradation of the endoplasmic reticulum (ER-phagy) has been found to play a critical role in human sensory neuropathy. So far, however, specific and efficient intervention means for ER-phagy remain unexplored. Herein, brefeldin A (BFA), a blocking agent on protein transport between the ER and Golgi, was screened from ER stress inducers. BFA was then delivered to the perinuclear area co-localized with the ER by a mesoporous silica nanoparticle-based drug-carrier functionalized with autophagy-inducing peptides of TAT-beclin 1 (MSNs-BFA), to evoke a perturbation of ER-phagy. The molecular mechanism of ER-phagy regulated by BFA was explored by biochemical evaluation including time-lapse live-cell fluorescence imaging. We found that MSNs-BFA treatment caused a lower mRNA/protein expression level of FAM134b even under a compensation of autophagic flux in U2OS cells, and resulted in ER-expansion. The fragmentation of the ER was blocked as a response to ER stress mediated by inactivation of the AKT/TSC/mTOR pathway. Our work developed an efficient external manipulation strategy to regulate ER-phagy and may contribute to the therapeutic application of autophagy-related major human diseases.

Bidirectional reporter assay using HAL promoter and TOPFLASH improves specificity in high-throughput screening of Wnt inhibitors.

Constitutive activation of Wnt signaling plays an important role in colorectal and liver tumorigenesis. Cell-based assays using synthetic TCF/LEF (T-cell factor/lymphoid enhancer factor) reporters, as readouts of ß-catenin/TCF-dependent transcriptional activity, have contributed greatly to the discovery of small molecules that modulate Wnt signaling. In the present study, we report a novel screening method, called a bidirectional dual reporter assay. Integrated transcriptome analysis identified a histidine ammonia-lyase gene (HAL) that was negatively regulated by ß-catenin/TCF-dependent transcriptional activity. We leveraged a promoter region of the HAL gene as another transcriptional readout of Wnt signaling. Cells stably expressing both an optimized HAL reporter and the TCF/LEF reporter enabled bidirectional reporter activities in response to Wnt signaling. Increased HAL reporter activity and decreased TCF/LEF reporter activity were observed simultaneously in the cells when ß-catenin/TCF7L2 was inhibited. Notably, this method could decrease the number of false positives observed when screening an inhibitor library compared with the conventional TCF/LEF assay. We found that Brefeldin A, a disruptor of the Golgi apparatus, inhibited the Wnt/ß-catenin signaling pathway. The utility of our system could be expanded to examine other disease-associated pathways beyond the Wnt/ß-catenin signaling pathway.

Connexin 43 Acts as a Proapoptotic Modulator in Cisplatin-Induced Auditory Cell Death.

AIMS: Gap junction coupling is known to play a role in intercellular communication by the Good Samaritan effect or bystander effect. Nonjunctional connexins (Cxs) may also play certain gap junction-independent roles in cell death or survival. The purpose of the present study was to investigate the role of junctional and nonjunctional Cxs in ototoxic drug-induced auditory cell death by focusing on Cx43 in the cochlea. RESULTS: Nonjunctional Cx43 conditions were prepared by low confluence culture (5 × 103/cm2) or a trafficking inhibitor, brefeldin A (BFA), in auditory cells, and short lengthened Cx43s with amino-terminal (NT; amino acids 1-256) or carboxy-terminal (CT; amino acids 257-382) were transfected into Cx-deficient HeLa cells to avoid gap junction formation. Knockdown of nonchannel Cx43 (small interfering RNA [siRNA]) inhibited Cis-diamminedichloroplatinum (cisplatin)-induced cell death regardless of gap junction formation; however, a gap junction blocker, 18 alpha-glycyrrhetinic acid (18α-GA), showed inhibitory effect only under the junctional condition. BFA did not show any additive influence on the inhibitory effect of siRNA Cx43. Shortened Cx43-transfected HeLa cells also resulted in a significant increase in cell death under cisplatin. In the animal studies with cisplatin-treated rats, hearing thresholds of auditory brainstem response were significantly preserved by a gap junction blocker, carbenoxolone, showing much more preserved stereocilia of hair cells in scanning electron microscopic findings. Innovation and Conclusion: Cx43 plays a proapoptotic role in cisplatin-induced auditory cell death in both junctional and nonjunctional conditions. Targeting the Cx-mediated signaling control may be helpful in designing new therapeutic strategies for drug-induced ototoxicity. Antioxid. Redox Signal. 25, 623-636.

