Apoptotic and Anti-Inflammatory Effects of Eupatorium japonicum Thunb. in Rheumatoid Arthritis Fibroblast-Like Synoviocytes.

Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease characterized by synovitis, hyperplasia, and the destruction of bone and cartilage. A variety of immunosuppressive biological agents have been developed because the pathogenesis of RA is related predominantly to the inflammatory response. However, rheumatoid arthritis fibroblast-like synovial cells (RAFLS), which are known to play an important role in RA progression, exhibit resistance to immunosuppressants through cancer-like properties. In this study, we identified a novel therapeutic compound for RA, which reduced inflammation and the abnormal proliferation of RAFLS in natural product library made from Korean native plants. Eupatorium japonicum Thunb. (EJT) extract, a component of the natural product library, most effectively reduced viability through the induction of ROS-mediated apoptosis in a dose-dependent manner. In addition, the increased ROS induced the expression of ATF4 and CHOP, key players in ER stress-mediated apoptosis. Interestingly, EJT extract treatment dose-dependently reduced the expression of IL-1ß and the transcription of MMP-9, which were induced by TNF-α treatment, through the inhibition of NF-κB and p38 activation. Collectively, we found that EJT extract exerted apoptotic effects through increases in ROS production and CHOP expression and exerted anti-inflammatory effects through the suppression of NF-κB activation, IL-1ß expression, and MMP-9 transcription.

Angelica gigas Nakai Has Synergetic Effects on Doxorubicin-Induced Apoptosis.

Natural products are valuable sources for drug discovery because they have a wide variety of useful chemical components and biological properties. A quick reevaluation of the potential therapeutic properties of established natural products was made possible by the recent development of the methodology and improvement in the accuracy of an automated high-throughput screening system. In this study, we screened natural product libraries to detect compounds with anticancer effects using HeLa cells. Of the 420 plant extracts screened, the extract of Angelica gigas Nakai (AGN) was the most effective in reducing cell viability of HeLa cells. Markers of apoptosis, such as exposure of phosphatidylserine and cleavage of caspase-7 and PARP, were increased by treatment with the AGN extract. Treatment of the AGN extract increased expression of PKR as well as ATF4 and CHOP, the unfolded protein response genes. In addition, cotreatment of doxorubicin and the AGN extract significantly increased doxorubicin-induced apoptosis in HeLa cells. Decursin and decursinol angelate, which were known to have anticancer effects, were the main components of the AGN extract. These results suggest that the extract of AGN containing, decursin and decursinol angelate, increases doxorubicin susceptibility.

New role of human ribosomal protein S3: Regulation of cell cycle via phosphorylation by cyclin-dependent kinase 2.

Human ribosomal protein S3 (hRpS3) is a component of the 40S ribosomal subunit that associated in protein synthesis. hRpS3 has additional ribosomal functions such as DNA repair, transcription, metastasis, and apoptosis via interaction with numerous signaling molecules and has different modifications. Cyclin-dependent kinases (CDKs) are heterodimeric serine/threonine protein kinases that regulate cell cycle progression. Among its members, the Cdk1-cyclin B complex is known to control cell progression in the G2/M phase, while Cdk2-cyclin E/A complexes function in G1/S and S/G2 transition. In our previous study, we observed interaction between hRpS3 and Cdk1. The present study investigated the interaction between hRpS3 and Cdk2. Cdk2 phosphorylated hRps3 at amino acid residues S6 and T221 during the S-phase. Furthermore, hRpS3 knockdown delayed cell cycle progression by modulating the expression of cell cycle-related proteins, including cyclin B1 and cyclin E1. These findings suggest that hRpS3 is involved in Cdk2-mediated cell cycle regulation.

MutY DNA Glycosylase Protects Cells From Tumor Necrosis Factor Alpha-Induced Necroptosis.

