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Article

Proteomic Analysis of Secretomes of Oncolytic Herpes Simplex Virus-Infected Squamous Cell Carcinoma Cells

Department of Oral and Maxillofacial Surgery, Osaka University Graduate School of Dentistry, Suita, Osaka 565-0871, Japan
*
Author to whom correspondence should be addressed.
Cancers 2018, 10(2), 28; https://doi.org/10.3390/cancers10020028
Submission received: 1 December 2017 / Revised: 3 January 2018 / Accepted: 15 January 2018 / Published: 23 January 2018
(This article belongs to the Special Issue Oncolytic Virotherapy)

Abstract

:
Oncolytic herpes simplex virus type 1 (HSV-1) strain RH2 induced immunogenic cell death (ICD) with the release and surface exposure of damage-associated molecular patterns (DAMPs) in squamous cell carcinoma (SCC) SCCVII cells. The supernatants of RH2-infected SCCVII cells also exhibited antitumor ability by intratumoral administration in SCCVII tumor-bearing mice. The supernatants of RH2-infected cells and mock-infected cells were concentrated to produce Med24 and MedC for proteomic analyses. In Med24, the up- and down-regulated proteins were observed. Proteins including filamin, tubulin, t-complex protein 1 (TCP-1), and heat shock proteins (HSPs), were up-regulated, while extracellular matrix (ECM) proteins were markedly down-regulated. Viral proteins were detected in Med 24. These results indicate that HSV-1 RH2 infection increases the release of danger signal proteins and viral gene products, but decreases the release of ECM components. These changes may alter the tumor microenvironment (TME) and contribute to enhancement of anti-tumor immunity against SCC.

1. Introduction

Oncolytic virotherapy is a novel therapeutic modality that directly induces the lysis of infected tumor cells, and subsequently enhances host immune responses [1,2,3,4]. Recent clinical studies demonstrated that oncolytic viral therapy increases cytokines, activates immune responses, and effectively destroys primary and metastatic lesions, even if the virus does not reach distant lesions. Therefore, oncolytic viruses are also considered to be immunotherapeutic agents [5]. Herpes simplex virus type 1 (HSV-1) is one of the most widely studied viruses for the treatment of patients with solid tumors. Oncolytic HSV-1 lacks the neurovirulence gene γ34.5, which is responsible for encephalitis, as a requisite gene modification for safety [6,7].
In microbial infection, pathogen-associated molecular patterns (PAMPs) that exist in diverse organisms, but not in the host, provide exogenous signal regarding the presence of pathogens in the immune system, thereby promoting immunity [8,9]. In contrast, cells release damage-associated molecular patterns (DAMPs) as endogenous signals that alert the immune system to respond to unexpected cell death, microbial invasion, and stress [3,10]. DAMPs may be proteins, nucleic acids, or metabolic products. Protein DAMPs include intracellular proteins such as high mobility group box 1 (HMGB1), heat shock protein (HSP), hyaluronic acid, calreticulin (CRT), and S100 protein [3,5]. When solid tumors are infected with oncolytic viruses, the virus mostly induces immunogenic cell death (ICD), resulting in the cell surface exposure of CRT and HSP and release of ATP, HMGB1, uric acid, other DAMPs, PAMPs, and tumor-associated antigens (TAAs) [3,11,12,13].
Previously, we reported that an injection of oncolytic HSV-1 strain RH2 into squamous cell carcinoma cell (SCC) SCCVII tumors in inbred mice enhanced systemic anti-tumor immunity [14], and that cell death caused by RH2 was immunogenic cell death (ICD) with the release of DAMPs such as ATP and HMGB1 [13]. We also indicated that intratumoral injection of the supernatants of RH2-infected cells suppressed tumor growth, even if infectivity was lost by ultraviolet irradiation, suggesting their ability to enhance antitumor immunity [13].
Secreted proteins comprise an important group of molecules encoded by about 10% of the human genome, and are able to reflect a broad variety of different conditions of the cell [15]. A proteomic analysis of human macrophages infected with HSV-1 demonstrated the release of proteins functionally related to metabolic processes, transport, stress responses, cell death, proteolysis, the extracellular matrix (ECM), and cell adhesion [16]. However, the characteristics of the secreted proteins, secretomes, of oncolytic HSV-1-infected tumor cells have not yet been studied. In the present study, we performed proteomic analysis of ecretomes of RH2-infected and mock-infected SCC cells, and compared the protein profiles.

