Shaoxiang Wang

Epidermal Growth Factor Receptor-PI3K Signaling Controls Cofilin Activity To Facilitate Herpes Simplex Virus 1 Entry into Neuronal Cells

ABSTRACT Herpes simplex virus type 1 (HSV-1) establishes latency in neurons and can cause severe disseminated infection with neurological impairment and high mortality. This neurodegeneration is thought to be tightly associated with virus-induced cytoskeleton disruption. Currently, the regulation pattern of the actin cytoskeleton and the involved molecular mechanisms during HSV-1 entry into neurons remain unclear. Here, we demonstrate that the entry of HSV-1 into neuronal cells induces biphasic remodeling of the actin cytoskeleton and an initial inactivation followed by the subsequent activation of cofilin, a member of the actin depolymerizing factor family that is critical for actin reorganization. The disruption of F-actin dynamics or the modulation of cofilin activity by mutation, knockdown, or overexpression affects HSV-1 entry efficacy and virus-mediated cell ruffle formation. Binding of the HSV-1 envelope initiates the epidermal growth factor receptor (EGFR)-phosphatidylinositide 3-kinase (PI3K) signaling pathway, which leads to virus-induced early cofilin phosphorylation and F-actin polymerization. Moreover, the extracellular signal-regulated kinase (ERK) kinase and Rho-associated, coiled-coil-containing protein kinase 1 (ROCK) are recruited as downstream mediators of the HSV-1-induced cofilin inactivation pathway. Inhibitors specific for those kinases significantly reduce the virus infectivity without affecting virus binding to the target cells. Additionally, lipid rafts are clustered to promote EGFR-associated signaling cascade transduction. We propose that HSV-1 hijacks cofilin to initiate infection. These results could promote a better understanding of the pathogenesis of HSV-1-induced neurological diseases. IMPORTANCE The actin cytoskeleton is involved in many crucial cellular processes and acts as an obstacle to pathogen entry into host cells. Because HSV-1 establishes lifelong latency in neurons and because neuronal cytoskeletal disruption is thought to be the main cause of HSV-1-induced neurodegeneration, understanding the F-actin remodeling pattern by HSV-1 infection and the molecular interactions that facilitate HSV-1 entry into neurons is important. In this study, we showed that HSV-1 infection induces the rearrangement of the cytoskeleton as well as the initial inactivation and subsequent activation of cofilin. Then, we determined that activation of the EGFR-PI3K-Erk1/2 signaling pathway inactivates cofilin and promotes F-actin polymerization. We postulate that by regulating actin cytoskeleton dynamics, cofilin biphasic activation could represent the specific cellular machinery usurped by pathogen infection, and these results will greatly contribute to the understanding of HSV-1-induced early and complex changes in host cells that are closely linked to HSV-1 pathogenesis. The actin cytoskeleton is involved in many crucial cellular processes and acts as an obstacle to pathogen entry into host cells. Because HSV-1 establishes lifelong latency in neurons and because neuronal cytoskeletal disruption is thought to be the main cause of HSV-1-induced neurodegeneration, understanding the F-actin remodeling pattern by HSV-1 infection and the molecular interactions that facilitate HSV-1 entry into neurons is important. In this study, we showed that HSV-1 infection induces the rearrangement of the cytoskeleton as well as the initial inactivation and subsequent activation of cofilin. Then, we determined that activation of the EGFR-PI3K-Erk1/2 signaling pathway inactivates cofilin and promotes F-actin polymerization. We postulate that by regulating actin cytoskeleton dynamics, cofilin biphasic activation could represent the specific cellular machinery usurped by pathogen infection, and these results will greatly contribute to the understanding of HSV-1-induced early and complex changes in host cells that are closely linked to HSV-1 pathogenesis.

Cofilin 1-Mediated Biphasic F-Actin Dynamics of Neuronal Cells Affect Herpes Simplex Virus 1 Infection and Replication