Numerous studies have implied that mutY DNA glycosylase (MYH) is involved in the repair of post-replicative mispairs and plays a critical role in the base excision repair pathway. Recent in vitro studies have shown that MYH interacts with tumor necrosis factor receptor type 1-associated death domain (TRADD), a key effector protein of tumor necrosis factor receptor-1 (TNFR1) signaling. The association between MYH and TRADD is reversed during tumor necrosis factor alpha (TNF-α)- and camptothecin (CPT)-induced apoptosis, and enhanced during TNF-α-induced survival. After investigating the role of MYH interacts with various proteins following TNF-α stimulation, here, we focus on MYH and TRADD interaction functions in necroptosis and its effects to related proteins. We report that the level of the MYH and TRADD complex was also reduced during necroptosis induced by TNF-α and zVAD-fmk. In particular, we also found that MYH is a biologically important necrosis suppressor. Under combined TNF-α and zVAD-fmk treatment, MYH-deficient cells were induced to enter the necroptosis pathway but primary mouse embryonic fibroblasts (MEFs) were not. Necroptosis in the absence of MYH proceeds via the inactivation of caspase-8, followed by an increase in the formation of the kinase receptor- interacting protein 1 (RIP1)-RIP3 complex. Our results suggested that MYH, which interacts with TRADD, inhibits TNF-α necroptotic signaling. Therefore, MYH inactivation is essential for necroptosis via the downregulation of caspase-8. J. Cell. Biochem. 118: 1827-1838, 2017. © 2017 Wiley Periodicals, Inc.

Functional interaction between hMYH and hTRADD in the TNF-α-mediated survival and death pathways of HeLa cells.

UNLABELLED: The tumor necrosis factor (TNF) signaling pathway is a classical immune system pathway that plays a key role in regulating cell survival and apoptosis. The TNF receptor-associated death domain (TRADD) protein is recruited to the death domain of TNF receptor 1 (TNFR1), where it interacts with TNF receptor-associated factor 2 (TRAF2) and receptor-interacting protein (RIP) for the induction of apoptosis, necrosis, nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB), and mitogen-activated protein (MAP) kinase activation. In this study, we found that the human MutY homolog (hMYH) interacted with human TRADD (hTRADD) via the C-terminal domain of hMYH. Moreover, under conditions promoting TNF-α-induced cell death or survival in HeLa cells, this interaction was weakened or enhanced, respectively. The interaction between hMYH and hTRADD was important for signaling pathways mediated by TNF-α. Our results also suggested that the hTRADD-hMYH association was involved in the nuclear translocation of NFκB and formation of the TNFR1-TRADD complex. Thus, this study identified a novel mechanism through which the hMYH-hTRADD interaction may affect the TNF-α signaling pathway. IMPLICATIONS: In HeLa cells, the hTRADD-hMYH interaction functioned in both cell survival and apoptosis pathways following TNF-α stimulation.

Phosphorylation of cyclin O, a novel cyclin family protein containing a cyclin-like domain, is involved in the activation of cyclin-dependent kinase 2.

Cell cycles, ordered series of events modulating cell growth and division, are tightly regulated by complexes containing cyclin-dependent kinases (CDKs) and cyclins. Cyclin O is a novel cyclin family protein which interacts with CDK2. However, the molecular effects of cyclin O on the activity of CDK2 have not been fully evaluated. In this study, an interaction between cyclin O and CDK2 was identified by co-immunoprecipitation and the effect of cyclin O on the kinase activity of CDK2 was investigated using cyclin O point mutants. Co-immunoprecipitation was achieved using using HEK293 human embryonic kidney cells which were transiently transfected with vectors expressing cyclin O and CDK2, which revealed that cyclin O interacted with CDK2, particularly with the active form of endogenous CDK2. Cyclin O was expressed as several different bands with molecular weights between 45 and 50 kDa, possibly due to different post-translational modifications. When co-expressed with CDK2, cyclin O appeared as a band with a molecular weight of 50 kDa. Treatment with calf intestinal phosphatase reduced the intensity of the uppermost band. Mass spectroscopic analysis of cyclin O co-expressed with CDK2 revealed that the 81st serine residue of cyclin O was phosphorylated. The in vitro kinase activity of CDK2 phosphorylating histone H1 was markedly increased in the cells overexpressing cyclin O. This activity was reduced in cells overexpressing cyclin O, in which the 81st serine had been replaced with alanine (S81A). These results suggest that cyclin O is a novel cyclin family protein that regulates CDK2 kinase activity, which is mediated by the phosphorylation of the 81st serine residue of cyclin O.