2. Results

2.1. Classification of Released Proteins of Med24 and MedC

The supernatants of RH2-infected SCCVII cells and mock-infected cells were harvested and concentrated to produce samples named Med24 and MedC. Med24 and MedC were sequentially analyzed, and isobaric tag for relative and absolute quantitation (iTRAQ) quantitation was performed for each protein. A total of 1567 proteins were detected in Med24 and 1344 in MedC, with 223 extracellular proteins being increased by RH2 infection. Based on the results of iTRAQ quantitation, MedC proteins spontaneously secreted from SCCVII cells were classified into three categories, i.e., high, medium, and low abundance groups. The numbers of proteins in the high, medium, and low abundance groups were 190, 262, and 892, respectively.
In order to examine the effects of RH2 infection on protein secretion, iTRAQ quantitation data for each protein was compared between Med24 and MedC. Protein release was found to be up- or down-regulated by RH2 infection. In the high abundance group, 29 proteins were up-regulated by more than 1.4 fold by RH2 infection (Table 1). However, a significant increase was observed in 16 proteins. They included filamin alpha, tubulin beta-4B, clathrin heavy chain, t-complex protein 1 (TCP-1) subunit beta, theta, alpha, gamma and delta, bifunctional purine biosynthesis protein (PURH), filamin-C, bifunctional glutamate/proline–tRNA ligase, plastin-3, fascin, coatomer subunit alpha, beta enolase, and tubulin beta-6 chain.
In the high abundance group of MedC, 50 proteins were down-regulated by more than 50% by RH2 infection (Table 2). They included ECM components such as fibronectin, thrombospodin-1, basement membrane-specific heparin sulfate proteoglycan core protein, fibrillin-1, chondroitin sulfate proteoglycan 4, SPARC, procollagen C, collagen alpha, and extracellular matrix protein 1. Other proteins, cathepsin B, cathepsin D, matrixmetalloproteinase-9, and 72 kDa type IV collagenase, were involved in the remodeling of ECM. Among all proteins listed, a significant difference was observed between the Med24 and MedC groups.

2.2. Alteration of DAMPs and DAMPs-Related Proteins in the Secretomes

In DAMPs, the numbers of high, medium, and low abundance proteins were 11, 6, and 3, respectively (Table 3). In the high abundance group, HSPs, actin cytoplasmic 1, annexin A1, annexin A2, galectin-1, peroxiredoxin-2, and histone H2B type 1 were observed. Only the levels of HSP90-beta, actin cytoplasmic 1, and galectin-3 were significantly higher in Med24 than in MedC. ATP-citrate, which is responsible for the production of ATP, a known DAMP, was also detected as a significantly elevated protein (Table 3).

2.3. Viral Proteins in the Secretomes

Twenty-nine viral proteins that were detected in Med24 were not present in MedC (Table 4). When viral proteins were categorized into three groups, the numbers of proteins in the high, medium, and low abundance groups were 8, 9, and 12, respectively. Major capsid protein unique sequence of the L component (UL) 19, tegument protein UL37, capsid protein virion polypeptide (VP) 23, glycoprotein D, and glycoprotein E were identified as viral structural proteins. In addition, ribonucleoside-diphosphate reductase, single-strand DNA-binding protein, DNA polymerase processivity subunit, transcriptional regulator infected cell protein (ICP) 4, exonuclease (UL12), multifunctional expression regulator (ICP27), and deoxyuridine triphosphatase were identified as high abundance proteins in Med24 (Table 4).

