Deficiency of the IRE1α-Autophagy Axis Enhances the Antitumor Effects of the Oncolytic Virus M1.

Oncolytic virotherapy is an emerging treatment modality that uses replication-competent viruses to destroy cancer cells. M1 is a naturally occurring alphavirus (Togaviridae) which shows potent oncolytic activities against many cancers. Accumulation of unfolded proteins during virus replication leads to a transcriptional/translational response known as the unfolded protein response (UPR), which might counteract the antitumor effect of the oncolytic virus. In this report, we show that either pharmacological or biological inhibition of IRE1α or PERK, but not ATF6, substantially increases the oncolytic effects of the M1 virus. Moreover, inhibition of IRE1α blocks M1 virus-induced autophagy, which restricts the antitumor effects of the M1 virus through degradation of viral protein, in glioma cells. In addition, IRE1α suppression significantly increases the oncolytic effect of M1 virus in an orthotopic glioma model. From a molecular pathology study, we found that IRE1α is expressed at lower levels in higher-grade gliomas, suggesting greater antitumor efficacy of the oncolytic virus M1. Taken together, these findings illustrate a defensive mechanism of glioma cells against the oncolytic virus M1 and identify possible approaches to enhance the oncolytic viral protein accumulation and the subsequent lysis of tumor cells.IMPORTANCE Although oncolytic virotherapy is showing great promise in clinical applications, not all patients are benefiting. Identifying inhibitory signals in refractory cancer cells for each oncolytic virus would provide a good chance to increase the therapeutic effect. Here we describe that infection with the oncolytic virus M1 triggers the unfolded protein response (UPR) and subsequent autophagy, while blocking the UPR-autophagy axis significantly potentiates the antitumor efficacy of M1 in vitro and in vivo A survey of cancer tissue banks revealed that IRE1α, a key element in the UPR pathway, is commonly downregulated in higher-grade human gliomas, suggesting favorable prospects for the application of M1. Our work provides a potential predictor and target for enhancement of the therapeutic effectiveness of the M1 virus. We predict that the mechanism-based combination therapy will promote cancer virotherapy in the future.

Liposome Encapsulation of Oncolytic Virus M1 To Reduce Immunogenicity and Immune Clearance in Vivo.

Oncolytic viral therapy is an attractive novel strategy for cancer therapy. As a natural alphavirus, oncolytic virus M1 is able to infect and kill various zinc finger antiviral protein (ZAP)-deficient tumor cells selectively, while leaving normal cells undamaged. However, M1 can trigger the production of neutralizing antibodies that dramatically weaken its antitumor effect. In order to attenuate immunogenicity of the therapeutic M1 virus, we encapsulated it into liposomes (referred to as M-LPO) using the thin-film hydration method. The effect of anti-M1 neutralizing antibody on M-LPO was examined in LoVo and Hep 3B cell lines. In the absence of neutralizing antibodies, treating cells with naked M1, blank liposomes (LPO), M-LPO, or a simple mixture of M1 and liposomes (LPO+M1) inhibited cell growth. In the presence of neutralizing antibodies, only M-LPO inhibited cell growth. After intravenous administration, M-LPO reduced the production of the M1-neutralizing antibody and the corresponding immune response. Analysis of the M-LPO uptake by cells was examined by confocal microscopy using M1 labeled with FITC and liposomal shells labeled with RhB. The results suggest that M1 may be released from liposomes before or after M-LPO internalization. Taken together, our results suggest that encapsulating oncolytic virus M1 in liposomes may reduce intrinsic viral immunogenicity for improved anticancer therapy.

Hexokinase 2-dependent hyperglycolysis driving microglial activation contributes to ischemic brain injury.

