A peptide derived from interleukin-10 exhibits potential anticancer activity and can facilitate cell targeting of gold nanoparticles loaded with anticancer therapeutics.

Human interleukin-10 (IL-10) is an immunosuppressive and anti-inflammatory cytokine, and its expression is upregulated in tumor tissues and serum samples of patients with various cancers. Because of its immunosuppressive nature, IL-10 has also been suggested to be a factor leading to tumor cells' evasion of immune surveillance and clearance by the host immune system. In this study, we refined a peptide with 20 amino acids, named NK20a, derived from the binding region of IL-10 on the basis of in silico analysis of the complex structure of IL-10 with IL-10Ra, the ligand binding subunit of the IL-10 receptor. The binding ability of the peptide was confirmed through in vitro biophysical biolayer interferometry and cellular experiments. The IL-10 inhibitory peptide exerted anticancer effects on lymphoma B cells and could abolish the suppression effect of IL-10 on macrophages. NK20a was also conjugated with gold nanoparticles to target the chemotherapeutic 5-fluorouracil (5-FU)-loaded nanoparticles to enhance the anticancer efficacy of 5-FU against the breast cancer cell line BT-474. Our study demonstrated that NK20a designed in silico with improved binding affinity to the IL-10 receptor can be used as a tool in developing anticancer strategies.

Therapeutic Peptide RF16 Derived from CXCL8 Inhibits MDA-MB-231 Cell Invasion and Metastasis.

Interleukin (IL)-8 plays a vital role in regulating inflammation and breast cancer formation by activating CXCR1/2. We previously designed an antagonist peptide, (RF16), to inhibits the activation of downstream signaling pathways by competing with IL-8 in binding to CXCR1/2, thereby inhibiting IL-8-induced chemoattractant monocyte binding. To evaluate the effect of the RF16 peptide on breast cancer progression, triple-negative MDA-MB-231 and ER-positive MCF-7 breast cancer cells were used to investigate whether RF16 can inhibit the IL-8-induced breast cancer metastasis. Using growth, proliferation, and invasiveness assays, the results revealed that RF16 reduced cell proliferation, migration, and invasiveness in MDA-MB-231 cells. The RF16 peptide also regulated the protein and mRNA expressions of epithelial-mesenchymal transition (EMT) markers in IL-8-stimulated MDA-MB-231 cells. It also inhibited downstream IL-8 signaling and the IL-8-induced inflammatory response via the mitogen-activated protein kinase (MAPK) and Phosphoinositide 3-kinase (PI3K) pathways. In the xenograft tumor mouse model, RF16 synergistically reinforces the antitumor efficacy of docetaxel by improving mouse survival and retarding tumor growth. Our results indicate that RF16 significantly inhibited IL-8-stimulated cell growth, migration, and invasion in MDA-MB-231 breast cancer cells by blocking the activation of p38 and AKT cascades. It indicated that the RF16 peptide may serve as a new supplementary drug for breast cancer.

A Multitarget Therapeutic Peptide Derived From Cytokine Receptors Based on in Silico Analysis Alleviates Cytokine-Stimulated Inflammation.

Septicemia is a severe inflammatory response caused by the invasion of foreign pathogens. Severe sepsis-induced shock and multiple organ failure are the two main causes of patient death. The overexpression of many proinflammatory cytokines, such as TNF-α, IL-1ß, and IL-6, is closely related to severe sepsis. Although the treatment of sepsis has been subject to many major breakthroughs of late, the treatment of patients with septic shock is still accompanied by a high mortality rate. In our previous research, we used computer simulations to design the multifunctional peptide KCF18 that can bind to TNF-α, IL-1ß, and IL-6 based on the binding regions of receptors and proinflammatory cytokines. In this study, proinflammatory cytokines were used to stimulate human monocytes to trigger an inflammatory response, and the anti-inflammatory ability of the multifunctional KCF18 peptide was further investigated. Cell experiments demonstrated that KCF18 significantly reduced the binding of proinflammatory cytokines to their cognate receptors and inhibited the mRNA and protein expressions of TNF-α, IL-1ß, and IL-6. It could also reduce the expression of reactive oxygen species induced by cytokines in human monocytes. KCF18 could effectively decrease the p65 nucleus translocation induced by cytokines, and a mice endotoxemia experiment demonstrated that KCF18 could reduce the expression of IL-6 and the increase of white blood cells in the blood stimulated by lipopolysaccharides. According to our study of tissue sections, KCF18 alleviated liver inflammation. By reducing the release of cytokines in plasma and directly affecting vascular cells, KCF18 is believed to significantly reduce the risk of vascular inflammation.

