Synergistic Anti-Cancer Effects of ERB-041 and Genistein through Estrogen Receptor Suppression-Mediated PI3K/AKT Pathway Downregulation in Canine Mammary Gland Tumor Cells.

Canine-mammary-gland tumors (CMTs) are prevalent in female dogs, with approximately 50% of them being malignant and often presenting as inoperable owing to their size or metastasis. Owing to poor outcomes, effective alternatives to conventional chemotherapy for humans are necessary. Two estrogen receptors, estrogen receptor alpha (ERα) and estrogen receptor beta (ERß), which act in opposition to each other, are involved, and CMT growth involves ERα through the phosphoinositide 3-kinases (PI3K)/AKT pathway. In this study, we aimed to identify the synergistic anti-cancer effects of ERB-041, an ERß agonist, and genistein, an isoflavonoid from soybeans known to have ERß-specific pseudo-estrogenic actions, on CMT-U27 and CF41.Mg CMT cell lines. ERB-041 and genistein synergistically inhibited cell proliferation and increased the number of annexin V-positive cells in both cell lines. Furthermore, we observed a synergistic increase in the Bax/Bcl-2 ratio and cleaved caspase-3 expression. Additionally, cell-cycle arrest occurred through the synergistic regulation of cyclin D1 and cyclin-dependent kinase 4 (CDK4). We also found a synergistic decrease in the expression of ERα, and the expression of proteins involved in the PI3K/AKT pathway, including p-PI3K, phosphatase and tensin homolog (PTEN), AKT, and mechanistic target of rapamycin (mTOR). In conclusion, ERB-041 and genistein exhibited a synergistic anticancer effect on CMTs, suggesting that cotreatment with ERB-041 and genistein is a promising treatment for CMTs.

Melatonin inhibits Japanese encephalitis virus replication and neurotoxicity via calcineurin-autophagy pathways.

BACKGROUND: Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that has no specific treatment except for supportive medical care. JEV is a neurotropic virus that affects the nervous system and triggers inflammation in the brain. METHODS: Melatonin is used as a sleep-inducing agent in neurophysiology and may serve as a protective agent against neurological and neurodegenerative diseases. Herein, we investigated the effects of melatonin and the critical roles of the serine/threonine protein phosphatase calcineurin during JEV infection in SK-N-SH neuroblastoma cells. RESULTS: Melatonin treatment decreased JEV replication and JEV-mediated neurotoxicity. Calcineurin activity was increased by JEV infection and inhibited by melatonin treatment. Through calcineurin regulation, melatonin decreased the JEV-mediated neuroinflammatory response and attenuated JEV-induced autophagy. CONCLUSIONS: Calcineurin inactivation has a protective effect in JEV-infected neuronal cells, and melatonin is a novel resource for the development of anti-JEV agents.

Anti-cancer effect of palmatine through inhibition of the PI3K/AKT pathway in canine mammary gland tumor CMT-U27 cells.

Canine mammary gland tumors (CMTs) are the most common and lethal cancers in female dogs. Dysregulated phosphoinositide 3-kinases (PI3K)/AKT pathway reportedly was involved in the growth and metastasis of CMTs. However, there are few studies on therapeutic strategies for targeting the PI3K pathway in CMTs. In this study, we aimed to determine whether palmatine, a natural isoquinoline alkaloid with anti-cancer properties, could inhibit the growth of CMTs and whether the inhibitory effect was mediated through the PI3K/AKT pathway. Our in vitro experiments on CMT-U27, a CMT cell line, showed that palmatine reduced cell proliferation and induced cell death. Western blotting results revealed that palmatine decreased the protein expression of PI3K, PTEN, AKT, and mechanistic target of rapamycin in the PI3K/AKT pathway, which was supported by the results of immunocytochemistry. Additionally, palmatine suppressed the migration and tube formation of canine aortic endothelial cells as well as the migration of CMT U27 cells. Our in vivo results showed that palmatine inhibited tumor growth in a CMT-U27 mouse xenograft model. We observed a decreased expression of proteins in the PI3K/AKT pathway in tumor tissues, similar to the in vitro results. Furthermore, palmatine significantly disrupted the tumor vasculature and inhibited metastasis to adjacent lymph nodes. In conclusion, our findings demonstrate that palmatine exerts anti-cancer effects against CMTs by inhibiting PI3K/AKT signaling pathway, suggesting that palmatine has potential as a canine-specific PI3K inhibitor for the treatment of CMTs.