Hyperglycolysis, observed within the penumbra zone during brain ischemia, was shown to be detrimental for tissue survival because of lactate accumulation and reactive oxygen species overproduction in clinical and experimental settings. Recently, mounting evidence suggests that glycolytic reprogramming and induced metabolic enzymes can fuel the activation of peripheral immune cells. However, the possible roles and details regarding hyperglycolysis in neuroinflammation during ischemia are relatively poorly understood. Here, we investigated whether overactivated glycolysis could activate microglia and identified the crucial regulators of neuroinflammatory responses in vitro and in vivo. Using BV 2 and primary microglial cultures, we found hyperglycolysis and induction of the key glycolytic enzyme hexokinase 2 (HK2) were essential for microglia-mediated neuroinflammation under hypoxia. Mechanistically, HK2 up-regulation led to accumulated acetyl-coenzyme A, which accounted for the subsequent histone acetylation and transcriptional activation of interleukin (IL)-1ß. The inhibition and selective knockdown of HK2 in vivo significantly protected against ischemic brain injury by suppressing microglial activation and IL-1ß production in male Sprague-Dawley rats subjected to transient middle cerebral artery occlusion (MCAo) surgery. We provide novel insights for HK2 specifically serving as a neuroinflammatory determinant, thus explaining the neurotoxic effect of hyperglycolysis and indicating the possibility of selectively targeting HK2 as a therapeutic strategy in acute ischemic stroke.

Activation of Cyclic Adenosine Monophosphate Pathway Increases the Sensitivity of Cancer Cells to the Oncolytic Virus M1.

Oncolytic virotherapy is a novel and emerging treatment modality that uses replication-competent viruses to destroy cancer cells. Although diverse cancer cell types are sensitive to oncolytic viruses, one of the major challenges of oncolytic virotherapy is that the sensitivity to oncolysis ranges among different cancer cell types. Furthermore, the underlying mechanism of action is not fully understood. Here, we report that activation of cyclic adenosine monophosphate (cAMP) signaling significantly sensitizes refractory cancer cells to alphavirus M1 in vitro, in vivo, and ex vivo. We find that activation of the cAMP signaling pathway inhibits M1-induced expression of antiviral factors in refractory cancer cells, leading to prolonged and severe endoplasmic reticulum (ER) stress, and cell apoptosis. We also demonstrate that M1-mediated oncolysis, which is enhanced by cAMP signaling, involves the factor, exchange protein directly activated by cAMP 1 (Epac1), but not the classical cAMP-dependent protein kinase A (PKA). Taken together, cAMP/Epac1 signaling pathway activation inhibits antiviral factors and improves responsiveness of refractory cancer cells to M1-mediated virotherapy.

Apoptosis of rat hepatic stellate cells induced by diallyl trisulfide and proteomics profiling in vitro.

Diallyl trisulfide (DATS), a major garlic derivative, inhibits cell proliferation and triggers apoptosis in a variety of cancer cell lines. However, the effects of DATS on hepatic stellate cells (HSCs) remain unknown. The aim of this study was to analyze the effects of DATS on cell proliferation and apoptosis, as well as the protein expression profile in rat HSCs. Rat HSCs were treated with or without 12 and 24 µg/mL DATS for various time intervals. Cell proliferation and apoptosis were determined using tetrazolium dye (MTT) colorimetric assay, bromodeoxyuridine (5-bromo-2'-deoxyuridine; BrdU) assay, Hoechst 33342 staining, electroscopy, and flow cytometry. Protein expression patterns in HSCs were systematically studied using 2-dimensional electrophoresis and mass spectrometry. DATS inhibited cell proliferation and induced apoptosis of HSCs in a time-dependent manner. We observed clear morphological changes in apoptotic HSCs and dramatically increased annexin V-positive - propidium iodide negative apoptosis compared with the untreated control group. Twenty-one significant differentially expressed proteins, including 9 downregulated proteins and 12 upregulated proteins, were identified after DATS administration, and most of them were involved in apoptosis. Our results suggest that DATS is an inducer of apoptosis in HSCs, and several key proteins may be involved in the molecular mechanism of apoptosis induced by DATS.

Identification and characterization of alphavirus M1 as a selective oncolytic virus targeting ZAP-defective human cancers.

