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-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.

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.

Antiviral activity of prion protein against Japanese encephalitis virus infection in vitro and in vivo.

Flaviviruses are a major cause of viral diseases worldwide, for which effective treatments have yet to be discovered. The prion protein (PrPc) is abundantly expressed in brain cells and has been shown to play a variety of roles, including neuroprotection, cell homeostasis, and regulation of cellular signaling. However, it is still unclear whether PrPc can protect against flaviviruses. In this study, we investigated the role of PrPc in regulating autophagy flux and its potential antiviral activity during Japanese encephalitis virus (JEV) infection. Our in vivo experiment showed that JEV was more lethal to the PrPc knocked out mice which was further supported by histological analysis, western blot and rtPCR results from infected mice brain samples. Role of PrPc against viral propagation in vitro was verified through cell survival study, protein expression and RNA replication analysis, and adenoviral vector assay by overexpressing PrPc. Further analysis indicated that after virus entry, PrPc inhibited autophagic flux that prevented JEV replication inside the host cell. Our results from in vivo and in vitro investigations demonstrate that prion protein effectively inhibited JEV propagation by regulating autophagy flux which is used by JEV to release its genetic material and replication after entering the host cell, suggesting that prion protein may be a promising therapeutic target for flavivirus infection.

An Antidepressant Drug Increased TRAIL Receptor-2 Expression and Sensitized Lung Cancer Cells to TRAIL-induced Apoptosis.

BACKGROUND: TRAIL has emerged as a promising therapeutic target due to its ability to selectively induce apoptosis in cancer cells while sparing normal cells. Autophagy, a highly regulated cellular recycling mechanism, is known to play a cell survival role by providing a required environment for the cell. Recent studies suggest that autophagy plays a significant role in increasing TRAIL resistance in certain cancer cells. Thus, regulating autophagy in TRAIL-mediated cancer therapy is crucial for its role in cancer treatment. OBJECTIVE: Our study explored whether the antidepressant drug desipramine could enhance the ability of TRAIL to kill cancer cells by inhibiting autophagy. METHODS: The effect of desipramine on TRAIL sensitivity was examined in various lung cancer cell lines. Cell viability was measured by morphological analysis, trypan blue exclusion, and crystal violet staining. Flow cytometry analysis was carried out to measure apoptosis with annexin V-PI stained cells. Western blotting, rtPCR, and immunocytochemistry were carried out to measure autophagy and death receptor expression. TEM was carried out to detect autophagy inhibition. RESULTS: Desipramine treatment increased the TRAIL sensitivity in all lung cancer cell lines. Mechanistically, desipramine treatment induced death receptor expression to increase TRAIL sensitivity. This effect was confirmed when the genetic blockade of DR5 reduced the effect of desipramine in enhanced TRAIL-mediated cell death. Further investigation revealed that desipramine treatment increased the LC3 and p62 levels, indicating the inhibition of lysosomal degradation of autophagy. Notably, TRAIL, in combination with either desipramine or the autophagy inhibitor chloroquine, exhibited enhanced cytotoxicity compared to TRAIL treatment alone. CONCLUSION: Our findings revealed the potential of desipramine to induce TRAIL-mediated cell death by autophagy impairment. This discovery suggests its therapeutic potential for inducing TRAIL-mediated cell death by increasing the expression of death receptors, which is caused by impairing autophagy.

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.

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.

Methyl Gallate Suppresses Tumor Development by Increasing Activation of Caspase3 and Disrupting Tumor Angiogenesis in Melanoma.

Methyl gallate is a phenolic compound mainly found in medicinal plants. It has been reported to its anticancer activity in various tumors. In this study, we aimed to demonstrate the antitumor effect of methyl gallate in the melanoma mouse model and B16F10 cells. Our results showed that methyl gallate decreased cell viability and induced apoptosis by increasing the expression of cleaved caspase3 in B16F10 cells and prevented cell migration and tube formation in human umbilical vein endothelial cells. In B16F10 cell-inoculated mice, methyl gallate not only decreased tumor volume by 30% but also significantly reduced tumor vessel density and pericyte coverage. Moreover, methyl gallate diminished by close to 50% the expression of cytokeratin and LYVE-1 in mouse right inguinal lymph nodes, indicating that methyl gallate could suppress metastasis. In conclusion, this study suggests that methyl gallate inhibits tumor development by inducing apoptosis and blocking tumor angiogenesis and metastasis and might be considered a therapeutic agent for melanoma.