Human MutY homolog induces apoptosis in etoposide-treated HEK293 cells.

Etoposide (ETP) treatment of ataxia telangiectasia mutated (ATM) and Rad3-related protein (ATR)-, topoisomerase-binding protein-1 (TopBP1) and human MutY homolog (hMYH)-depleted cells results in a significant reduction in apoptotic signaling. The association between ATR or TopBP1 and hMYH increased following ETP treatment. In hMYH knockdown cells, the interaction between ATR and TopBP1 decreased following ETP treatment. We suggest that hMYH functions as a sensor of ETP-induced apoptosis. The results suggest that in the absence of hMYH, cells are unable to recognize the damage signal and the ATR pathway is not activated.

Arresting cancer proliferation by controlling the surface crystallinity of carbon materials without generating reactive oxygen species.

This study demonstrated that the surface crystallinity of carbon nanostructures is an additional independent factor that should be considered for the inhibition of cancer proliferation without activating reactive oxygen species (ROS). In addition, cytotoxic evaluation of both proliferating cancer cells and fully differentiated nerve cells (i.e. non-proliferative) showed selective cytotoxicity: single-walled and highly crystalline carbon nanostructures aggressively inhibited the proliferation of glioma cancer cells, but exhibited no notable cytotoxicity effects on differentiated nerve cells. Although single-wall carbon nanotubes have been shown to elicit potent proinflammatory responses by means of trigger ROS, our results demonstrated that highly crystalline carbon structures can be utilized as a selective antiproliferative agent against brain tumor cells without increasing the ROS level and without significant cytotoxic effects to adjacent nerve cells.

Palmitate promotes the paracrine effects of macrophages on vascular smooth muscle cells: the role of bone morphogenetic proteins.

Saturated fatty acids are known to activate macrophages and induce vascular inflammation. Although cytokines from activated macrophage influence other vascular cells, the influence of saturated fatty acids on the paracrine effect of macrophages is not fully understood yet. Here we examined the impact of palmitate on the effect of macrophages on vascular smooth muscle cells (SMCs) and their mediators. SMCs proliferation increased significantly after treatment with conditioned media from palmitate-stimulated RAW264.7 cells. SMC migration was found to be greater after treatment with palmitate-conditioned media. SM α-actin and SM22α were decreased in SMCs treated with palmitate-conditioned media. When stimulated with palmitate, RAW264.7 cells secreted more bone morphogenetic protein (BMP)2 and BMP4 into the cell culture media. SMC proliferation, migration, and phenotypic changes were attenuated after treatment of neutralizing antibodies against BMPs or knockdown of BMPs with siRNA. The influences of these proteins were further confirmed by direct treatment of recombinant BMP2 and BMP4 on SMCs. Particularly, the effects of BMPs on SMC migration on phenotypic change were obvious, whereas their effect on SMC proliferation seemed not significant or modest. In conclusion, palmitate promoted macrophages' paracrine effects on SMC proliferation, migration, and phenotypic change. The effect of stimulated macrophages was mediated, at least in part, by BMP2 and BMP4. These results suggest a novel mechanism linking saturated fatty acids and the progression of vascular diseases that is possibly mediated by BMPs from macrophages.

Ubiquitin ligase CHIP induces TRAF2 proteasomal degradation and NF-κB inactivation to regulate breast cancer cell invasion.

