Introduction
Human cytomegalovirus (HCMV), a member of the herpes virus family, is the leading viral cause of congenital abnormalities and mental impairment in newborns, and contributes significantly to morbidity and mortality rates in immunocom-promised persons. HCMV can be transmitted via multiple pathways, including saliva, sexual contact, placental transfer, breast-feeding and blood transfusion (1). Over one-third of transplant recipients are infected with HCMV, and the mortality rate of these patients is as high as 85%, and 40% of patients with acquired immunodeficiency syndrome, who have a secondary infection of HCMV exhibit retinopathy, gastrointestinal damage and central nervous system disorders (2,3). In addition, latent HCMV infection is closely associated with cardiovascular disease, stroke, diabetes and other chronic diseases (4,5). However, the cellular and molecular mechanisms of the persistent infection, latency and reactivation of HCMV remain to be fully elucidated (2). Following the widespread introduction of anti-HCMV drugs, including ganciclovir, valganciclovir and foscarnet, there has been a significant reduction in the number of HCMV infections, however, these drugs have been demonstrated to cause potentially serious side effects, exhibit low bioavailability and the inability to eliminate carrier states, and issues with drug resistance, patient compliance and cost, further limiting their effectiveness and wide application (3). Therefore, the development of safe and effective anti-HCMV drugs with novel mechanisms of action is urgently required.
Curcumin is a traditional Chinese medicine, which is extracted from the rhizome of the herb Curcuma longa (6). Curcumin has a low toxicity and has been widely used for the prevention of viral infection and other diseases, including cancer (7–9). Our previous study demonstrated that curcumin has significant anti-HCMV activity and improves the survival rate of host cells by inhibiting the HCMV DNA replication, apoptosis and inflammation induced by HCMV infection (10). However, the mechanism of anti-HCMV activity of curcumin remains to be fully elucidated.
In the development of novel drugs, bioinformatics is capable of identifying the key molecular targets in the pathological process and confirming the association between the structure and function of the drug. Bioinformatics has now become an important biopharmaceutical tool, which reduces the requirement for manpower and resources. The widespread application of bioinformatics has significantly accelerated the understanding and the development of novel drugs for several types of disease (11). Our previous studies have successfully applied bioinformatics to screen drugs for the treatment of leukemia (12) and aplastic anemia (13). In the present study, the anti-HCMV mechanism of curcumin was determined using bioinformatics, which was further confirmed by subsequent experiments.
Materials and methods
Reagents and kits
Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), SYBR Green, penicillin, streptomycin, L-glutamine, trypsin, dimethyl sulfoxide (DMSO), Triton X-100, phenylmethanesulfonyl fluoride (PMSF) and β-mercaptoethanol were obtained from Sigma-Aldrich (St. Louis, MO, USA). Rabbit anti-heat shock protein 90 (Hsp90) antibody was purchased from Cell Signaling Technology (Boston, MA, USA). Goat anti-rabbit secondary antibody was obtained from Invitrogen Life Technologies (Carlsbad, CA, USA).
Cell culture and virus propagation
Human embryonic lung fibroblast (HELF) cells were purchased from the National Platform of Experimental Cell Resources for Sci-Tech (Beijing, China) and were cultured in DMEM, supplemented with 10% FBS, 100 U/m penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine at 37°C in a humidified atmosphere of 95% air and 5% CO2. The HCMV laboratory strain, AD169, was purchased from American Type Culture Collection (Manassas, VA, USA) and prepared in the HELF cells maintained in DMEM, containing 2% FBS (maintenance medium).
Antiviral compounds
Curcumin was obtained from the National Institute for Food and Drug Control (Beijing, China) and stored as a 50 mM stock solution in DMSO for in vitro use. The high, middle and low doses of curcumin used in the present study were 2, 1 and 0.5 µM, respectively. Intravenous formulations of 0.2 mM ganciclovir (GCV; Sigma-Aldrich) and 0.5 µM geldanamycin (Sigma-Aldrich) in DMSO were used as references.