Calcineurin, Calcium-Dependent Serine-Threonine Phosphatase Activation by Prion Peptide 106-126 Enhances Nuclear Factor-κB-Linked Proinflammatory Response through Autophagy Pathway.

Prion diseases are mortal neurodegenerative pathologies that are caused by the accumulation of abnormal prion protein (PrPSc) in the brain. Recent advances reveal that calcineurin may play a critical role in regulating nuclear factor kappa B (NF-κB) in the calcium-calmodulin pathway. However, the exact mechanism by calcineurin remains unclear. In the present study, we observed that the prion peptide induces calcineurin and autophagy activation. Also, NF-κB and proinflammatory cytokines like interleukin (IL)-6 and tumor necrosis factor (TNF)-α are upregulated upon exposure to prion peptide in human neuroblastoma. The results show that the prion peptide induces calcineurin activation, leading to the activation of NF-κB transcription factor via autophagy signaling. Expression of TNF-α and IL-6 was increased by calcineurin activation and blocked by calcineurin inhibitor and autophagy inhibitor treatments. Collectively, these findings indicate that calcineurin activation mediated by prion protein induces NF-κB-driven neuroinflammation via autophagy pathway, suggesting that calcineurin and autophagy may be possible therapeutic targets for neuroinflammation in neurodegeneration diseases including prion disease.

Calcineurin Activation by Prion Protein Induces Neurotoxicity via Mitochondrial Reactive Oxygen Species.

Prion diseases are caused by PrPsc accumulation in the brain, which triggers dysfunctional mitochondrial injury and reactive oxygen species (ROS) generation in neurons. Recent studies on prion diseases suggest that endoplasmic reticulum (ER) stress induced by misfolding proteins such as misfolded prion protein results in activation of calcineurin. Calcineurin is a calcium-related protein phosphatase of type 2B that exists in copious quantities in the brain and acts as a critical nodal component in the control of cellular functions. To investigate the relationship between calcineurin and intracellular ROS, we assessed the alteration of CaN and ROS induced by prion peptide (PrP) 106-126. Human prion peptide increased mitochondrial ROS by activating calcineurin, and the inhibition of calcineurin activity protected mitochondrial function and neuronal apoptosis in neuronal cells. These results suggest that calcineurin plays a pivotal role in neuronal apoptosis by mediating mitochondrial injury and ROS in prion diseases.

Sensitizing TRAIL­resistant A549 lung cancer cells and enhancing TRAIL­induced apoptosis with the antidepressant amitriptyline.

Tumor necrosis factor­related apoptosis­inducing ligand (TRAIL) is a cytokine with the potential to induce cancer cell­specific apoptosis with minimal toxicity to normal cells. Therefore, the resistance of certain cancer cells to TRAIL is a major concern and agents that can either enhance TRAIL capabilities or overcome TRAIL resistance are necessary for the development of cancer treatments. The present study investigated whether the antidepressant drug amitriptyline could sensitize TRAIL­resistant A549 lung cancer cells and enhance TRAIL­induced apoptosis. Antidepressants are usually prescribed to cancer patients to relieve emotional distress, such as depression or dysthymia. The present study revealed for the first time, to the best of our knowledge, that amitriptyline increased death receptor (DR) 4 and 5 expression, a requirement for TRAIL­induced cell death. Genetic inhibitors of DR4 and DR5 significantly reduced amitriptyline­enhanced TRAIL­mediated apoptosis. Additionally, the present study explored whether blocking autophagy increased DR4 and DR5 expression. Blocking autophagy flux with the final stage autophagy inhibitor chloroquine (CQ) also upregulated DR4 and DR5 expression. TRAIL in combination with amitriptyline or CQ significantly increased the expression of apoptosis­indicator proteins cleaved caspase­8 and caspase­3. The expression levels of LC3­II and p62 were significantly higher in amitriptyline­treated cells, which confirmed that amitriptyline blocks autophagy by inhibiting the fusion of autophagosomes with lysosomes. Overall, the present results contributed to understanding the mechanism responsible for the synergistic anticancer effect of amitriptyline and TRAIL and also presented a novel mechanism involved in DR4 and DR5 upregulation.

The antidiabetic drug troglitazone protects against PrP (106­126)­induced neurotoxicity via the PPARγ­autophagy pathway in neuronal cells.