Transcriptional factor nuclear factor-kappaB (NF-κB) plays a crucial role in human breast cancer cell invasion and metastasis. The carboxyl terminus of Hsc70-interacting protein (CHIP) is a U-box-type ubiquitin ligase that induces ubiquitination and proteasomal degradation of its substrate proteins. In this study, we investigated the role of CHIP in the NF-κB pathway in the invasion of MDA-MB-231 cells, a highly aggressive breast cancer cell line. We showed that overexpression of CHIP significantly inhibits the invasion of the MDA-MB-231 cells. The overexpression of CHIP suppressed expression of urokinase plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP-9) in MDA-MB-231 cells. Moreover, CHIP strongly inhibited the nuclear localization and the transcriptional activity of NF-κB. The activation of the IkappaB kinase complex (IKK) was also blocked by CHIP overexpression. Importantly, CHIP overexpression resulted in a significant decrease in the level of TNF receptor-associated factor 2 (TRAF2), an upstream key player in the NF-κB pathway. However, the level of TRAF2 was restored after treatment with a proteasome inhibitor, MG-132. Moreover, CHIP overexpression promoted the ubiquitination of TRAF2. We also found cell invasion significantly decreased in cells transfected with TRAF2 small interfering RNA (siRNA). In contrast, when CHIP expression was suppressed by siRNA in poorly invasive MCF-7 cells, cell invasion significantly increased in conjunction with enhanced NF-κB activation and TRAF2 levels. Taken together, these results suggest that CHIP regulates NF-κB-mediated cell invasion via the down-regulation of TRAF2.

Induction of apoptotic cell death by synthetic naringenin derivatives in human lung epithelial carcinoma A549 cells.

Although flavonoids, which are both qualitatively and quantitatively one of the largest groups of natural products, exhibit a variety of beneficial health effects, the exact molecular mechanism of the cellular activities is still not fully explained and there currently exists a lack of evidence for any relationship between the structure-activity relationship and apoptosis-inducing activity. In order to determine the importance of the OH group or substitution of the 5 or carbon-7 in the diphenylpropane skeleton of flavonoids, we originally synthesized several modified naringenin derivatives, including 7-O-benzyl naringenin (KUF-1) and 7-O-(MeO-L-Leu-D-Pro-carbonylmethyl) naringenin (KUF-7). Treatment with KUF-1 or KUF-7 resulted in significant apoptosis-inducing effects concomitant with chromatin condensation, caspase activation, and intracellular ROS production. Our data indicate that originally synthesized naringenin derivatives, KUF-1 and KUF-7 differentially regulate the apoptosis of A549 cells via intracellular ROS production coupled with the concomitant activation of the caspase cascade signaling pathway, thereby implying that hydroxylation or substitution at Carbon-7 is critical for the apoptosis-inducing activity of flavonoids.

Genistein-induced apoptosis of human breast cancer MCF-7 cells involves calpain-caspase and apoptosis signaling kinase 1-p38 mitogen-activated protein kinase activation cascades.

The molecular mechanisms of genistein-induced apoptosis of human breast cancer MCF-7 cells were investigated. Genistein showed 50% cell growth inhibition at IC50=27.5+/-0.8 micromol/l in 24 h incubation under 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay conditions. Genistein is known to express both cell growth activity at nanomolar concentrations and anti-cell growth activity at micromolar concentrations. It was found that genistein at 100 micromol/l concentration effectively induced apoptosis of MCF-7 cells in 24 h. Genistein-induced apoptosis involved activation of calpain, caspase 7 and poly(ADP ribose) polymerase. Dantrolene, an inhibitor of Ca release from the endoplasmic reticulum, inhibited genistein-induced activation of calpain and caspase 7, in addition to effectively negating genistein-induced apoptosis. MCF-7 cells treated with genistein also showed increased phosphorylation of p38 mitogen-activated protein kinase, whereas no effect was observed for extracellular signal-regulating kinase 1/2. Phosphorylation of apoptosis signaling kinase 1, an upstream regulator of p38 mitogen-activated protein kinase, was also increased by genistein treatment. Genistein-induced phosphorylation of apoptosis signaling kinase 1 and p38 mitogen-activated protein kinase was diminished by the presence of dantrolene. These results suggest that genistein-induced apoptosis in MCF-7 cells is mediated through calpain-caspase 7 and apoptosis signaling kinase 1-p38 mitogen-activated protein kinase activation cascades that involve Ca release from the endoplasmic reticulum.