Controlled Dual Drug Release and In Vitro Cytotoxicity of Electrospun Poly(lactic-co-glycolic acid) Nanofibers Encapsulated with Micelles.

In order to realize controlled dual release of two hydrophobic drugs with distinct rates in a vehicle, novel poly(lactic-co-glycolic acid) (PLGA) composite nanofibers encapsulated with micelles were successfully fabricated by "emulsion-electrospinning." Brefeldin A (BFA) was firstly embedded in monomethoxy-poly(ethylene glycol)-b-poly(L-lactide) (MePEG-PLLA) micelles. By means of "emulsion-electrospinning," paclitaxel (PTX) and polymeric micelles contained BFA were successfully loaded into the electrospun PLGA composite nanofibers. The in vitro release results demonstrated that the location of the drugs in the electrospun fibers determined their release profiles. BFA had a long-term and sustained release while PTX had a relatively rapid release in the dual drugs delivery system. In vitro cytotoxicity studies revealed that the composite nanofibers with two drugs restrained HepG-2 cells more efficiently. These results strongly suggested that the electrospun composite nanofibers containing polymeric micelles can be used as an effective controlled dual release of hydrophobic drugs and were suitable for postoperative chemotherapy of cancers.

Integrative chemical proteomics and cell biology methods to study endocytosis and vesicular trafficking in Arabidopsis.

We present a comprehensive approach combining proteomics and cell biology to study vesicular trafficking in plants. Within this approach, we exploit chemical compounds inhibiting particular vesicular trafficking events in plant cells. Treatment of plants with these relatively specific inhibitors results in intracellular accumulation of proteins being transported by vesicles as well as in a change in abundance of regulatory proteins. Such pharmacological inhibition allows for identification of key proteins, and for further detailed functional investigation using cell biological, molecular biological, and biochemical methods used for validation of proteomic results.

Controlled release of brefeldin A from electrospun PEG-PLLA nanofibers and their in vitro antitumor activity against HepG2 cells.

In this work, PEG-PLLA electrospun fibers were developed as a new controlled release system for macrolide antibiotic drug brefeldin A (BFA). SEM and XRD analyses of the BFA-loaded PEG-PLLA fibers revealed that the average diameter of fibers was below 950 nm with smooth surfaces, and the drug was well incorporated into the fibers in amorphous form. The release profiles of BFA in PBS were measured by HPLC, demonstrating that the controlled release of BFA could be gained for long time. The in vitro antitumor activity against human liver carcinoma HepG2 cells of the fibers containing 3%, 6%, 9%, 12% and 15% BFA were examined by MTT method, and the results showed that cell growth inhibition rates at 72 h were 64%, 77%, 80%, 81% and 85%, respectively. These results strongly suggested that the BFA/PEG-PLLA fibers had an effect of controlled release of BFA and were suitable for postoperative chemotherapy of cancers.

Β-adrenergic receptor stimulation induces endoplasmic reticulum stress in adult cardiac myocytes: role in apoptosis.

Accumulation of misfolded proteins and alterations in calcium homeostasis induces endoplasmic reticulum (ER) stress, leading to apoptosis. In this study, we tested the hypothesis that ß-AR stimulation induces ER stress, and induction of ER stress plays a pro-apoptotic role in cardiac myocytes. Using thapsigargin and brefeldin A, we demonstrate that ER stress induces apoptosis in adult rat ventricular myocytes (ARVMs). ß-AR-stimulation (isoproterenol; 3h) significantly increased expression of ER stress proteins, such as GRP-78, Gadd-153, and Gadd-34, while activating caspase-12 in ARVMs. In most parts, these effects were mimicked by thapsigargin. ß-AR stimulation for 15 min increased PERK and eIF-2α phosphorylation. PERK phosphorylation remained higher, while eIF-2α phosphorylation declined thereafter, reaching to ~50% below basal levels at 3 h after ß-AR stimulation. This decline in eIF-2α phosphorylation was prevented by ß1-AR, not by ß2-AR antagonist. Forskolin, adenylyl cyclase activator, simulated the effects of ISO on eIF-2α phosphorylation. Salubrinal (SAL), an ER stress inhibitor, maintained eIF-2α phosphorylation and inhibited ß-AR-stimulated apoptosis. Furthermore, inhibition of caspase-12 using z-ATAD inhibited ß-AR-stimulated and thapsigargin-induced apoptosis. In vivo, ß-AR stimulation induced ER stress in the mouse heart as evidenced by increased expression of GRP-78 and Gadd-153, activation of caspase-12, and dephosphorylation of eIF-2α. SAL maintained phosphorylation of eIF-2α, inhibited activation of caspase-12, and decreased ß-AR-stimulated apoptosis in the heart. Thus, ß-AR stimulation induces ER stress in cardiac myocytes and in the heart, and induction of ER stress plays a pro-apoptotic role.