Prion diseases, which involve the alteration of cellular prion protein into a misfolded isoform, disrupt the central nervous systems of humans and animals alike. Prior research has suggested that peroxisome proliferator­activator receptor (PPAR)γ and autophagy provide some protection against neurodegeneration. PPARs are critical to lipid metabolism regulation and autophagy is one of the main cellular mechanisms by which cell function and homeostasis is maintained. The present study examined the effect of troglitazone, a PPARγ agonist, on autophagy flux in a prion peptide (PrP) (106­126)­mediated neurodegeneration model. Western blot analysis confirmed that treatment with troglitazone increased LC3­II and p62 protein expression, whereas an excessive increase in autophagosomes was verified by transmission electron microscopy. Troglitazone weakened PrP (106­126)­mediated neurotoxicity via PPARγ activation and autophagy flux inhibition. A PPARγ antagonist blocked PPARγ activation as well as the neuroprotective effects induced by troglitazone treatment, indicating that PPARγ deactivation impaired troglitazone­mediated protective effects. In conclusion, the present study demonstrated that troglitazone protected primary neuronal cells against PrP (106­126)­induced neuronal cell death by inhibiting autophagic flux and activating PPARγ signals. These results suggested that troglitazone may be a useful therapeutic agent for the treatment of neurodegenerative disorders and prion diseases.

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.

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.

Inhibition of autophagy flux by sertraline attenuates TRAIL resistance in lung cancer via death receptor 5 upregulation.

Tumor necrosis factor­related apoptosis­inducing ligand (TRAIL) is a potential target for cancer therapy, owing to its ability to selectively kill cancer cells without causing significant toxicity to normal cells. However, due to the lack of death receptor expression, cancer cells can become highly resistant to TRAIL. Hence, it is vital to develop agents that restore TRAIL efficacy. Sertraline is an antidepressant drug with anticancer properties. To the best of our knowledge, this is the first study to demonstrate that sertraline inhibits autophagic flux and increases the expression of death receptor 5 (DR5) on TRAIL­resistant lung cancer cells. Inhibition of autophagy using autophagy inhibitors 3­methyladenine and chloroquine upregulated the expression of DR5 and enhanced TRAIL­induced apoptosis, as confirmed by the increase of pro­apoptotic proteins caspase­8 and caspase­3. Silencing DR5 expression using DR5 small interfering RNA prevented sertraline­induced TRAIL­mediated apoptosis, indicating the role of DR5 in TRAIL­mediated apoptosis. Overall, sertraline enhanced TRAIL­mediated apoptosis via the downregulation of AMP­activated protein kinase phosphorylation, resulting in the inhibition of autophagic flux, upregulation of DR5 expression, and activation of the apoptotic caspase cascade. These data suggested that sertraline could be used to sensitize human lung cancer cells to TRAIL, while also serving as a therapeutic option in cancer patients with depression.

Downregulation of c­FLIP and upregulation of DR­5 by cantharidin sensitizes TRAIL­mediated apoptosis in prostate cancer cells via autophagy flux.

Tumor necrosis factor (TNF)­related apoptosis­inducing ligand (TRAIL), a type II transmembrane protein, is a part of the TNF superfamily of cytokines. Cantharidin, a type of terpenoid, is extracted from the blister beetles (Mylabris genus) used in Traditional Chinese Medicine. Cantharidin elicits antibiotic, antiviral and antitumor effects, and can affect the immune response. The present study demonstrated that a cantharidin and TRAIL combination treatment regimen elicited a synergistic outcome in TRAIL­resistant DU145 cells. Notably, it was also identified that cantharidin treatment initiated the downregulation of cellular FLICE­like inhibitory protein (c­FLIP) and upregulation of death receptor 5 (DR­5), and sensitized cells to TRAIL­mediated apoptosis by initiating autophagy flux. In addition, cantharidin treatment increased lipid­modified microtubule­associated proteins 1A/1B light chain 3B expression and significantly attenuated sequestosome 1 expression. Attenuation of autophagy flux by a specific inhibitor such as chloroquine and genetic modification using ATG5 small interfering RNA abrogated the cantharidin­mediated TRAIL­induced apoptosis. Overall, the results of the present study revealed that cantharidin effectively sensitized cells to TRAIL­mediated apoptosis and its effects are likely to be mediated by autophagy, the downregulation of c­FLIP and the upregulation of DR­5. They also suggested that the combination of cantharidin and TRAIL may be a successful therapeutic strategy for TRAIL­resistant prostate cancer.