Oncolytic virotherapy is a growing treatment modality that uses replicating viruses as selective antineoplastic agents. Safety and efficacy considerations dictate that an ideal oncolytic agent would discriminate between normal and cancer cells on the basis of common genetic abnormalities in human cancers. Here, we identify a naturally occurring alphavirus (M1) as a novel selective killer targeting zinc-finger antiviral protein (ZAP)-deficient cancer cells. In vitro, in vivo, and ex vivo studies showed potent oncolytic efficacy and high tumor tropism of M1. We showed that the selectivity depends on ZAP deficiency by systematic identification. A large-scale multicenter pathology study using tissue microarrays reveals that ZAP is commonly deficient in human cancers, suggesting extensive application prospects for M1. Additionally, M1 killed cancer cells by inducing endoplasmic reticulum stress-mediated apoptosis. Our report provides novel insights into potentially personalized cancer therapy using oncolytic viruses.

The major cholesterol metabolite cholestane-3ß,5α,6ß-triol functions as an endogenous neuroprotectant.

Overstimulation of NMDA-type glutamate receptors is believed to be responsible for neuronal death of the CNS in various disorders, including cerebral and spinal cord ischemia. However, the intrinsic and physiological mechanisms of modulation of these receptors are essentially unknown. Here we report that cholestane-3ß,5α,6ß-triol (triol), a major metabolite of cholesterol, is an endogenous neuroprotectant and protects against neuronal injury both in vitro and in vivo via negative modulation of NMDA receptors. Treatment of cultured neurons with triol protects against glutamate-induced neurotoxicity, and administration of triol significantly decreases neuronal injury after spinal cord ischemia in rabbits and transient focal cerebral ischemia in rats. An inducible elevation of triol is associated with ischemic preconditioning and subsequent neuroprotection in the spinal cord of rabbits. This neuroprotection is effectively abolished by preadministration of a specific inhibitor of triol synthesis. Physiological concentrations of triol attenuate [Ca(2+)]i induced by glutamate and decrease inward NMDA-mediated currents in cultured cortical neurons and HEK-293 cells transiently transfected with NR1/NR2B NMDA receptors. Saturable binding of [(3)H]triol to cerebellar granule neurons and displacement of [(3)H]MK-801 binding to NMDA receptors by triol suggest that direct blockade of NMDA receptors may underlie the neuroprotective properties. Our findings suggest that the naturally occurring oxysterol, the major cholesterol metabolite triol, functions as an endogenous neuroprotectant in vivo, which may provide novel insights into understanding and developing potential therapeutics for disorders in the CNS.

Determination of neuroprotective oxysterols in Calculus bovis, human gallstones, and traditional Chinese medicine preparations by liquid chromatography with mass spectrometry.

So far, the components responsible for the neuroprotective effects of Calculus bovis are unclear. Cholesterol, one of the major components in Calculus bovis, is easily oxidized into oxysterols, which possess direct or indirect neuroprotective effects proved by our and others' previous studies. Therefore, a liquid chromatography with mass spectrometry method coupled with ultrasonic extraction and solid-phase extraction was developed for the determination of neuroprotective oxysterols in Calculus bovis, human gallstones, and traditional Chinese medicine preparations. Chromatographic separation was achieved on a C18 column with isocratic elution at a flow rate of 1 mL/min. The established method showed good linearity (R(2) > 0.998), sensitivity with low limits of detection (0.06-0.39 µg/g), acceptable precisions (relative standard deviations ≤ 7.4%), stability (relative standard deviations ≤ 5.9%), and satisfactory accuracy (92.4-102.9%) for all analytes identified by different retention times, which could be applied for the determination of oxysterols. Five kinds of oxysterols proved to function as neuroprotectants were detected at different concentrations. Among them, 7ß-hydroxycholesterol and cholestane-3ß,5α,6ß-triol were rather abundant in the samples. It could be concluded that the potential neuroprotective components in Calculus bovis may be these oxysterols.

Triptolide inhibits proliferation and invasion of malignant glioma cells.