Mechanosensitive hyaluronan secretion: stimulus-response curves and role of transcription-translation-translocation in rabbit joints.

Joint movement was recently shown to stimulate the secretion of the lubricant hyaluronan (HA); also, exercise therapy and intra-articular hyaluronan injections are used to treat moderate osteoarthritis. The present study quantifies the stimulus-response curves for HA secretion in vivo and reports a role of transcription-translation-translocation in the secretory response. After washing out endogenous HA from anaesthetized, cannulated rabbit knees, the joints were cycled passively at various frequencies and durations, with or without intra-articular inhibitors of protein synthesis and Golgi processing. Newly secreted HA was harvested for analysis after 5 h. Joints displayed graded, non-linear stimulus-response curves to both duration and frequency of movement; 1 min duration per 15 min or a frequency of 0.17 Hz raised HA secretion by 42-54%, while rapid (1.5 Hz) or prolonged cycling (9 min per 15 min) raised it by 110-130%. Movement-stimulated secretion and phorbol ester-stimulated secretion were partly inhibited by the translation inhibitor cycloheximide, by the transcription-translation inhibitors actinomycin D and puromycin and by the Golgi translocation inhibitor brefeldin A. There is thus a graded coupling between HA secretion and cyclic joint movement that depends partly on new protein synthesis. This is likely to be important for joint homeostasis, providing protection during repetitive cycling and potentially contributing to exercise therapy for osteoarthritis.

Cutting edge: re-evaluating the in vivo cytokine responses of CD8+ T cells during primary and secondary viral infections.

Virus-specific CD8(+) T cells produce IFN-gamma after Ag contact and, in the absence of this cytokine, the host often cannot eradicate infection. However, our ability to identify cells that are actively expressing this critical effector function in vivo is limited, because the protein is rapidly secreted. In this study, we describe a simple approach that circumvents the need for ex vivo Ag stimulation and allows the enumeration of CD8(+) T cells that are actively synthesizing IFN-gamma in vivo during primary and secondary virus infections. The proportion of Ag-specific primary CD8(+) T cells producing IFN-gamma peaks at 5 days postinfection, when the T cell population is still expanding exponentially. In vivo IFN-gamma synthesis by memory cells is explosive, peaking at approximately 12 h after secondary infection and terminating hours thereafter. This technique will be useful when evaluating in vivo immune cell activity in many situations, including a variety of noninfectious (e.g., autoimmune) diseases.

Effect of brefeldin A and lysosomotropic reagents on intracellular trafficking of epidermal growth factor and transferrin in Madin-Darby canine kidney epithelial cells.

To regulate intracellular sorting of epidermal growth factor (EGF) or transferrin (Tf), the effect of brefeldin A (BFA) or lysosomotropic reagents was investigated. To examine the effect of them on the net transcellular transport of 125I-EGF or 125I-Tf, their transcytosis was investigated in the presence or absence of reagents. For the investigation of their fate after internalization, radiolabeled ligands were internalized at 37 degreesC, followed by extensive washing and subsequent incubation at 37 degreesC in the ligand-free medium (pulse-chase study). BFA enhanced transcytosis of 125I-Tf, but had no effect on 125I-EGF. Kinetic analysis in the pulse-chase study showed that BFA does not affect cell-surface binding or intracellular sorting of EGF, while it only increases the transcytosis rate constant of Tf. From the lysosomotropic reagents study, both ammonium chloride and monensin suppressed transcytosis and recycling as well as the degradation of EGF, while both chloroquine and bafilomycin A selectively suppressed the degradation process with only a minimal effect on transcytosis, resulting in an increase in the amount transcytosed. It is concluded the that enhancement effect of BFA on transcytosis depends upon the type of receptor targeted. Lysosomotropic reagents can be divided into two types as far as the specificity of the effect on the net amount of EGF transcytosed in Madin-Darby canine kidney (MDCK) cells is concerned.