Combinatorial Virtual Screening Revealed a Novel Scaffold for TNKS Inhibition to Combat Colorectal Cancer.

The abnormal Wnt signaling pathway leads to a high expression of ß-catenin, which causes several types of cancer, particularly colorectal cancer (CRC). The inhibition of tankyrase (TNKS) activity can reduce cancer cell growth, invasion, and resistance to treatment by blocking the Wnt signaling pathway. A pharmacophore search and pharmacophore docking were performed to identify potential TNKS inhibitors in the training databases. The weighted MM/PBSA binding free energy of the docking model was calculated to rank the databases. The reranked results indicated that 26.98% of TNKS inhibitors that were present in the top 5% of compounds in the database and near an ideal value ranked 28.57%. The National Cancer Institute database was selected for formal virtual screening, and 11 potential TNKS inhibitors were identified. An enzyme-based experiment was performed to demonstrate that of the 11 potential TNKS inhibitors, NSC295092 and NSC319963 had the most potential. Finally, Wnt pathway analysis was performed through a cell-based assay, which indicated that NSC319963 is the most likely TNKS inhibitor (pIC50 = 5.59). The antiproliferation assay demonstrated that NSC319963 can decrease colorectal cancer cell growth; therefore, the proposed method successfully identified a novel TNKS inhibitor that can alleviate CRC.

Interleukin-10: A double-edged sword in breast cancer.

Breast cancer (BC) is a frequently diagnosed cancer among women worldwide. Currently, BC can be divided into different subgroups according to the presence of the following hormone receptors: estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2. Each of these subgroups has different treatment strategies. However, the presence of new metastatic lesions and patient deterioration suggest resistance to a given treatment. Various lines of evidence had shown that cytokines are one of the important mediators of tumor growth, invasion, metastasis, and treatment resistance. Interleukin-10 (IL-10) is an immunoregulatory cytokine, and acts as a poor prognostic marker in many cancers. The anti-inflammatory IL-10 blocks certain effects of inflammatory cytokines. It also antagonizes the co-stimulatory molecules on the antigen-presenting cells. Here, we review the current knowledge on the function and molecular mechanism of IL-10, and recent findings on how IL-10 contributes to the progression of BC.

Albendazole-Schisandrin B Co-Therapy on Angiostrongylus cantonensis-Induced Meningoencephalitis in Mice.

Abstract: Currently, Angiostrongylus cantonensis infections are predominantly treated with albendazole. However, the use of albendazole can provoke certain neurological symptoms as a result of the immune response triggered by the dead worms. Therefore, treatment usually involves co-administration of corticosteroids to limit the inflammatory reaction. Corticosteroids play a useful role in suppressing inflammation in the brain; however, long-term usage or high dosage may make it problematic.Schisandrin B, an active ingredient from Schisandra chinensis, has been shown to have anti-inflammatory effects on the brain. This study aimed to investigate the effects and potential of schisandrin B in combination with albendazole to treat Angiostrongylus-induced meningoencephalitis. Here, we show that albendazole-schisandrin B co-treatment suppressed neuroinflammation in Angiostrongylus-infected mice and increased the survival of the mice. Accordingly, albendazole-schisandrin B co-treatment significantly inhibited inflammasome activation, pyroptosis, and apoptosis. The sensorimotor functions of the mice were also repaired after albendazole-schisandrin B treatment. Immune response was shown to shift from Th2 to Th1, which reduces inflammation and enhances immunity against A. cantonensis. Collectively, our study showed that albendazole-schisandrin B co-therapy may be used as an encouraging treatment for Angiostrongylus-induced meningoencephalitis.

Internal water channel formation in CXCR4 is crucial for Gi-protein coupling upon activation by CXCL12.