Malignant glioma is the most devastating and aggressive tumor in brain, characterized by rapid proliferation and diffuse invasion. Chemotherapy and radiotherapy are the pivotal strategies after surgery; however, high drug resistance of malignant glioma and the blood-brain barrier usually render chemotherapy drugs ineffective. Here, we find that triptolide, a small molecule with high lipid solubility, is capable of inhibiting proliferation and invasion of malignant glioma cells effectively. In both investigated malignant glioma cell lines, triptolide repressed cell proliferation via inducing cell cycle arrest in G0/G1 phase, associated with downregulation of G0/G1 cell cycle regulators cyclin D1, CDK4, and CDK6 followed by reduced phosphorylation of retinoblastoma protein (Rb). In addition, triptolide induced morphological change of C6 cells through downregulation of protein expression of MAP-2 and inhibition of activities of GTPases Cdc42 and Rac1/2/3, thus significantly suppressing migratory and invasive capacity. Moreover, in an in vivo tumor model, triptolide delayed growth of malignant glioma xenografts. These findings suggest an important inhibitory action of triptolide on proliferation and invasion of malignant glioma, and encourage triptolide as a candidate for glioma therapy.

The effect of the fibrinolytic enzyme FIIa from Agkistrodon acutus venom on acute pulmonary thromboembolism.

AIM: To evaluate the effects of the fibrinolytic enzyme FII(a) from Agkistrodon acutus venom on acute pulmonary thromboembolism (APT) in animal models. METHODS: Both rabbit and dog APT models were used. For the rabbit APT model, the thrombi weight before and after administration was measured. Central venous pressure (CVP) and mean arterial pressure (MAP) were measured before and 15, 30, 60, and 120 min after the injection of the blood clot. Partial thromboplastin time (APTT), prothrombin time (PT), platelet count, and fibrinogen concentration were measured using auto analyzers. Plasminogen activity was measured based on chromogenic substrates. In the dog APT model, pulmonary blood flow was recorded using pulmonary angiography. RESULTS: Intravenous administration of FIIa (0.1-5.0 mg/kg) improved the APT-induced hemodynamic derangements and reduced thrombi weight. The angiography evidence also showed that the pulmonary emboli had almost disappeared after FII(a) infusion. FII(a) (0.1, 0.5, or 1.0 mg/kg) did not impair the coagulation pathways, although very high doses of FII(a) (5.0 mg/kg) could stimulate the production of plasminogen and result in impairment of the pathways. CONCLUSION: FII(a) could effectively protect against APT via degradation of thrombi with less activation of plasminogen, and may provide a novel fibrinolytic enzyme for targeting the main pathological processes of the disease.

Real-Time Visualization and Quantification of Oncolytic M1 Virus In Vitro and In Vivo.

Alphavirus M1 is a promising oncolytic virus for cancer therapy. Here, we constructed a fluorescent reporter virus for real-time visualization and quantification of M1 virus both in vitro and in vivo. The reporter-encoding M1 virus maintained the characteristics of parental virus in the aspects of structure, replication capacity, the feature to induce cytopathic cell death, and the property of tumor targeting. The fluorescence is positively correlated with virus replication both in vitro and in vivo. More importantly, the reporter can be stably expressed for at least 10 generations in a serial passage assay. In summary, we successfully constructed stable and authentic reporter viruses for studying M1 virus and provided a feasible technical route for gene modification of oncolytic virus M1.

Aspirin inhibits proliferation of gemcitabine-resistant human pancreatic cancer cells and augments gemcitabine-induced cytotoxicity.