Acacetin-induced apoptosis of human breast cancer MCF-7 cells involves caspase cascade, mitochondria-mediated death signaling and SAPK/JNK1/2-c-Jun activation.

The mechanism of acacetin-induced apoptosis of human breast cancer MCF-7 cells was investigated. Acacetin caused 50% growth inhibition (IC50) of MCF-7 cells at 26.4% 0.7% M over 24 h in the MTT assay. Apoptosis was characterized by DNA fragmentation and an increase of sub-G1 cells and involved activation of caspase-7 and PARP (poly-ADP-ribose polymerase). Maximum caspase 7 activity was observed with 100 microM acacetin for 24 h. Caspase 8 and 9 activation cascades mediated the activation of caspase 7. Acacetin caused a reduction of Bcl-2 expression leading to an increase of the Bax:Bcl-2 ratio. It also caused a loss of mitochondrial membrane potential that induced release of cytochrome c and apoptosis inducing factor (AIF) into the cytoplasm, enhancing ROS generation and subsequently resulting in apoptosis. Pretreatment of cells with N-acetylcysteine (NAC) reduced ROS generation and cell growth inhibition, and pretreatment with NAC or a caspase 8 inhibitor (Z-IETD-FMK) inhibited the acacetin-induced loss of mitochondrial membrane potential and release of cytochrome c and AIF. Stress-activated protein kinase/c-Jun NH4-terminal kinase 1/2 (SAPK/ JNK1/2) and c-Jun were activated by acacetin but extracellular-regulated kinase 1/2 (Erk1/2) nor p38 mitogen-activated protein kinase (MAPK) were not. Our results show that acacetin-induced apoptosis of MCF-7 cells is mediated by caspase activation cascades, ROS generation, mitochondria-mediated cell death signaling and the SAPK/JNK1/2-c-Jun signaling pathway, activated by acacetin-induced ROS generation.

Alzheimer's disease drug discovery from herbs: neuroprotectivity from beta-amyloid (1-42) insult.

OBJECTIVE: To comparatively evaluate selected herbs for their ability to protect neuronal cells from direct betaA(1-42) insult. DESIGN: Twenty-seven (27) herbs were selected, extracted with aqueous methanol (90%) and chloroform, and the extracts were evaluated for their ability to protect PC12 rat pheochromocytoma and primary neuronal cells from betaA(1-42) insult using both 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide reduction assay and lactate dehydrogenase efflux assay. RESULTS: Curcuma aromatia (ul-keum) and Zingiber officinale (ginger) extracts effectively protected cells from betaA(1-42) insult, followed by Ginkgo biloba (ginkgo), Polygonatum sp. (King Solomon's seal), Cinnamum cassia (Chinese cinnamon), Rheum coreanum (Korean rhubarb), Gastrodia elata (gastrodia), and Scutellaria baicalensis (skullcap). Several extracts showed cytotoxicity at high concentration (approximately 150 microg/mL), whereas other extracts did not at all protect cells from betaA(1-42) insult. CONCLUSION: Selective herbs may be potentially important resources to discover drug candidates against the onset of Alzheimer's disease.

T-type calcium channel trigger p21ras signaling pathway to ERK in Cav3.1-expressed HEK293 cells.