Prion peptide-mediated calcium level alteration governs neuronal cell damage through AMPK-autophagy flux.

BACKGROUND: The distinctive molecular structure of the prion protein, PrPsc, is established only in mammals with infectious prion diseases. Prion protein characterizes either the transmissible pathogen itself or a primary constituent of the disease. Our report suggested that prion protein-mediated neuronal cell death is triggered by the autophagy flux. However, the alteration of intracellular calcium levels, AMPK activity in prion models has not been described. This study is focused on the effect of the changes in intracellular calcium levels on AMPK/autophagy flux pathway and PrP (106-126)-induced neurotoxicity. METHODS: Western blot and Immunocytochemistry was used to detect AMPK and autophagy-related protein expression. Flow cytometry and a TdT-mediated biotin-16-dUTP nick-end labeling (TUNEL) assay were used to detect the percentage of apoptotic cells. Calcium measurement was employed using fluo-4 by confocal microscope. RESULTS: We examined the effect of calcium homeostasis alterations induced by human prion peptide on the autophagy flux in neuronal cells. Treatment with human prion peptide increased the intracellular calcium concentration and induced cell death in primary neurons as well as in a neuronal cell line. Using pharmacological inhibitors, we showed that the L-type calcium channel is involved in the cellular entry of calcium ions. Inhibition of calcium uptake prevented autophagic cell death and reduction in AMP-activated protein kinase (AMPK) activity induced by human prion peptide. CONCLUSION: Our data demonstrated that prion peptide-mediated calcium inflow plays a pivotal role in prion peptide-induced autophagic cell death, and reduction in AMPK activity in neurons. Altogether, our results suggest that calcium influx might play a critical role in neurodegenerative diseases, including prion diseases. Video Abstract.

Naringenin exerts anticancer effects by inducing tumor cell death and inhibiting angiogenesis in malignant melanoma.

Malignant melanoma is one of the most deadly skin cancer, due to its aggressive proliferation and metastasis. Naringenin, abundantly present in citrus fruits, has widely studied in cancer therapy. In this study, we investigated whether naringenin also has anticancer effects against B16F10 murine and SK-MEL-28 human melanoma cells. Moreover, we assessed the effects of naringenin treatment on angiogenesis of HUVECs and ex vivo sprouting of microvessels.Naringenin inhibited tumor cell proliferation and migration in a dose-dependent manner in B16F10 and SK-MEL-28 cells, which is supported by the results that phosphorylation of ERK1/2 and JNK MAPK decreased. Furthermore, naringenin induced cell apoptosis. Western blot analysisshowed naringenin treatment significantly upregulated the protein expression of activated cas3 and PARP in B16F10 and SK-MEL-28 cells. In addition, in vitro and ex vivo angiogenesis assays demonstrated that naringenin treatment potently suppressed EC migration, tube formation, and sprouting of microvessels. RT-PCR analysis showed that naringenin treatment significantly reduced the mRNA expression of Tie2, but did not inhibit the expression of Ang2. In conclusion, present study demonstrates the anticancer effects of naringenin by its induction of tumor cell death and inhibition of angiogenesis in malignant melanoma, suggesting that naringenin has potential as a safe and effective therapeutic agent to treat melanoma.

Cardiac glycoside sensitized hepatocellular carcinoma cells to TRAIL via ROS generation, p38MAPK, mitochondrial transition, and autophagy mediation.