Chemokine receptor CXCR4 is a major drug target for numerous diseases because of its involvement in the regulation of cell migration and the developmental process. In this study, atomic-level molecular dynamics simulations were used to determine the activation mechanism and internal water formation of CXCR4 in complex with chemokine CXCL12 and Gi-protein. The results indicated that CXCL12-bound CXCR4 underwent transmembrane 6 (TM6) outward movement and a decrease in tyrosine toggle switch by eliciting the breakage of hydrophobic layer to form a continuous internal water channel. In the GDP-bound Gαi-protein state, the rotation and translation of the α5-helix of Gαi-protein toward the cytoplasmic pocket of CXCR4 induced an increase in interdomain distance for GDP leaving. Finally, an internal water channel formation model was proposed based on our simulations for CXCL12-bound CXCR4 in complex with Gαi-protein upon activation for downstream signaling. This model could be useful in anticancer drug development.

A potential peptide derived from cytokine receptors can bind proinflammatory cytokines as a therapeutic strategy for anti-inflammation.

Chronic inflammation is a pivotal event in the pathogenesis of cardiovascular diseases, including atherosclerosis, restenosis, and coronary artery disease. The efficacy of current treatment or preventive strategies for such inflammation is still inadequate. Thus, new anti-inflammatory strategies are needed. In this study, based on molecular docking and structural analysis, a potential peptide KCF18 with amphiphilic properties (positively charged and hydrophobic residues) derived from the receptors of proinflammatory cytokines was designed to inhibit cytokine-induced inflammatory response. Simulations suggested that KCF18 could bind to cytokines simultaneously, and electrostatic interactions were dominant. Surface plasmon resonance detection showed that KCF18 bound to both tumor necrosis factor-α (TNF-α) and interleukin-6, which is consistent with MM/PBSA binding free energy calculations. The cell experiments showed that KCF18 significantly reduced the binding of proinflammatory cytokines to their cognate receptors, suppressed TNF-α mRNA expression and monocyte binding and transmigration, and alleviated the infiltration of white blood cells in a peritonitis mouse model. The designed peptide KCF18 could remarkably diminish the risk of vascular inflammation by decreasing plasma cytokines release and by directly acting on the vascular endothelium. This study demonstrated that a combination of structure-based in silico design calculations, together with experimental measurements can be used to develop potential anti-inflammatory agents.

The In Vitro and In Vivo Wound Healing Properties of the Chinese Herbal Medicine "Jinchuang Ointment".

"Jinchuang ointment" is a traditional Chinese herbal medicine complex for treatment of incised wounds. For more than ten years, it has been used at China Medical University Hospital (Taichung, Taiwan) for the treatment of diabetic foot infections and decubitus ulcers. Three different cases are presented in this study. "Jinchuang" ointment is a mixture of natural product complexes from nine different components, making it difficult to analyze its exact chemical compositions. To further characterize the herbal ingredients used in this study, the contents of reference standards present in a subset of the ointment ingredients (dragon's blood, catechu, frankincense, and myrrh) were determined by HPLC. Two in vitro cell based assay platforms, wound healing and tube formation, were used to examine the biological activity of this medicine. Our results show that this herbal medicine possesses strong activities including stimulation of angiogenesis, cell proliferation, and cell migration, which provide the scientific basis for its clinically observed curative effects on nonhealing diabetic wounds.

Peptides derived from CXCL8 based on in silico analysis inhibit CXCL8 interactions with its receptor CXCR1.

Chemokine CXCL8 is crucial for regulation of inflammatory and immune responses via activating its cognate receptor CXCR1. In this study, molecular docking and binding free energy calculations were combined to predict the initial binding event of CXCL8 to CXCR1 for peptide drug design. The simulations reveal that in the initial binding, the N-loop of CXCL8 interacts with the N-terminus of CXCR1, which is dominated by electrostatic interactions. The derived peptides from the binding region of CXCL8 are synthesized for further confirmation. Surface plasmon resonance analyses indicate that the CXCL8 derived peptide with 14 residues is able to bind to the receptor CXCR1 derived peptide with equilibrium KD of 252 µM while the peptide encompassing a CXCL8 K15A mutation hardly binds to CXCR1 derived peptide (KD = 1553 µM). The cell experiments show that the designed peptide inhibits CXCL8-induced and LPS-activated monocytes adhesion and transmigration. However, when the peptides were mutated on two lysine residues (K15 and K20), the inhibition effects were greatly reduced indicating these two amino acids are key residues for the initial binding of CXCL8 to CXCR1. This study demonstrates that in silico prediction based functional peptide design can be effective for developing anti-inflammation drugs.