3. Discussion

Proteomic analysis has been applied to HSV-1 virion-incorporated host proteins, cellular proteins interacting with viral proteins such as ICP27, ICP8, and VP16, and protein profiles in HSV-1-infected cells [17,18,19]. Miettinen et al. [16] characterized the secretome of HSV-1-infected human primary macrophage using high-throughput quantitative proteomics, and identified 516 distinct proteins from macrophages secretome upon HSV-1 infection, and the secretion of 411 proteins was >2-fold increased upon interferon β (IFN-β) priming and/or HSV-1 infection. Based on our previous findings that the supernatants of RH2-infected cells suppressed the tumor growth in mice [13], we hypothesized that the supernatants contained immunostimulatory proteins, and performed proteomic analysis of the secretomes. Since the amounts of proteins detectable in the secretomes were important, we classified MedC proteins into three categories based on the results of iTRAQ quantitation, and then determined whether they were up-regulated or down-regulated by RH2 infection. When the high abundance proteins that increased by more than 1.4 fold following RH2 infection were selected, 29 proteins fulfilled this criterion. However, a significant increase was only observed in 16 proteins, including filamin alpha, tubulin beta-4B, clathrin heavy chain, and TCP-1s (Table 1).
Actin-binding proteins, filamin and pastin-3, and the actin-bundling protein, fascin, were identified as up-regulated high abundance proteins. An increase in tubulin, which is involved in the polymerization of microtubules, was demonstrated. Autoantibodies against filamin C were elevated in the patients with low-grade glioma and those against tubulin increased in the patients with nasopharyngeal carcinoma and neuroblastoma [20,21,22]. Clathrin heavy chain is one of the most important proteins known to be involved in the secretion and transport of vesicles [23]. Seliger et al. [24] examined potential candidate biomarkers and novel targets for T-cell-based immunotherapies of renal carcinoma, and identified human leukocyte antigen class I ligands, including clathrin heavy chain. Filamin, tubulin, and clathrin may be potential immunogenic antigens in specific cancers.
We also identified TCP-1 family members as the major up-regulated proteins in Med24 secretomes. Chaperonin-containing TCP-1 (CCT), known as the TCP-1 ring complex (TRiC), forms an oligomer that consists of distinct subunits (alpha, beta, gamma, delta, epsilon, zeta, eta, and theta) occupying a fixed position within the two back-to-back chaperonin rings [25]. CCT was originally identified as a molecular chaperone that is required for the folding of the highly abundant cytoskeletal proteins, actin and tubulin [26]. The genes for CCT alpha and CCT beta were amplified in breast cancer and necessary for cancer growth and proliferation [27]. CCT beta and CCT epsilon were overexpressed in both hepatocellular and colorectal cancers [28,29]. Gao et al. [30] reported that CCT5 (CCT epsilon) induced an autoantibody response in non-small cell lung cancer sera, and showed higher expression in cancer tissues. In the case of melanoma immunotherapy, mutated TCP 1-zeta-6A (CCT6A) antigen had previously been identified and proved to be immunogenic in a melanoma patient, so mutated CCT6A peptide was used as a model for patient-specific neoantigen [31]. Therefore, TCP-1 subunits identified in the present study may act as TAAs required for capable of activating tumor-specific immunity.
In secretomes of HSV-1-infected macrophages, endogenous danger signal proteins were demonstrated as up-regulated proteins. They included HSPs, annexins, S100-A proteins, galectins, and thioredoxin superfamily members [16]. The HSP family comprises proteins that act as molecular chaperones and catalyze the folding of proteins. HSPs bind to potential antigens under necrotic conditions and deliver them to various antigen-presenting cells [32]. We found 20 proteins that are known as DAMPs (Table 3). High abundance DAMPs including heat shock cognate 71 kDa protein, HSP90-alpha, HSP 70 kDa protein 4, HSP60, annexin A1, and peroxiredoxin-2 increased by RH2 infection, but the fold change observed was less than 1.4 fold. The levels of HSP90-beta, actin cytoplasmic 1, and galectin-3 were significantly higher in Med24 than in MedC. ATP is a known DAMP released extensively during ICD [4]. We also confirmed that ATP-citrate synthase and ATP synthase were up-regulated proteins as DAMPs-related proteins. The spatiotemporally coordinated emission of specific DAMPs promotes the recruitment of antigen presenting cells to sites of ongoing ICD, their ability to take up dead cell-derived particulate materials, as well as their capacity to prime an adaptive immune response [33]. Up-regulated DAMPs and DAMPs-related proteins observed in the present study were considered to be involved in the augmentation of antitumor immunity, operating as adjuvants.