A major concern in the clinical application of tumor necrosis factor related apoptosis-inducing ligand (TRAIL) in tumors is the development of resistance. Therefore, agents that can potentially restore TRAIL sensitivity are important therapeutic targets for cancer treatment. Herein, we evaluated lanatoside c and digoxin, both of which are widely used cardiac glycosides (CGs), for their ability to sensitize human hepatocellular carcinoma cells (Huh-7 and HepG2) through TRAIL-induced apoptosis. CGs functionalize TRAIL as shown by its effect on intracellular reactive oxygen species (ROS) generation, which damages mitochondrial integrity and thereby confers intrinsic apoptotic caspase cascade during combined treatment. Caspase activation is dependent on ROS as shown by the ability of CGs to generate ROS and the ROS-N-acetylcysteine (NAC) relationship, which inhibits apoptosis during cotreatment by preventing the formation of caspase-8 and -3. Furthermore, CGs triggered p38MAPK phosphorylation and NAC pre-exposure blocked p38MAPK phosphorylation, which demonstrated that p38MAPK was dependent upon ROS generation. Additionally, CGs were found to be potent inducers of AMPK-mediated protective autophagy as pharmacological and genetic autophagy inhibition reached the higher threshold of TRAIL-mediated apoptosis. Finally, CGs downregulated the expression of the antiapoptotic protein Bcl-2 and increased the translocation of proapoptotic protein cytochrome c, thereby inducing apoptosis. Collectively, these results indicate that CGs potentiate the enhanced cytotoxic capacity to TRAIL through ROS generation, p38MAPK phosphorylation, cell survival protein downregulation, and protective autophagy inhibition.

Antihypertensive drug-candesartan attenuates TRAIL resistance in human lung cancer via AMPK-mediated inhibition of autophagy flux.

Angiotensin II type 1 receptor blockers (ARBs) are widely used as antihypertensive drugs. Candesartan is an ARB that has also been known for its anticancer effects but the exact molecular mechanism is remaining elusive. In this research, we showed for the first time that candesartan treatment significantly sensitized human lung adenocarcinoma cells to Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis by targeting TRAIL-DR5. TRAIL selectively kills cancer cells by binding to death receptors on the cell membrane, beyond the levels causing minimal toxicity in normal cells. However, some non-small-cell lung carcinoma (NSCLC) patients are resistant to TRAIL treatment in clinical trials due to inactivation of the death receptors during cytoprotective autophagy. The molecular mechanisms underlying candesartan-induced TRAIL-mediated apoptosis involved the downstream of AMPK phosphorylation resulting inhibition of autophagy flux, recruitment of death receptor 5 (DR5) and activation of apoptotic caspase cascade. Candesartan treatment also inhibits the expression of anti-apoptotic protein c-FLIP. Furthermore, blocking DR5 signaling using DR5 siRNA negatively regulated the apoptotic pathway and also induced autophagy flux, demonstrating the cytoprotective role of autophagy responsible for treatment resistance. This suggests that candesartan can be used to sensitize tumors to TRAIL treatment and may represent a useful strategy for human adenocarcinoma patients to overcome TRAIL resistance. Candesartan in combination with TRAIL also could be a novel therapeutic treatment for patients presenting both conditions of hypertension and lung cancer.

CCL2, but not its receptor, is essential to restrict immune privileged central nervous system-invasion of Japanese encephalitis virus via regulating accumulation of CD11b(+) Ly-6C(hi) monocytes.

Japanese encephalitis virus (JEV) is a re-emerging zoonotic flavivirus that poses an increasing threat to global health and welfare due to rapid changes in climate and demography. Although the CCR2-CCL2 axis plays an important role in trafficking CD11b(+) Ly-6C(hi) monocytes to regulate immunopathological diseases, little is known about their role in monocyte trafficking during viral encephalitis caused by JEV infection. Here, we explored the role of CCR2 and its ligand CCL2 in JE caused by JEV infection using CCR2- and CCL2-ablated murine models. Somewhat surprisingly, the ablation of CCR2 and CCL2 resulted in starkly contrasting susceptibility to JE. CCR2 ablation induced enhanced resistance to JE, whereas CCL2 ablation highly increased susceptibility to JE. This contrasting regulation of JE progression by CCR2 and CCL2 was coupled to central nervous system (CNS) infiltration of Ly-6C(hi) monocytes and Ly-6G(hi) granulocytes. There was also enhanced expression of CC and CXC chemokines in the CNS of CCL2-ablated mice, which appeared to induce CNS infiltration of these cell populations. However, our data revealed that contrasting regulation of JE in CCR2- and CCL2-ablated mice was unlikely to be mediated by innate natural killer and adaptive T-cell responses. Furthermore, CCL2 produced by haematopoietic stem cell-derived leucocytes played a dominant role in CNS accumulation of Ly-6C(hi) monocytes in infected bone marrow chimeric models, thereby exacerbating JE progression. Collectively, our data indicate that CCL2 plays an essential role in conferring protection against JE caused by JEV infection. In addition, blockage of CCR2, but not CCL2, will aid in the development of strategies for prophylactics and therapeutics of JE.