Drug genome similarity analysis of transcription
The mechanism of action of curcumin was predicted from pharmacogenomics databases, using bioinformatics to perform genome-wide transcriptional analysis of similarity. Using curcumin as a keyword, associated gene expression information was identified in various gene expression databases, including Gene Expression Omnious, BodyMap (Human and Mouse gene expression database), ArrayExpress Database (Public repository for microarray-based gene expression data), RNA Abundance Database, Stanford Microarray Database, Human Gene Expression and ChipDB, a searchable database of gene expression. These databases are open access database resources and can be accessed online. According to the data in the GenBank and internet platforms, the cloned drug genome sequences of curcumin were transferred to the above online databases to determine the expression information.
Molecular docking simulation
ChemBio Office (2010) was used to draw the molecular structure of curcumin, and the resultant files were saved in the standard delay format. MOE (2010) was applied to analyze the three-dimensional minimum energy conformations. The docking parameters in MOE were set as follows: The placement method, the first scoring function rescoring 1 and the saved poses were Triangle Matcher, London dG and 30, respectively. In addition, the refinement, the second refinement scoring function rescoring 2 and the saved poses were set to force field, none and 10, respectively (7).
Western blotting
The HELF cells were seeded at a density of 1×106/ml in tissue culture flasks and cultured for 12 h. The cells were subsequently infected with HCMV at 100 times the median tissue culture infective dose/0.1 ml. Following incubation at 37°C for 2 h under a humidified atmosphere of 95% air and 5% CO2, the supernatant was removed and replaced with maintenance medium, with or without curcumin. Following an additional 48 h at 37°C, the cells were harvested and washed twice with cold phosphate-buffered saline and lysed in cold radioimmunoprecipitation buffer, containing 25 mM Tris-HCl (pH 7.6), 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 1 mM PMSF and 0.1% sodium dodecyl sulfate (SDS), on ice for 30 min, followed by centrifugation at 9,600 × g at 4°C for 10 min. Protein quantification was performed using a bicinchoninic acid protein assay (Pierce, Rockford, IL, USA). Equal quantities of protein (50 µg) from the different groups were denatured in SDS sample buffer and separated by 8% SDS-polyacrylamide gel electrophoresis. The proteins were transferred onto polyvinylidene difluoride membranes in buffer, containing 25 mM Tris, 192 mM glycine and 20% methanol. The membranes were blocked with Tris-buffered saline, containing TBS; 137 mM NaCl and 20 mM Tris-HCl (pH 7.5), containing 0.1% Tween 20 and 5% dried non-fat milk powder at room temperature for 2 h. Following blocking, the blots were incubated with primary antibodies against rabbit anti-human Hsp 90 (mAb; Cell Signaling Technologies, Danvers, MA, USA; cat. no. 4877) and mouse anti-human β-actin (mAb, ZSJB-Bio, Beijing, China; cat. no. 131023) at 1:1,000 in TBS containing 0.05% Tween-20 (TBST) at 4°C overnight. The membranes were subsequently washed with TBST three times. The blots were then incubated with Alexa Fluor®680 goat anti-mouse IgG and goat anti-rabbit IgG at 1:10,000 (Invitrogen Life Technologies, Grand Island, NY, USA) in TBST. After washing the membranes with TBS, the signals were detected using Odyssey infrared laser imaging system (LI-COR Biosciences, Inc,. Lincoln, NE, USA), according to the manufacturer's instructions. For densitometric analysis of western blotting images, density profiles of the bands were measured using ImageJ software (National Institute of Health, Bethesda, MA, USA).
Statistical analysis
The data were analyzed using Microsoft Office Excel 2003 (Microsoft, Redmond, WA, USA) and expressed as the mean ± standard error of the mean. Group comparisons were evaluated using one-way analysis of variance, and significant differences between treatments were determined using the post-hoc test with Tukey-Kramer's honestly significant difference simultaneous pairwise mean comparison. P<0.05 was considered to indicate a statistically significant difference.