ABSTRACT Herpes simplex virus 1 (HSV-1) invades the nervous system and causes pathological changes. In this study, we defined the remodeling of F-actin and its possible mechanisms during HSV-1 infection of neuronal cells. HSV-1 infection enhanced the formation of F-actin-based structures in the early stage of infection, which was followed by a continuous decrease in F-actin during the later stages of infection. The disruption of F-actin dynamics by chemical inhibitors significantly reduced the efficiency of viral infection and intracellular HSV-1 replication. The active form of the actin-depolymerizing factor cofilin 1 was found to increase at an early stage of infection and then to continuously decrease in a manner that corresponded to the remodeling pattern of F-actin, suggesting that cofilin 1 may be involved in the biphasic F-actin dynamics induced by HSV-1 infection. Knockdown of cofilin 1 impaired HSV-1-induced F-actin assembly during early infection and inhibited viral entry; however, overexpression of cofilin 1 did not affect F-actin assembly or viral entry during early infection but decreased intracellular viral reproduction efficiently. Our results, for the first time, demonstrated the biphasic F-actin dynamics in HSV-1 neuronal infection and confirmed the association of F-actin with the changes in the expression and activity of cofilin 1. These results may provide insight into the mechanism by which HSV-1 productively infects neuronal cells and causes pathogenesis.

SNX-2112, an Hsp90 inhibitor, induces apoptosis and autophagy via degradation of Hsp90 client proteins in human melanoma A-375 cells

SNX-2112 is an Hsp90 inhibitor which is currently undergoing multiple phase 1 clinical trials; however, its mechanism of action needs to be further elaborated. Here we investigated the effects of SNX-2112 in A-375 cells. SNX-2112 induced the degradation of multiple Hsp90 client proteins, activated both the mitochondrial-mediated and death receptor-mediated apoptotic pathways, downregulated Bcl-2 and Bcl-xL, upregulated Bid, cleaved caspase-9, caspase-7, caspase-3 and PARP, and activated caspase-8. The general caspase inhibitor, z-VAD-fmk, did not completely abolish SNX-2112-induced cell death. SNX-2112 induced autophagy in a time- and dose-dependent manner via Akt/mTOR/p70S6K inhibition. SNX-2112 induces significant apoptosis and autophagy in human melanoma A-375 cells, and may be an effective targeted therapy agent.

Induction of apoptosis in K562 cells by dicyclohexylammonium salt of hyperforin through a mitochondrial-related pathway.

Hyperforin is an abundant phloroglucinol-type constituent isolated from the extract of the flowering upper portion of the plant Hypericum perforatum L. The dicyclohexylammonium salt of hyperforin (DCHA-HF) has exhibited antitumor and antiangiogenic activities in various cancer cells. Here, the antitumor effects of DCHA-HF on the chronic myeloid leukemia K562 cell line were investigated for the first time. DCHA-HF exhibited dose- and time-dependent inhibitory activities against K562 cells, with IC(50) values of 8.6 and 3.2 μM for 48 h and 72 h of treatment, respectively, which was more effective than that of the hyperforin. In contrast, little cytotoxic activity was observed with DCHA-HF on HUVECs. DCHA-HF treatment resulted in induction of apoptosis as evidenced from DNA fragmentation, nuclear condensation and increase of early apoptotic cells by DAPI staining analysis, TUNEL assay and Annexin V-FITC/PI double-labeled staining analysis, respectively. Moreover, DCHA-HF elicited dissipation of mitochondrial transmembrane potential that commenced with the release of cytochrome c through down-regulation of expression of anti-apoptotic proteins and up-regulation of expression of pro-apoptotic proteins. DCHA-HF treatment induced activation of the caspase 3, 8, and 9 cascade and subsequent PARP cleavage, and DCHA-HF-induced apoptosis was significantly inhibited by caspase inhibitors. Treated cells were arrested at the G1 phase of the cell cycle and the expression of p53 and p27(Kip1), two key regulators related to cell cycle and apoptosis, was up-regulated. These results suggest that DCHA-HF inhibits K562 cell growth by inducing caspase-dependent apoptosis mediated by a mitochondrial pathway and arresting the cell cycle at the G1 phase. Therefore, DCHA-HF is a potential chemotherapeutic antitumor drug for chronic myeloid leukemia therapy.

The Hsp90 inhibitor SNX-2112, induces apoptosis in multidrug resistant K562/ADR cells through suppression of Akt/NF-κB and disruption of mitochondria-dependent pathways.