CCR5 ameliorates Japanese encephalitis via dictating the equilibrium of regulatory CD4(+)Foxp3(+) T and IL-17(+)CD4(+) Th17 cells.

BACKGROUND: CCR5 is a CC chemokine receptor involved in the migration of effector leukocytes including macrophages, NK, and T cells into inflamed tissues. Also, the role of CCR5 in CD4(+)Foxp3(+) regulatory T cell (Treg) homing has recently begun to grab attention. Japanese encephalitis (JE) is defined as severe neuroinflammation of the central nervous system (CNS) following infection with mosquito-borne flavivirus JE virus. However, the potential contribution of CCR5 to JE progression via mediating CD4(+)Foxp3(+) Treg homing has not been investigated. METHODS: Infected wild-type (Ccr5(+/+)) and CCR5-deficient (Ccr5(-/-)) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, NK- and JEV-specific T cell responses were analyzed. Adoptive transfer of CCR5(+)CD4(+)Foxp3(+) Tregs was used to evaluate the role of Tregs in JE progression. RESULTS: CCR5 ablation exacerbated JE without altering viral burden in the extraneural and CNS tissues, as manifested by increased CNS infiltration of Ly-6C(hi) monocytes and Ly-6G(hi) granulocytes. Compared to Ccr5(+/+) mice, Ccr5(-/-) mice unexpectedly showed increased responses of IFN-γ(+)NK and CD8(+) T cells in the spleen, but not CD4(+) T cells. More interestingly, CCR5-ablation resulted in a skewed response to IL-17(+)CD4(+) Th17 cells and correspondingly reduced CD4(+)Foxp3(+) Tregs in the spleen and brain, which was closely associated with exacerbated JE. Our results also revealed that adoptive transfer of sorted CCR5(+)CD4(+)Foxp3(+) Tregs into Ccr5(-/-) mice could ameliorate JE progression without apparently altering the viral burden and CNS infiltration of IL-17(+)CD4(+) Th17 cells, myeloid-derived Ly-6C(hi) monocytes and Ly-6G(hi) granulocytes. Instead, adoptive transfer of CCR5(+)CD4(+)Foxp3(+) Tregs into Ccr5(-/-) mice resulted in increased expression of anti-inflammatory cytokines (IL-10 and TGF-ß) in the spleen and brain, and transferred CCR5(+) Tregs were found to produce IL-10. CONCLUSIONS: CCR5 regulates JE progression via governing timely and appropriate CNS infiltration of CD4(+)Foxp3(+) Tregs, thereby facilitating host survival. Therefore, this critical and extended role of CCR5 in JE raises possible safety concerns regarding the use of CCR5 antagonists in human immunodeficiency virus (HIV)-infected individuals who inhabit regions in which both HIV and flaviviruses, such as JEV and West Nile virus, are endemic.

Blockage of indoleamine 2,3-dioxygenase regulates Japanese encephalitis via enhancement of type I/II IFN innate and adaptive T-cell responses.