We constructed a new cell line which stably expressed Cav3.1 and Kir2.1 subunits in HEK293 cells (HEK293/Cav3.1/Kir2.1) in order to investigate the unknown cellular signaling pathways of T-type voltage-dependent calcium channels. The new cell line has a stable resting membrane potential and can activate T-type Ca(2+) channels by KCl-mediated depolarization. We showed that Cav3.1 activation resulted in the level of p21(ras)-GTP in the cells being rapidly decreased during the first 2 min, and then recovering between 2 min and 15 min. The kinetics of ERK activation following Cav3.1 stimulation was also investigated. ERK activation was decreased from 2 min to 5 min after KCl stimulation, which means that Cav3.1 activation reduced ERK activity in the very early stages of activation. In addition, similar results for Cav3.1 activation were also shown in the case of Sos1, Grb2, and Shc, which means that Cav3.1 activation triggers p21(ras) and that this signal is transferred to ERK by Sos1, Grb2, and Shc.

Molecular cloning and characterization of an NADPH quinone oxidoreductase from Kluyveromyces marxianus.

NAD(P)H quinone oxidoreductase is a ubiquitous enzyme that is known to directly reduce quinone substrates to hydroquinones by a two-electron reaction. We report the identification of NADPH quinone oxidoreductase from Kluyveromyces marxianus (KmQOR), which reduces quinone substrates directly to hydroquinones. The KmQOR gene was sequenced, expressed in Escherichia coli, purified, and characterized. The open-reading frame of the KmQOR gene consists of 1143 nucleotides, encoding a 380 amino acid polypeptide. The nucleotide sequence of the KmQOR gene was assigned to EMBL under accession number AY040868. The M(r) that was determined by SDS-PAGE for the protein subunit was about 42 kDa, and the molecular mass of the native KmQOR was 84 kDa, as determined by column calibration, indicating that the native protein is a homodimer. The KmQOR protein efficiently reduced 1,4-benzoquinone, whereas no activities were found for menadiones and methoxyquinones. These observations, and the result of an extended sequence analysis of known NADPH quinone oxidoreductase, suggest that KmQOR possesses a different action mechanism.

Association of hepatitis B virus polymerase with promyelocytic leukemia nuclear bodies mediated by the S100 family protein p11.

Hepatitis B virus (HBV) polymerase (Pol) interacts with cellular chaperone proteins and thereby performs multiple functions necessary for viral replication. Yeast two-hybrid analysis was applied to identify additional cellular targets required for HBV Pol function. HBV Pol interacted with S100A10 (p11), a Ca(2+)-modulated protein previously shown to bind to annexin II. The interaction between HBV Pol and p11 was confirmed by co-immunoprecipitation of the two proteins synthesized either in vitro or in transfected cells and by inhibition of the DNA polymerase activity of HBV Pol by p11. Immunofluorescence analysis of transfected human cell lines revealed that, although most HBV Pol and p11 was restricted to the cytoplasm, a small proportion of each protein colocalized as nuclear speckles; HBV Pol was not detected in the nucleus in the absence of p11. The HBV Pol-p11 nuclear speckles coincided with nuclear bodies containing the promyelocytic leukemia protein PML. Furthermore, the association of HBV Pol-p11 with PML was increased by exposure of cells to EGTA and inhibited by valinomycin. These results suggest a role for p11 in modulation of HBV Pol function and implicate PML nuclear bodies and intracellular Ca(2+) in viral replication.

Expression of the active human and duck hepatitis B virus polymerases in heterologous system of Pichia methanolica.

We expressed the Hepatitis B virus polymerase (HBV P protein) using a recently introduced yeast system, Pichia methanolica. HBV (1-680 amino acids) and Duck Hepatitis B virus (DHBV, 1-780 amino acids) polymerase were expressed and showed DNA dependent DNA polymerase (DDDP). The DHBV polymerase had RNA dependent DNA polymerase (RDDP) and RNase H activities. We present a new simplified way of obtaining active viral P protein using the yeast expression system. The viral P proteins proved to be stable and were not aggregated in the yeast system.