Antimycobacterial Activities of Endolysins Derived From a Mycobacteriophage, BTCU-1.

The high incidence of Mycobacterium infection, notably multidrug-resistant M. tuberculosis infection, has become a significant public health concern worldwide. In this study, we isolate and analyze a mycobacteriophage, BTCU-1, and a foundational study was performed to evaluate the antimycobacterial activity of BTCU-1 and its cloned lytic endolysins. Using Mycobacterium smegmatis as host, a mycobacteriophage, BTCU-1, was isolated from soil in eastern Taiwan. The electron microscopy images revealed that BTCU-1 displayed morphology resembling the Siphoviridae family. In the genome of BTCU-1, two putative lytic genes, BTCU-1_ORF7 and BTCU-1_ORF8 (termed lysA and lysB, respectively), were identified, and further subcloned and expressed in Escherichia coli. When applied exogenously, both LysA and LysB were active against M. smegmatis tested. Scanning electron microscopy revealed that LysA and LysB caused a remarkable modification of the cell shape of M. smegmatis. Intracellular bactericidal activity assay showed that treatment of M. smegmatis-infected RAW 264.7 macrophages with LysA or LysB resulted in a significant reduction in the number of viable intracellular bacilli. These results indicate that the endolysins derived from BTCU-1 have antimycobacterial activity, and suggest that they are good candidates for therapeutic/disinfectant agents to control mycobacterial infections.

beta-Naphthoflavone protects from peritonitis by reducing TNF-alpha-induced endothelial cell activation.

ß-Naphthoflavone (ß-NF), a ligand of the aryl hydrocarbon receptor, has been shown to possess anti-oxidative properties. We investigated the anti-oxidative and anti-inflammatory potential of ß-NF in human microvascular endothelial cells treated with tumor necrosis factor-alpha (TNF-α). Pretreatment with ß-NF significantly inhibited TNF-α-induced intracellular reactive oxygen species, translocation of p67(phox), and TNF-α-induced monocyte binding and transmigration. In addition, ß-NF significantly inhibited TNF-α-induced ICAM-1 and VCAM-1 expression. The mRNA expression levels of the inflammatory cytokines TNF-α and IL-6 were reduced by ß-NF, as was the infiltration of white blood cells, in a peritonitis model. The inhibition of adhesion molecules was associated with suppressed nuclear translocation of NF-κB p65 and Akt, and suppressed phosphorylation of ERK1/2 and p38. The translocation of Egr-1, a downstream transcription factor involved in the MEK-ERK signaling pathway, was suppressed by ß-NF treatment. Our findings show that ß-NF inhibits TNF-α-induced NF-kB and ERK1/2 activation and ROS generation, thereby suppressing the expression of adhesion molecules. This results in reduced adhesion and transmigration of leukocytes in vitro and prevents the infiltration of leukocytes in a peritonitis model. Our findings also suggest that ß-NF might prevent TNF-α-induced inflammation.

RHBDL2 is a critical membrane protease for anoikis resistance in human malignant epithelial cells.

Anoikis resistance allows metastatic tumor cells to survive in a homeless environment. Activation of epithelial growth factor receptor (EGFR) signaling is one of the key mechanisms for metastatic tumor cells to resist anoikis, yet the regulation mechanisms of homeless-triggered EGFR activation in metastatic tumor cells remain unclear. Rhomboid-like-2 (RHBDL2), an evolutionally conserved intramembrane serine protease, can cleave the EGF ligand and thus trigger EGFR activation. Herein, we demonstrated that RHBDL2 overexpression in human epithelial cells resulted in promotion of cell proliferation, reduction of cell adhesion, and suppression of anoikis. During long-term suspension cultures, increased RHBDL2 was only detected in aggressive tumor cell lines. Treatment with the rhomboid protease inhibitor or RHBDL2 shRNA increased cleaved caspase 3, a marker of apoptosis. Finally, inhibition of EGFR activation increased the cleaved caspase 3 and attenuated the detachment-induced focal adhesion kinase phosphorylation. Taken together, these findings provide evidence for the first time that RHBDL2 is a critical molecule in anoikis resistance of malignant epithelial cells, possibly through the EGFR-mediated signaling. Our study demonstrates RHBDL2 as a new therapeutic target for cancer metastasis.