BACKGROUND: Japanese encephalitis (JE), a leading cause of viral encephalitis, is characterized by extensive neuroinflammation following infection with neurotropic JE virus (JEV). Indoleamine 2,3-dioxygenase (IDO) has been identified as an enzyme associated with immunoregulatory function. Although the regulatory role of IDO in viral replication has been postulated, the in vivo role of IDO activity has not been fully addressed in neurotropic virus-caused encephalitis. METHODS: Mice in which IDO activity was inhibited by genetic ablation or using a specific inhibitor were examined for mortality and clinical signs after infection. Neuroinflammation was evaluated by central nervous system (CNS) infiltration of leukocytes and cytokine expression. IDO expression, viral burden, JEV-specific T-cell, and type I/II interferon (IFN-I/II) innate responses were also analyzed. RESULTS: Elevated expression of IDO activity in myeloid and neuron cells of the lymphoid and CNS tissues was closely associated with clinical signs of JE. Furthermore, inhibition of IDO activity enhanced resistance to JE, reduced the viral burden in lymphoid and CNS tissues, and resulted in early and increased CNS infiltration by Ly-6C(hi) monocytes, NK, CD4(+), and CD8(+) T-cells. JE amelioration in IDO-ablated mice was also associated with enhanced NK and JEV-specific T-cell responses. More interestingly, IDO ablation induced rapid enhancement of type I IFN (IFN-I) innate responses in CD11c(+) dendritic cells (DCs), including conventional and plasmacytoid DCs, following JEV infection. This enhanced IFN-I innate response in IDO-ablated CD11c(+) DCs was coupled with strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7, STAT1), and antiviral ISG genes (Mx1, Mx2, ISG49, ISG54, ISG56). IDO ablation also enhanced the IFN-I innate response in neuron cells, which may delay the spread of virus in the CNS. Finally, we identified that IDO ablation in myeloid cells derived from hematopoietic stem cells (HSCs) dominantly contributed to JE amelioration and that HSC-derived leukocytes played a key role in the enhanced IFN-I innate responses in the IDO-ablated environment. CONCLUSIONS: Inhibition of IDO activity ameliorated JE via enhancement of antiviral IFN-I/II innate and adaptive T-cell responses and increased CNS infiltration of peripheral leukocytes. Therefore, our data provide valuable insight into the use of IDO inhibition by specific inhibitors as a promising tool for therapeutic and prophylactic strategies against viral encephalitis caused by neurotropic viruses.

Amelioration of Japanese encephalitis by blockage of 4-1BB signaling is coupled to divergent enhancement of type I/II IFN responses and Ly-6C(hi) monocyte differentiation.

BACKGROUND: Japanese encephalitis (JE), a neuroinflammation caused by zoonotic JE virus, is the major cause of viral encephalitis worldwide and poses an increasing threat to global health and welfare. To date, however, there has been no report describing the regulation of JE progression using immunomodulatory tools for developing therapeutic strategies. We tested whether blocking the 4-1BB signaling pathway would regulate JE progression using murine JE model. METHODS: Infected wild-type and 4-1BB-knockout (KO) mice were examined daily for mortality and clinical signs, and neuroinflammation in the CNS was evaluated by infiltration of inflammatory leukocytes and cytokine expression. In addition, viral burden, JEV-specific T cell, and type I/II IFN (IFN-I/II) innate responses were analyzed. RESULTS: Blocking the 4-1BB signaling pathway significantly increased resistance to JE and reduced viral burden in extraneural tissues and the CNS, rather than causing a detrimental effect. In addition, treatment with 4-1BB agonistic antibody exacerbated JE. Furthermore, JE amelioration and reduction of viral burden by blocking the 4-1BB signaling pathway were associated with an increased frequency of IFN-II-producing NK and CD4(+) Th1 cells as well as increased infiltration of mature Ly-6C(hi) monocytes in the inflamed CNS. More interestingly, DCs and macrophages derived from 4-1BB KO mice showed potent and rapid IFN-I innate immune responses upon JEV infection, which was coupled to strong induction of PRRs (RIG-I, MDA5), transcription factors (IRF7), and antiviral ISG genes (ISG49, ISG54, ISG56). Further, the ablation of 4-1BB signaling enhanced IFN-I innate responses in neuron cells, which likely regulated viral spread in the CNS. Finally, we confirmed that blocking the 4-1BB signaling pathway in myeloid cells derived from hematopoietic stem cells (HSCs) played a dominant role in ameliorating JE. In support of this finding, HSC-derived leukocytes played a dominant role in generating the IFN-I innate responses in the host. CONCLUSIONS: Blocking the 4-1BB signaling pathway ameliorates JE via divergent enhancement of IFN-II-producing NK and CD4(+) Th1 cells and mature Ly-6C(hi) monocyte infiltration, as well as an IFN-I innate response of myeloid-derived cells. Therefore, regulation of the 4-1BB signaling pathway with antibodies or inhibitors could be a valuable therapeutic strategy for the treatment of JE.

Melatonin regulates the autophagic flux via activation of alpha-7 nicotinic acetylcholine receptors.