AIM: To investigate whether aspirin is able to augment gemcitabine-induced cytotoxicity in human pancreatic cancer cells. METHODS: Two gemcitabine-insensitive human pancreatic cancer cell lines, PANC-1 and Capan-1, were used. Cells were treated with either aspirin or gemcitabine alone or both of them. Cell growth and apoptosis were determined by MTT assay, Annexin V or Hoechest 33258 staining. Cell cycle distribution was examined by flow cytometry. Western blot with specific phosphorylated protein antibodies was used to detect the activation of protein kinase. RT-PCR and Western blot were applied to assess the transcription and protein level for cyclin D1 and Bcl-2. RESULTS: Aspirin alone significantly inhibits the proliferation of PANC-1 cells by causing cell cycle arrest at G(1) phase. Aspirin potentiates the anti-survival effect of gemcitabine as well as its pro-apoptotic effect in PANC-1 cells, although aspirin per se does not trigger apoptosis. Aspirin inhibits GSK-3beta activation and suppresses the expression of its downstream gene products (cyclin D1 and Bcl-2), which are implicated in proliferation, survival and chemoresistance of pancreatic cancer. The effects of aspirin on Capan-1, were similar to that on PANC-1. CONCLUSION: Our results suggest that aspirin inhibits the proliferation of gemcitabine-resistant pancreatic cancer cells and augments the antisurvival effect of gemcitabine, probably by suppressing the activity of GSK-3beta and its downstream gene products.

A small-molecule triptolide suppresses angiogenesis and invasion of human anaplastic thyroid carcinoma cells via down-regulation of the nuclear factor-kappa B pathway.

Anaplastic thyroid carcinoma (ATC) is among the most aggressive malignancies known and is characterized with rapid growth, early invasion, and complete refractoriness to current therapies. Here we report that triptolide, a small molecule from a Chinese herb, could potently inhibit proliferation in vitro, angiogenesis in vivo, and invasion in a Matrigel model in human ATC cell line TA-K cells at nanomolar concentrations. We further elucidate that triptolide inhibits the nuclear factor-kappaB (NF-kappaB) transcriptional activity via blocking the association of p65 subunit with CREB-binding protein (CBP)/p300 in the early stage and via decreasing the protein level of p65 in the late stage. Expression of the NF-kappaB targeting genes cyclin D1, vascular endothelial growth factor, and urokinase-type plasminogen activator is significantly reduced by triptolide in both TA-K and 8505C human ATC cell lines, which are well known to be critical for proliferation, angiogenesis, and invasion in solid tumors. Our findings suggest that triptolide may function as a small molecule inhibitor of tumor angiogenesis and invasion and may provide novel mechanistic insights into the potential therapy for human ATC.

Negative regulation of miR-1275 by H3K27me3 is critical for glial induction of glioblastoma cells.

Activation of the cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway induces glial differentiation of glioblastoma (GBM) cells, but the mechanism by which microRNA (miRNA) regulate this process remains poorly understood. In this study, by performing miRNA genomics and loss- and gain-of-function assays in dibutyryl-cAMP-treated GBM cells, we identified a critical negative regulator, hsa-miR-1275, that modulates a set of genes involved in cancer progression, stem cell maintenance, and cell maturation and differentiation. Additionally, we confirmed that miR-1275 directly and negatively regulates the protein expression of glial fibrillary acidic protein (GFAP), a marker of mature astrocytes. Of note, tri-methyl-histone H3 (Lys27) (H3K27me3), downstream of the PKA/polycomb repressive complex 2 (PRC2) pathway, accounts for the downregulation of miR-1275. Furthermore, decreased miR-1275 expression and induction of GFAP expression were also observed in dibutyryl-cAMP-treated primary cultured GBM cells. In a patient-derived glioma stem cell tumor model, a cAMP elevator and an inhibitor of H3K27me3 methyltransferase inhibited tumor growth, induced differentiation, and reduced expression of miR-1275. In summary, our study shows that epigenetic inhibition of miR-1275 by the cAMP/PKA/PRC2/H3K27me3 pathway mediates glial induction of GBM cells, providing a new mechanism and novel targets for differentiation-inducing therapy.

Purification and characterization of a novel antinociceptive toxin from Cobra venom (Naja naja atra).