Neferine treatment enhances the TRAIL­induced apoptosis of human prostate cancer cells via autophagic flux and the JNK pathway.

Prostate cancer (PCa) is a common type of cancer among males, with a relatively high mortality rate. Tumor necrosis factor­related apoptosis­inducing ligand (TRAIL), a member of the tumor necrosis factor (TNF) family, initiates the apoptosis of certain cancer cells. Neferine, a primary ingredient of bisbenzylisoquinoline alkaloids, has various antitumor activities. The present study examined the effects of neferine treatment on human PCa cells. Human prostate cancer (DU145) cells were treated with neferine for 18 h, and subsequently treated with TRAIL for 2 h. Combined treatment with neferine and TRAIL significantly decreased cell viability compared to treatment with TRAIL alone. Furthermore, neferine treatment decreased the expression of p62 and increased LC3B­II expression, as assessed by western blot analysis and immunocytochemistry. It was alsp demonstrated that neferine and TRAIL act synergistically to trigger autophagy in PCa cells, as revealed by autophagosome formation, LC3B­II accumulation demonstrated by transmission electron microscopy (TEM) analysis and phosphorylated c­Jun N­terminal kinase (p­JNK) upregulation. When the autophagic flux was attenuated by the inhibitor, chloroquine, or by genetically modified ATG5 siRNA, the enhancement of TRAIL­induced autophagy by neferine­induced was also attenuated. Furthermore, treatment with the JNK inhibitor, SP600125, distinctly increased the viability of the cells treated with neferine and TRAIL. On the whole, the findings of the present study demonstrate that neferine treatment effectively promotes TRAIL­mediated cell death and this effect likely occurs via the autophagic flux and the JNK pathway.

Human prion protein-mediated calcineurin activation induces neuron cell death via AMPK and autophagy pathway.

It is usually accepted that prion proteins induce apoptosis in nerve cells. However, the mechanisms of PrPSc-neurotoxicity are not completely clear. Calcineurin is a Ca2+/calmodulin-dependent phosphatase. It activates autophagy, and may represent a link between deregulation of Ca2+ homeostasis and neuronal cell death. In this study, the effect of calcineurin activation mediated by human prion protein induced neuronal cell death via AMPK dephosphorylation and autophagy, was investigated. Synthetic peptides of PrP (PrP 106-126) increased calcineurin activity, without changing the levels of this protein phosphatase. Furthermore, these peptides reduced the levels of AMPK phosphorylation at threonine residue 172 and in autophagy activation. Calcineurin inhibitor, FK506, prevented this effect. The data showed that PrP-treated neurons had lower levels of AMPK than control neurons. This decrease in AMPK levels was matched via activation of autophagy. FK506 prevented the changes in AMPK and autophagy levels induced by PrP peptides. Taken together, the data demonstrated that prion peptides triggered an apoptotic cascade via calcineurin activation, which mediated AMPK dephosphorylation and autophagy activation. Therefore, these data suggest that therapeutic strategies targeting calcineurin inhibition might facilitate the management of neurodegenerative disorders including prion disease.

Duloxetine Enhances TRAIL-mediated Apoptosis via AMPK-mediated Inhibition of Autophagy Flux in Lung Cancer Cells.

BACKGROUND/AIM: The antidepressant duloxetine is known as a serotonin-norepinephrine reuptake inhibitor, used for treating depression and anxiety. TRAIL selectively induces cell death in a variety of tumor cells by binding to its membrane death receptor (DR). The aim of the study was to examine whether duloxetine affects TRAIL-mediated apoptosis. MATERIALS AND METHODS: Cell viability and apoptosis was measured by morphological image, crystal violet staining, MTT and LDH assay. Immunocytochemistry and western blotting techniques were applied to detect autophagy and apoptosis indicator proteins. TEM assay was used to determine the autophagy. RESULTS: Duloxetine treatment considerably sensitizes human lung adenocarcinoma cells to TRAIL-mediated apoptosis by targeting TRAIL-DR5. Treatment with duloxetine inhibited AMPK phosphorylation and resulted in increased p62 and microtubule-associated protein 1A/1B light chain 3B-II levels, indicating inhibition of autophagy flux. Blockade of DR5 with DR5-specific small-interfering RNA negatively regulated the apoptotic effect. CONCLUSION: Clinical administration of TRAIL in combination with duloxetine may serve as a therapeutic approach for the treatment of TRAIL-resistant lung cancer cells.

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.