Our previous study suggested that melatonin-mediated neuroprotective effects are related with the activation of autophagy. However, the mechanism of melatonin-mediated autophagic activation in prion-mediated mitochondrial damage is not reported. Alpha-7 nicotinic acetylcholine receptors (α7nAchR) is a member of nicotinic acetylcholine receptors, and α7nAchR activation regulates via melatonin. Thus, we hypothesized that melatonin-mediated neuroprotective effect related with to autophagy pathway as a result of α7nAchR regulation. Inactivation of α7nAchR inhibited melatonin-mediated autophagic activation and protective effect against prion-mediated mitochondrial neurotoxicity. Also, knockdown of ATG5 blocked the melatonin-mediated neuroprotection and did not influence to the activation of α7nAchR caused by melatonin. This report is the first study demonstrating that melatonin-mediated autophagic activation regulates via modulation of α7nAchR signals, and upregulation of α7nAchR signals induced by melatonin plays a pivotal role in neuroprotection of prion-mediated mitochondrial neurotoxicity. Our results suggested that regulator of α7 nAChR signals including melatonin may have used for neuroprotective strategies for the neurodegenerative disorders including prion diseases.

Utilization of a Modified Phage E Protein Lysis System Accounts for Increased Biomass in Salmonella Gallinarum Ghosts.

A major limiting issue of bacterial ghost technology involves the stable maintenance of Phix174 lysis gene E expression. Unwanted leaky expression of gene E in the absence of induction temperature results in reduced biomass production of host bacterium, consequently leading to the lower yield of bacterial ghost. To mitigate the leaky expression status of lysis gene E, we utilized a novel E-lysis system in which gene E is located between sense λpR promoter with a CI857 regulator and antisense ParaBAD promoter with the AraC regulator. In the presence of L-arabinose at 28 C, unwanted transcription of lysis gene E from λpR promoter is repressed by a simultaneous transcription event from ParaBAD promoter by means of anti-sense RNA-mediated inhibition. Tight repression of lysis gene E in the absence of induction temperature resulted in higher bacterial cell number in culture suspension and, consequently, higher production of Salmonella Gallinarum (SG) ghost biomass. The safety and protective efficacy of the SG ghost vaccine were further examined in chickens. All of the immunized chickens showed significantly higher mucosal and systemic antibody responses accompanied by a potent antigen-specific lymphocyte proliferative response. Vaccination of chickens with SG ghost preparation offered efficient protection against wild-type SG challenge.

Evaluation of Salmonella Gallinarum ghost formulated with Montanide™ ISA 70 VG adjuvant as a vaccine against fowl typhoid.

Escherichia coli heat-labile enterotoxin B subunit (LTB) protein is a potent adjuvant. Salmonella Gallinarum ghosts carrying LTB (S. Gallinarum-LTB ghosts) were genetically constructed using a plasmid, pJHL187-LTB, designed for the co-expression of the LTB and E lysis proteins. This study evaluates the immunopotentiating effects of Montanide™ ISA 70 VG on S. Gallinarum-LTB ghost vaccination against fowl typhoid. Five-week-old layer chickens were injected intramuscularly with sterile PBS (non-immunised control, Group A), S. Gallinarum-LTB ghost (Group B) or S. Gallinarum-LTB ghost emulsified with Montanide™ ISA 70 VG adjuvant (Group C). Chickens from both Groups B and C showed significant induction of antigen-specific systemic IgG response compared to controls; in addition, Group C showed enhanced induction of systemic IgG response compared to Group B. We observed significant induction of antigen-specific lymphocyte proliferative response and increased mRNA levels of Th1 cytokines (IFN-γ and IL2) in both Groups B and C. Furthermore, in the challenge experiment with a virulent strain of S. Gallinarum, Group C showed higher survival rates compared with other groups. These results indicate that vaccination with the S. Gallinarum-LTB ghost in combination with Montanide™ ISA 70 VG may enhance the protective immunity against fowl typhoid.

Endogenous MMP-9 and not MMP-2 promotes rheumatoid synovial fibroblast survival, inflammation and cartilage degradation.