Dextromethorphan attenuates LPS-induced adhesion molecule expression in human endothelial cells.

OBJECTIVE: This study examines the effect of Dextromethorphan (d-3-methoxy-17-methylmorphinan; DXM), a commonly used cough-suppressing drug, on the expression of VCAM-1 and ICAM-1 in human umbilical vein endothelial cells (HUVECs) stimulated with lipopolysaccharide (LPS). METHODS: The effect of DXM on expression of cell adhesion molecules induced by LPS was evaluated by monocyte bindings in vitro and ex vivo and transmigration assays. The signaling pathways involved in the inflammation inhibitory effect of DXM were analyzed by Western blot and immunofluorescent stain. RESULTS: Pretreatment of HUVECs with DXM inhibited LPS-induced adhesion of THP-1 cells in vitro and ex vivo, and reduced transendothelial migration of these cells. Furthermore, treatment of HUVECs with DXM can significantly decrease LPS-induced expression of ICAM-1 and VCAM-1. DXM abrogated LPS-induced phosphorylation of ERK and Akt. The translocation of early growth response gene-1 (Egr-1), a downstream transcription factor involved in the mitogen-activated kinase (MEK)-ERK signaling pathway, was suppressed by DXM treatment. Furthermore, DXM inhibited LPS-induced IκBα degradation and nuclear translocation of p65. CONCLUSIONS: Dextromethorphan inhibits the adhesive capacity of HUVECs by reducing the LPS-induced ICAM-1 and VCAM-1 expression via the suppression of the ERK, Akt, and NF-κB signaling pathways. Thus, DXM is a potential anti-inflammatory therapeutic that may modulate atherogenesis.

The epidermal growth factor-like domain of CD93 is a potent angiogenic factor.

Human CD93, an epidermal growth factor (EGF)-like domain containing transmembrane protein, is predominantly expressed in the vascular endothelium. Studies have shown that AA4, the homolog of CD93 in mice, may mediate cell migration and angiogenesis in endothelial cells. Soluble CD93 has been detected in the plasma of healthy individuals. However, the role of soluble CD93 in the endothelium remains unclear. Recombinant soluble CD93 proteins with EGF-like domains (rCD93D123, with domains 1, 2, and 3; and rCD93D23, with domains 2 and 3) were generated to determine their functions in angiogenesis. We found that rCD93D23 was more potent than rCD93D123 in stimulating the proliferation and migration of human umbilical vein endothelial cells (HUVECs). Production of matrix-metalloproteinase 2 increased after the HUVECs were treated with rCD93D23. Further, in a tube formation assay, rCD93D23 induced cell differentiation of HUVECs through phosphoinositide 3-kinase/Akt/endothelial nitric oxide synthase and extracellular signal-regulated kinases-1/2 signaling. Moreover, rCD93D23 promoted blood vessel formation in a Matrigel-plug assay and an oxygen-induced retinopathy model in vivo. Our findings suggest that the soluble EGF-like domain containing CD93 protein is a novel angiogenic factor acting on the endothelium.

Dextromethorphan reduces oxidative stress and inhibits atherosclerosis and neointima formation in mice.

AIMS: Macrophage-related oxidative stress plays an important role in the inflammatory process in atherosclerosis. Recently, dextromethorphan (DXM), a common cough-suppressing ingredient with a high safety profile, was found to inhibit the activation of microglia, the resident macrophage in the nervous system. We investigated whether DXM could reduce macrophage production of cytokines and superoxide and the resultant influence on atherosclerosis formation in mice. METHODS AND RESULTS: We used in vitro and in vivo studies to evaluate the DXM inhibitory effect on oxidative stress. Dextromethorphan pretreatment significantly suppressed the production of tumour necrosis factor-alpha, monocyte chemoattractant protein-1, interleukin-6, interleukin-10, and superoxide in macrophage cell culture after stimulation. Indeed, DXM reduced macrophage nicotinamide adenine dinucleotide phosphate oxidase activity by decreasing membrane translocation of p47(phox) and p67(phox) through the inhibition of protein kinase C and extracellular signal-regulated kinase activation. The anti-atherosclerosis effect of DXM was tested using two animal models, apolipoprotein E (apoE)-deficient mice and a mouse carotid ligation model. Dextromethorphan treatment (10-40 mg/kg/day) for 10 weeks in apoE-deficient mice significantly reduced superoxide production in their polymorphonuclear leukocytes and aortas. It significantly decreased the severity of aortic atherosclerosis in the apoE-deficient mice and decreased carotid neointima formation after ligation in C57BL/6 mice. CONCLUSION: Our data show that DXM, with its novel effect in reducing oxidative stress, significantly reduces atherosclerosis and neointima formation in mice.