Heat shock protein 90 (Hsp90) serves as an ATP-dependent molecular chaperone for numerous cell signaling proteins, including many oncogenes and clinically validated cancer targets that are involved in cell proliferation and survival. Recent studies have shown that the Hsp90 inhibitor, SNX-2112, effectively inhibits tumor cell growth and angiogenesis in hematological and solid tumors. However, little is known about the effects of SNX-2112 on leukemias that are resistant to chemotherapy, which is emerging as a major clinical problem. In this study, the effects of SNX-2112 on the multidrug-resistant human chronic myeloid leukemia (CML) K562/ADR cell line were investigated. We observed that SNX-2112 exhibited dose- and time-dependent inhibitory activities against K562/ADR cells. These effects included the induction of apoptosis and secondary necrosis in addition to cell cycle arrest at the G1 and G2 phases. Furthermore, SNX-2112-induced apoptosis was predominantly mediated by the mitochondrial pathway, initiated by the release of cytochrome c and the participation of Bcl-2 family proteins. SNX-2112 also induced the activation of the caspase-3, -8 and -9 cascade and the subsequent cleavage of PARP in K562/ADR cells. Moreover, the inactivation of the Akt and NF-κB signaling pathways may be involved in SNX-2112-induced apoptosis. The expression levels of P-glycoprotein (P-gp) and several chaperons related to drug resistance and apoptosis were also shown to be inhibited, including the Grp78 and Hsp90 isoforms, Grp94 and Trap1. Taken together, these results provide a possible molecular mechanism for the anti-cancer effect of SNX-2112 on K562/ADR cells and provide new insights into the future application of SNX-2112 as a therapeutic agent for anti-multidrug-resistant leukemias.

BJ-B11, a novel Hsp90 inhibitor, induces apoptosis in human chronic myeloid leukemia K562 cells through the mitochondria-dependent pathway.

In the past few years heat shock protein 90 (Hsp90) inhibitors have been reported to possess significant antitumor activity. We investigated, for the first time, the antitumor activity of a novel Hsp90 inhibitor 2-(4-acetyloxycyclohexylamino)-4-(3, 6, 6-trimethyl-4-oxo-4, 5, 6, 7-tetrahydro-1H-indazol-1-yl)-benzamide (BJ-B11) and the molecular mechanism underlying the apoptosis it induces in human chronic myeloid leukemia K562 cells. The results revealed that BJ-B11 triggered growth inhibition in K562 cells and other malignant cell lines in vitro with only minor toxicity in a normal human cell line. BJ-B11 inhibited the proliferation of K562 cells in a concentration- and time-dependent manner, with IC(50) values of 1.1 ± 0.2 μM and 0.4 ± 0.1 μM after 48 and 72 h incubations respectively. This most likely results from cell cycle arrest at the G(0)/G(1) phase and the induction of apoptosis. In addition, BJ-B11 degraded the Hsp90 client proteins Bcr-Abl and Akt, induced activation of caspase-9 and caspase-3, and subsequent cleavage of PARP. The caspase signals may originate from mitochondrial dysfunction, which is supported by the finding of cytochrome c release. In addition, inactivation of the Akt signaling pathway may be involved in the process of BJ-B11-induced apoptosis. Taken together, our data provide a putative molecular mechanism for the anticancer effect of BJ-B11 on K562 cells, and suggest a potential application for BJ-B11 in chronic myeloid leukemia therapy.

The Hsp90 inhibitor SNX-2112 induces apoptosis of human hepatocellular carcinoma cells: The role of ER stress

Heat shock protein 90 (Hsp90) has been predicted to be involved in hepatocellular carcinoma (HCC) therapy; however, the mechanisms of action remain elusive. SNX-2112 is an Hsp90 inhibitor showing broad antitumor activity. Here we aim to determine the role of the endoplasmic reticulum (ER) stress in SNX-2112-induced apoptosis in HCC cells. In general, three HCC cells (i.e., HepG2, Huh7, and SK-Hep1) were used in our experiments. The cell viability was determined by the CCK-8 assay. The apoptosis was analyzed using flow cytometry, laser scanning confocal microscopy (LSM) and Western blotting. The efficacy and mechanisms of action of SNX-2112 were also evaluated in a mouse xenograft model. We found that SNX-2112 showed stronger inhibition on cell growth than 17-AAG, a classical Hsp90 inhibitor. SNX-2112 treatment led to the caspase-dependent apoptosis. Interestingly, SNX-2112 decreased the expression levels of the ER chaperone proteins calnexin and immunoglobulin binding protein (BiP). It also inhibited all three ER stress sensors, namely, inositol-requiring gene 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF-6) in vitro and/or in vivo. However, the ER stress inducer tunicamycin strongly enhanced SNX-2112-induced apoptosis, whereas the IRE1 knockdown did not. Taken together, we for the first time indicated the possible apoptotic pathways of SNX-2112 in HCC cells, raising the possibility that the induction of ER stress might be favorable for SNX-2112-induced apoptosis.