OBJECTIVE: The aim of this study was to investigate the effect of endogenous matrix metalloproteinases 2 and 9 (MMP-2 and MMP-9) on the invasive characteristics of RA synovial fibroblasts. METHODS: Synovial fibroblasts isolated from patients with RA or OA were treated with MMP small interfering RNA (siRNA), inhibitors and recombinant proteins or TNF-α, with or without cartilage explants. Cell viability and proliferation were measured by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide and 5-bromo-2-deoxyuridine (BrdU) proliferation assays, respectively; apoptosis by an in situ cell death detection kit; migration and invasion by CytoSelect invasion assay, scratch migration and collagen gel assays; cartilage degradation by 1,9-dimethylmethylene blue assay; and inflammatory mediators and MMPs by ELISA, western blot and zymography. RESULTS: MMP-2 was expressed by both OA and RA synovial fibroblasts, whereas only RA synovial fibroblasts expressed MMP-9. Suppressing MMP-2 or MMP-9 reduced RA synovial fibroblast proliferation equally. However, MMP-9 siRNA had greater effects compared with MMP-2 siRNA on promoting apoptosis and suppressing RA synovial fibroblast viability, migration and invasion. Suppression/inhibition of MMP-9 also decreased the production of IL-1ß, IL-6, IL-8 and TNF-α, inactivated nuclear factor κB (NF-κB), extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK) and suppressed RA synovial fibroblast-mediated cartilage degradation. In contrast, suppression/inhibition of MMP-2 stimulated TNF-α and IL-17 secretion and activated NF-κB, while recombinant MMP-2 (rMMP-2) inactivated NF-κB and suppressed RA synovial fibroblast-mediated cartilage degradation. Results using specific inhibitors and rMMPs provided supportive evidence for the siRNA results. CONCLUSION: Endogenous MMP-2 or MMP-9 contribute to RA synovial fibroblast survival, proliferation, migration and invasion, with MMP-9 having more potent effects. Additionally, MMP-9 stimulates RA synovial fibroblast-mediated inflammation and degradation of cartilage, whereas MMP-2 inhibits these parameters. Overall, our data indicate that MMP-9 derived from RA synovial fibroblasts may directly contribute to joint destruction in RA.

Melatonin-mediated ß-catenin activation protects neuron cells against prion protein-induced neurotoxicity.

Activation of ß-catenin in neurons regulates mitochondrial function and protects against protein misfolding disorders, including Alzheimer's disease and Huntington's disease. Melatonin, a natural secretory product of the pineal gland, exerts neuroprotective effects through the activation of ß-catenin. In this study, melatonin increased ß-catenin protein expression and activation in human neuroblastoma cell lines SH-SY5Y cells. Melatonin also inhibited PrP (106-126)-induced neurotoxicity and the inhibition attenuated by treatment of ß-catenin inhibitor ICG-001. Activation of ß-catenin blocked PrP (106-126)-mediated downregulation of anti-apoptotic protein survivin and Bcl-2. Reduction of mitochondrial membrane potential, translocation of Bax, and cytochrome c release which induced by PrP (106-126) treatment were inhibited by ß-catenin activation, which contributed to prevented PrP (106-126)-induced neuronal cell death. In conclusion, ß-catenin activation by melatonin prevented PrP (106-126)-induced neuronal cell death through regulating anti-apoptotic proteins and mitochondrial pathways. These results also suggest the therapeutic value of Wnt/ß-catenin signaling in prion-related disorders as influenced by melatonin.

Overcoming hypoxic-resistance of tumor cells to TRAIL-induced apoptosis through melatonin.

A solid tumor is often exposed to hypoxic or anoxic conditions; thus, tumor cell responses to hypoxia are important for tumor progression as well as tumor therapy. Our previous studies indicated that tumor cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cell apoptosis under hypoxic conditions. Melatonin inhibits cell proliferation in many cancer types and induces apoptosis in some particular cancer types. Here, we examined the effects of melatonin on hypoxic resistant cells against TRAIL-induced apoptosis and the possible mechanisms of melatonin in the hypoxic response. Melatonin treatment increased TRAIL-induced A549 cell death under hypoxic conditions, although hypoxia inhibited TRAIL-mediated cell apoptosis. In a mechanistic study, hypoxia inducible factor-1α and prolyl-hydroxylase 2 proteins, which increase following exposure to hypoxia, were dose-dependently down-regulated by melatonin treatment. Melatonin also blocked the hypoxic responses that reduced pro-apoptotic proteins and increased anti-apoptotic proteins including Bcl-2 and Bcl-xL. Furthermore, melatonin treatment reduced TRAIL resistance by regulating the mitochondrial transmembrane potential and Bax translocation. Our results first demonstrated that melatonin treatment induces apoptosis in TRAIL-resistant hypoxic tumor cells by diminishing the anti-apoptotic signals mediated by hypoxia and also suggest that melatonin could be a tumor therapeutic tool by combining with other apoptotic ligands including TRAIL, particularly in solid tumor cells exposed to hypoxia.