Retinoic acid prevents Chlamydia pneumoniae-induced foam cell development in a mouse model of atherosclerosis.

Chlamydia pneumoniae, a common respiratory pathogen, has been associated with cardiovascular disease. C. pneumoniae infection accelerates atherosclerotic lesion development in hyperlipidemic animals. Retinoic acid, an anti-oxidant, inhibits infection of endothelial cells by C. pneumoniae. The present study demonstrated that retinoic acid suppresses the acceleration of foam cell lesion development induced by C. pneumoniae in hyperlipidemic C57BL/6J mice. Retinoic acid treatment had no effect on foam cell lesion development in uninfected animals. Lung infection and duration was decreased in treated mice, suggesting one mechanism by which retinoic acid reduces C. pneumoniae-accelerated foam cell lesion formation in hyperlipidemic mice.

Identification and characterization of Chlamydia pneumoniae-specific proteins that activate tumor necrosis factor alpha production in RAW 264.7 murine macrophages.

Chlamydia pneumoniae is a common respiratory pathogen, which activates macrophages to induce inflammatory cytokines that may promote atherosclerosis. However, the antigens that induce macrophage activation have not been well defined. In the current study, three chlamydial proteins which are recognized during human infection, outer membrane protein 2 (OMP2) and two 53-kDa proteins (Cpn 0980 and Cpn 0809), were investigated to determine whether they activate macrophages and, if they do, what mechanism they use for this activation. It was shown that these three proteins could (i) induce expression of tumor necrosis factor alpha (TNF-alpha) and tissue factor and (ii) induce phosphorylation of p44/42 mitogen-activated protein kinases (MAPK) and activation of early growth response factor 1 (Egr-1). Control proteins, the N-terminal fragment of polymorphic membrane protein 8 and the thioredoxin portion of the fusion protein, had no effect on macrophages. Treatment of cells with a MEK1/2 inhibitor, U0126, dramatically reduced the phosphorylation of ERK, activation of Egr-1, and expression of TNF-alpha in macrophages treated with recombinant proteins. Toll-like receptors (TLRs) act as sensors for microbial antigens and can signal via the MAPK pathway. Chlamydial protein-induced expression of TNF-alpha was significantly reduced in macrophages lacking TLR2 or TLR4. These findings suggest that C. pneumoniae may activate macrophages through OMP2, Cpn 0980, and Cpn 0809 in addition to cHSP60 and that activation occurs via TLR2 or TLR4, Egr-1, and MAPK pathways.

Inoculation of Chlamydia pneumoniae or Chlamydia trachomatis with ligands that inhibit attachment to host cells reduces infectivity in the mouse model of lung infection: implication for anti-adhesive therapy.

Previous studies have shown that the chlamydial glycan contains a high-mannose oligosaccharide, which mediates attachment and infectivity of the organism. Removal of the glycan decreases infectivity in vitro and in vivo. The present study demonstrates that simultaneous inoculation of chlamydial organisms and a ligand that prevents glycan binding reduces lung burden in infected animals.

Inhibition of bovine herpesvirus-4 replication in endothelial cells by arsenite.

The effect of arsenite pretreatment on bovine herpesvirus-4 (BHV-4) replication in bovine arterial endothelial (BAE) cells was studied. BHV-4 infectivity, including IE-2 expression, DNA replication and viral yield, were significantly reduced at nontoxic concentrations of arsenite in which cellular DNA synthesis or cell viability of BAE cells were not affected under resting and confluent conditions. This effect was accompanied by the induction of heat shock protein 70 (HSP70) and an interrupted cell cycle (causing cell cultures to accumulate at the S and G2/M phases). Actinomycin D inhibited the induction of HSP70 and reduced arsenite antiviral activity. In conclusion, cellular stress response induced by arsenite in BAE cells inhibited replication of BHV-4, and probably resulted from the induction of HSP70 and interference of cell cycle progression.