Snake venoms have demonstrated antinociceptive activity, and certain isolated neurotoxins have demonstrated significant analgesia in animal models. Here we report a novel analgesic toxin which was isolated from Naja naja atra and was given the name 'najanalgesin'. The LD(50) of the crude venom and najanalgesin were 0.89mg/kg and 2.69mg/kg, respectively. We used the writhing test and hot plate test to evaluate the antinociceptive properties of the crude venom and najanalgesin after intraperitoneal (ip) administration. The analgesic mechanism of najanalgesin was also studied. The response latency time was significantly prolonged in the hot plate test after ip administration of the crude venom of Naja naja atra (0.111-0.445mg/kg) in a dose-dependent manner. Najanalgesin (1mg/kg) elicited almost the same antinociceptive effect as that of the crude venom of Naja naja atra at the dose of 0.445mg/kg and remained for 6h after intraperitoneal injection, shown by hot plate test. The percentage of increase in the latency time for the venom and the najanalgesin 3h after drug administration was 96.2% and 112%, respectively. The number of writhes decreased to almost 1/3, 1/6, and 1/12 of the NS (physiological saline) group after intraperitoneal administration of najanalgesin at 0.25, 0.5, and 1.0mg/kg, respectively. Pretreatment with atropine (1mg/kg) or naloxone (3mg/kg) blocked the antinociception of najanalgesin in the hot plate test. Based on the sequence information, najanalgesin is found to be highly homologous with the conventional CTXs (cardiotoxins). To our knowledge, no study had previously reported that a toxin which was homologous with CTXs possessed the antinociceptive activity. Thus, this is the first report that the antinociceptive effect induced by najanalgesin is mediated by cholinergic and opioidergic mechanisms.

DNA-PK inhibition synergizes with oncolytic virus M1 by inhibiting antiviral response and potentiating DNA damage.

Oncolytic virotherapy is a promising therapeutic strategy that uses replication-competent viruses to selectively destroy malignancies. However, the therapeutic effect of certain oncolytic viruses (OVs) varies among cancer patients. Thus, it is necessary to overcome resistance to OVs through rationally designed combination strategies. Here, through an anticancer drug screening, we show that DNA-dependent protein kinase (DNA-PK) inhibition sensitizes cancer cells to OV M1 and improves therapeutic effects in refractory cancer models in vivo and in patient tumour samples. Infection of M1 virus triggers the transcription of interferons (IFNs) and the activation of the antiviral response, which can be abolished by pretreatment of DNA-PK inhibitor (DNA-PKI), resulting in selectively enhanced replication of OV M1 within malignancies. Furthermore, DNA-PK inhibition promotes the DNA damage response induced by M1 virus, leading to increased tumour cell apoptosis. Together, our study identifies the combination of DNA-PKI and OV M1 as a potential treatment for cancers.

Selective Antagonism of Bcl-xL Potentiates M1 Oncolysis by Enhancing Mitochondrial Apoptosis.

Oncolytic virotherapy is a novel and intriguing treatment strategy for cancer therapy. However, the clinical potential of oncolytic virus as single agent is limited. M1 virus is a promising oncolytic virus that has been tested in preclinical studies. In this study, we investigated the effect of the combination use of M1 virus and Bcl-2 family inhibitors. A chemical compounds screening including ten Bcl-2 family inhibitors demonstrated that pan-Bcl-2 inhibitors selectively augmented M1 virus oncolysis in cancer cells at very low doses. The mechanism of the enhanced antitumor effect of pan-Bcl-2 inhibitors with M1 virus is mainly due to the inhibition of Bcl-xL, which synergizes with M1-induced upregulation of Bak to trigger apoptosis. In xenograft mouse models and patient-derived tumor tissues, the combination of M1 and pan-Bcl-2 inhibitors significantly inhibited tumor growth and prolonged survival, suggesting the potential therapeutic value of this strategy. These findings offer insights into the synergy between Bcl-xL inhibition and oncolytic virus M1 as a combination anticancer treatment modality.

Inhibition of the mevalonate pathway enhances cancer cell oncolysis mediated by M1 virus.