Melatonin-mediated calcineurin inactivation attenuates amyloid beta-induced apoptosis.

Alzheimer's disease (AD) is the most common age-related progressive neurodegenerative disorder. The accumulation of amyloid beta-peptide is a neuropathological marker of AD. While melatonin is recognized to have protective effects on aging and neurodegenerative disorders, the therapeutic effect of melatonin on calcineurin in AD is poorly understood. In this study, we examined the effect and underlying molecular mechanisms of melatonin treatment on amyloid beta-mediated neurotoxicity in neuroblastoma cells. Melatonin treatment decreased calcineurin and autophagy in neuroblastoma cells. Electron microscopy images showed that melatonin inhibited amyloid beta-induced autophagic vacuoles. The increase in the amyloid beta-induced apoptosis rate was observed more in PrPC-expressing ZW cells than in PrPC-silencing Zpl cells. Taken together, the results suggest that by mitigating the effect of calcineurin and autophagy flux activation, melatonin could also rescue amyloid beta-induced neurotoxic effects. These findings may be relevant to therapy for neurodegenerative diseases, including AD.

Gingerol-induced hypoxia-inducible factor 1 alpha inhibits human prion peptide-mediated neurotoxicity.

Prion diseases are a family member of neurodegenerative disorders caused by the accumulation of misfolded-prion proteins (scrapie form of PrP, PrP(Sc)). The accumulation of PrP(Sc) in the brain leads to neurotoxicity by the induction of mitochondrial-apoptotic pathways. Recent studies implicated gingerol in protection against neurodegeneration. However, the basis of the neuroprotection in prion disease remains unclear. Thus, we investigated the influence of gingerol on prion peptide-induced neuronal damage. Gingerol blocked PrP(106-126)-mediated neurotoxicity by protecting mitochondrial function. Moreover, the protective effect of gingerol against PrP(106-126)-induced mitochondrial damage was associated with hypoxia-inducible factor 1 alpha (HIF-1α) expression. Gingerol-induced HIF-1α expression inhibited the PrP(106-126)-induced mitochondrial dysfunction. On the other hand, inhibition of gingerol-induced HIF-1 α expression attenuated the gingerol-mediated neuroprotective effect. Here, we demonstrate for the first time that treatment with gingerol prevents prion peptide-mediated neuronal cell death and that the neuroprotection is induced by HIF-1α-mediated signals. This study suggests that treatment with gingerol may provide a novel therapeutic strategy for prion-mediated neurotoxicity.

Antitumor Effects of Esculetin, a Natural Coumarin Derivative, against Canine Mammary Gland Tumor Cells by Inducing Cell Cycle Arrest and Apoptosis.

Mammary gland tumors are the most common neoplasms in female dogs, of which 50% are malignant. Esculetin, a coumarin derivative, reportedly induces death in different types of cancer cells. In this study, we explore the anticancer effects of esculetin against CMT-U27 and CF41.mg canine mammary gland tumor cells. Esculetin significantly inhibited the viability and migration of both CMT-U27 and CF41.mg cells in a dose- and time-dependent manner. Flow cytometric analysis and terminal deoxynucleotidyl transferase dUTP nick-end labeling assay revealed increased numbers of annexin-V-positive cells and DNA fragmentation. Furthermore, a cell cycle analysis demonstrated that esculetin blocked the cell progression at the G0/G1 phase and the S phase in CMT-U27 and CF41.mg cells. These results were supported by a Western blot analysis, which revealed upregulated protein expression of cleaved caspase-3, a marker of apoptosis, and downregulated cyclin-dependent kinase 4 and cyclin D1 protein, the cell cycle regulators. In conclusion, this novel study proves that esculetin exerts in vitro antitumor effects by inducing apoptosis and cell cycle arrest in canine mammary gland tumors.