Results
Curcumin genome similarity analysis of transcription
To investigate the pharmacogenomics of curcumin, a comparative analysis of 3,000 clinical drugs was performed, which revealed that the gene expression profiles of curcumin had a positive association with several drugs (Table I). Among these drugs, the pharmacogenomics of curcumin were most similar to that of the antiviral drug, geldanamycin. Geldanamycin exerts an antiviral effect, predominantly by specifically binding to Hsp90 (14). This analysis suggested that curcumin may inhibit HCMV by targeting Hsp90.
Molecular docking simulation of curcumin with Hsp90
To confirm the above hypothesis, a molecular docking simulation of curcumin with Hsp90 was performed, which revealed that curcumin fit well into the binding pocket of Hsp90, among which hydrogen bonds, hydrophobic interactions and conjugation maintained the predominant driving force of adhesion (Fig. 1). As shown in Fig. 2, a carbonyl oxygen atom of curcumin acted as a hydrogen bond acceptor to form a hydrogen bond with Tyr139 of Hsp90. In addition, Trp162 and Phe22 of Hsp90 were conjugated with curcumin (Fig. 2). Furthermore, Ile26, Leu107, Ile104, Phe170, Leu103, Ala111 and other hydrophobic amino acids of Hsp90 formed hydrophobic interactions with curcumin (Fig. 2).
Curcumin reduces the protein expression of Hsp90α
The present study investigated the effect of curcumin on the protein expression of Hsp90α using western blotting. In contrast to the mock-treated cells, infection with HCMV in the curcumin-treated cells led to a significant increase in the protein expression of Hsp90α (Fig. 3; P<0.05). As expected, treatment with geldanamycin, but not GCV, markedly eliminated the induction of the protein expression of Hsp90α by HCMV. Notably, curcumin also inhibited the upregulated protein expression of Hsp90α by HCMV in a dose-dependent manner (Fig. 3).
Discussion
In the present study, it was demonstrated that the pharmacogenomics of curcumin were most similar to that of geldanamycin, which is an inhibitor of Hsp90 and exerts a wide-spectrum antiviral role (15). The in vitro experiments revealed that curcumin significantly inhibited the protein expression of Hsp90α. This data suggested that the inhibition of Hsp90 may be one of the mechanisms by which curcumin inhibits HCMV and improves the survival rate of the host cells.
Hsp90 is a highly conserved and essential molecular chaperone at the center of a large protein-folding network (16). It is one of the most abundant chaperone proteins in eukaryotic and prokaryotic cells, and is formed by Hsp90α and Hsp90β monomer polymerization in higher eukaryotic organisms (17–19). Dysregulation of Hsp90α is closely associated with the occurrence and development of different types of cancer (17). Therefore, targeting Hsp90 is a promising strategy for the development anticancer drugs.
Hsp90 is a leading member of the heat shock protein family and can form complexes with a variety of proteins. Hsp90 is involved in improving cellular stress tolerance (18), regulating protein conformations (19) and in client protein maturation following transduction (20). Accumulating evidence has demonstrated that Hsp90 and its client proteins cannot only alter the micro-environment of a cell through involvement in cell signaling transduction, cell cycle regulation and cell apoptosis (21), but they also promote viral infections of herpes simplex virus 1, Epstein-Barr virus or intestinal virus by being involved in viral penetration (22), DNA polymerase positioning (14), expression of key proteins (23) and DNA injury repair (24). In agreement with these observations, it was previously revealed that curcumin inhibits the proliferation of HCMV and increases the survival rate of host cells by inhibiting HCMV DNA replication, apoptosis and inflammation (10). The present study used molecular docking simulation analysis to demonstrate that curcumin fit well into the binding pocket of Hsp90. Notably, curcumin eliminated the induction of Hsp90α by HCMV infection in the HELF cells, indicating that Hsp90 is involved in the process of viral infection (25–27), and targeting Hsp90 offers a novel promising strategy for the prevention and treatment of infection with viruses, including HCMV.
In conclusion, the present study indicated that the anti-HCMV activity of curcumin is possibly due to its inhibitory effects on Hsp90α. These results suggested that Hsp90 may be a novel target for the treatment of infectious diseases caused by HCMV.