Oncolytic virus is an attractive anticancer agent that selectively lyses cancer through targeting cancer cells rather than normal cells. Although M1 virus is effective against several cancer types, certain cancer cells present low sensitivity to it. Here we identified that most of the components in the cholesterol biosynthesis pathway are downregulated after M1 virus infection. Further functional studies illustrate that mevalonate/protein farnesylation/ras homolog family member Q (RHOQ) axis inhibits M1 virus replication. Further transcriptome analysis shows that RHOQ knockdown obviously suppresses Rab GTPase and ATP-mediated membrane transporter system, which may mediate the antiviral effect of RHOQ. Based on this, inhibition of the above pathway significantly enhances the anticancer potency of M1 virus in vitro, in vivo, and ex vivo. Our research provides an intriguing strategy for the rational combination of M1 virus with farnesyl transferase inhibitors to enhance therapeutic efficacy.

Exogenous hydrogen sulfide eliminates spatial memory retrieval impairment and hippocampal CA1 LTD enhancement caused by acute stress via promoting glutamate uptake.

Acute stress impairs the hippocampus-dependent spatial memory retrieval, and its synaptic mechanisms are associated with hippocampal CA1 long-term depression (LTD) enhancement in the adult rats. Endogenous hydrogen sulfide (H2S) is recognized as a novel gasotransmitter and has the neural protective roles. However, very little attention has been paid to understanding the effects of H2S on spatial memory retrieval impairment. We observed the protective effects of NaHS (a donor of H2S) against spatial memory retrieval impairment caused by acute stress and its synaptic mechanisms. Our results showed that NaHS abolished spatial memory retrieval impairment and hippocampal CA1 LTD enhancement caused by acute stress, but not by glutamate transporter inhibitor l-trans-pyrrolidine-2,4-dicarboxylic (tPDC), indicating that the activation of glutamate transporters is necessary for exogenous H2S to exert its roles. Moreover, NaHS restored the decreased glutamate uptake in the hippocampal CA1 synaptosomal fraction caused by acute stress. Dithiothreitol (DTT, a disulfide reducing agent) abolished a decrease in the glutamate uptake caused by acute stress, and NaHS eradicated the decreased glutamate uptake caused by 5,5'-dithio-bis(2-nitrobenzoic)acid (DTNB, a thiol oxidizing agent), collectively, revealing that exogenous H2S increases glutamate uptake by reducing disulfide bonds of the glutamate transporters. Additionally, NaHS inhibited the increased expression level of phosphorylated c-Jun-N-terminal kinase (JNK) in the hippocampal CA1 region caused by acute stress. The JNK inhibitor SP600125 eliminated spatial memory retrieval impairment, hippocampal CA1 LTD enhancement and the decreased glutamate uptake caused by acute stress, indicating that exogenous H2S exerts these roles by inhibiting the activation of JNK signaling pathway.

Targeting VCP enhances anticancer activity of oncolytic virus M1 in hepatocellular carcinoma.

Oncolytic virotherapy is rapidly progressing through clinical evaluation. However, the therapeutic efficacy of oncolytic viruses in humans has been less than expected from preclinical studies. We describe an anticancer drug screen for compounds that enhance M1 oncolytic virus activity in hepatocellular carcinoma (HCC). An inhibitor of the valosin-containing protein (VCP) was identified as the top sensitizer, selectively increasing potency of the oncolytic virus up to 3600-fold. Further investigation revealed that VCP inhibitors cooperated with M1 virus-suppressed inositol-requiring enzyme 1α (IRE1α)-X-box binding protein 1 (XBP1) pathway and triggered irresolvable endoplasmic reticulum (ER) stress, subsequently promoting robust apoptosis in HCC. We show that VCP inhibitor improved the oncolytic efficacy of M1 virus in several mouse models of HCC and primary HCC tissues. Finally, this combinatorial therapeutic strategy was well tolerated in nonhuman primates. Our study identifies combined VCP inhibition and oncolytic virus as a potential treatment for HCC and demonstrates promising therapeutic potential.