Inhibition of herpes simplex virus type 1 entry by chloride channel inhibitors tamoxifen and NPPB

Herpes simplex virus type 1 (HSV-1) infection is very common worldwide and can cause significant health problems from periodic skin and corneal lesions to encephalitis. Appearance of drug-resistant viruses in clinical therapy has made exploring novel antiviral agents emergent. Here we show that chloride channel inhibitors, including tamoxifen and 5-nitro-2-(3-phenyl-propylamino) benzoic acid (NPPB), exhibited extensive antiviral activities toward HSV-1 and ACV-resistant HSV viruses. HSV-1 infection induced chloride ion influx while treatment with inhibitors reduced the increase of intracellular chloride ion concentration. Pretreatment or treatment of inhibitors at different time points during HSV-1 infection all suppressed viral RNA synthesis, protein expression and virus production. More detailed studies demonstrated that tamoxifen and NPPB acted as potent inhibitors of HSV-1 early entry step by preventing viral binding, penetration and nuclear translocation. Specifically the compounds appeared to affect viral fusion process by inhibiting virus binding to lipid rafts and interrupting calcium homeostasis. Taken together, the observation that tamoxifen and NPPB can block viral entry suggests a stronger potential for these compounds as well as other ion channel inhibitors in antiviral therapy against HSV-1, especially the compound tamoxifen is an immediately actionable drug that can be reused for treatment of HSV-1 infections.

SNX-25a, a novel Hsp90 inhibitor, inhibited human cancer growth more potently than 17-AAG.

17-Allylamino-17-demethoxygeldanamycin (17-AAG), a typical Hsp90 inhibitor derived from geldanamycin (GA), has entered Phase III clinical trials for cancer therapy. However, it has several significant limitations such as poor solubility, limited bioavailability and unacceptable hepatotoxicity. In this study, the anticancer activity and mechanism of SNX-25a, a novel Hsp90 inhibitor, was investigated comparing with that of 17-AAG. We showed that SNX-25a triggered growth inhibition more sensitively than 17-AAG against many human cancer cells, including K562, SW-620, A375, Hep-2, MCF-7, HepG2, HeLa, and A549 cell lines, especially at low concentrations (<1 μM). It showed low cytotoxicity in L-02, HDF and MRC5 normal human cells. Compared with 17-AAG, SNX-25a was more potent in arresting the cell cycle at G2 phase, and displayed more potent effects on human cancer cell apoptosis and Hsp90 client proteins. It also exhibited a stronger binding affinity to Hsp90 than 17-AAG using molecular docking. Considering the superiority effects on Hsp90 affinity, cell growth, cell cycle, apoptosis, and Hsp90 client proteins, SNX-25a is supposed as a potential anticancer agent that needs to be explored in detail.

The selective Hsp90 inhibitor BJ-B11 exhibits potent antitumor activity via induction of cell cycle arrest, apoptosis and autophagy in Eca-109 human esophageal squamous carcinoma cells

BJ-B11 is a selective heat shock protein 90 (Hsp90) inhibitor that has been reported to possess significant antitumor activity in multiple types of cancer cells; however, the mechanism of action needs to be further clarified. We investigated, for the first time, the antitumor activity and the molecular mechanism underlying growth inhibition in Eca-109 cells. The results revealed that BJ-B11 inhibited the proliferation of Eca-109 cells in a time- and concentration-dependent manner, with 50% inhibitory concentration (IC(50)) values of 0.31±0.01 µM after 48-h incubation. BJ-B11 induced concentration-dependent G2/M cell cycle arrest and apoptosis. The cleavage of caspase 3 and PARP signals detected might originate from mitochondrial dysfunction, which was supported by the results of reactive oxygen species (ROS) production, cytochrome c release and the mitochondrial membrane potential (MMP) reduction. The general caspase inhibitor Z-VAD-fmk did not completely abolish BJ-B11-induced cell death. Furthermore, inhibition of the Akt/mTOR/p70S6K signaling pathway might be involved in the process of BJ-B11-induced autophagy, which was characterized by the production of autophagic vacuoles and upregulation of LC3-II protein in a time- and concentration-dependent manner. Meanwhile, the general autophagy inhibitor 3-MA decreased the apoptotic ratio. Furthermore, BJ-B11 induced the polymerization of cytoskeleton β-tubulin and F-actin. Taken together, our results suggest that the growth inhibition of Eca-109 cells induced by BJ-B11 may result from the induction of G2/M cell cycle arrest, apoptosis and autophagy.