Exosomes are small membrane-derived vesicles secreted by many types of normal and tumor cells [34]. Xiang et al. [35] reported the presence of DAMPs, such as HSPs, annexins, and histones, in exosomes, indicating the role of microvesicles as a form of excretion. DAMPs observed in the secretomes of MedC may be partly contained in exosomes. However, since oncolytic viruses including RH2 have been shown to induce apoptosis, necroptosis, autophagy, and pyroptosis [3,5,36,37,38], increases in the secretion of DAMPs may be mostly due to the disintegration of the cell membrane by virus-mediated cell death.
Another result of the present study is the marked decrease in the release of proteins constituting the tumor microenvironment (TME). In the present study, 50 proteins were selected as down-regulated proteins that were reduced to less than 0.5 (Table 2). They included fibronectin, thrombospondin-1, basement membrane-specific heparan sulfate proteoglycan core protein, fibrillin-1, 72 kDa type IV collagenase, procollagen C, and extracellular matrix protein 1. Since tumor and tumor stroma create the TME, which efficiently promotes tumor progression and supports evasion from antitumor immunity [39,40], oncolytic HSV-1 may exert its antitumor effects by decreasing the release of ECM components.
Using the secretomes of HSV-1-infected macrophages, 5 envelope proteins, 3 tegument proteins, alkaline nuclease, and deoxyuridine 5′-triphosphate nucleotidohydrolase were identified [16]. In the present study, viral structural components, i.e., the tegument, nucleocapsid, and envelope, were also detected in the supernatants of HSV-1-infected SCCVII cells. Furthermore, viral enzymes responsible for viral replication such as ribonucleoside-diphosphate reductase, single-strand DNA-binding protein, DNA polymerase processivity subunit, deoxyuridine triphosphatase, and thymidine kinase were identified. This result indicated that these cytoplasmic and nuclear viral proteins were passively released from dying cells. These proteins have been suggested to act as PAMPs, which promote inflammatory reactions and induce the infiltration of innate and adaptive immune cells.
Immunological abnormalities associated with TME inhibit the priming of antitumor adaptive immunity or tolerize tumor-specific CD4+ and CD8+ T cells [40,41] (Figure 1A). Oncolytic virotherapy is supposed to release DAMPs, PAMPs, and TAA triggering proinflammatory cytokine release, DC maturation, and cytotoxic CD8+ T cell proliferation, and overcome TME-associated immunosuppression by reducing secretion of stromal components [5,40,42,43] (Figure 1B). Using the supernatants of RH2-infected SCC cells, we herein demonstrated that DAMPs increased, and ECM proteins decreased. Viral gene products were also included. This may promote the immunological effects of the oncolytic HSV-1 on SCC cells. As a next step, it is important to confirm the role of each secreted protein in the antitumor effects of oncolytic HSV-1 by specific deletion.

4. Materials and Methods

4.1. Cells and Virus

SCCVII cells derived from the cutaneous SCC of C3H mice were cultured with Eagle’s minimal essential medium supplemented with 10% calf serum at 37 °C in a humidified atmosphere with 5% CO2 [13,14]. HSV-1 RH2 lacking the γ34.5 gene and with multiple mutations including glycoprotein B were grown in Vero cell monolayers and infectivity was assessed using a plaque assay on a Vero cell monolayer [14,44].

4.2. Preparation of Concentrated Supernatants of RH2-Infected Cells

SCCVII cells were infected with RH2 at a multiplicity of infection of 10. After being incubated for 60 min for virus adsorption, cell monolayers were washed twice with phosphate-buffered saline, covered with serum-free medium, and cultured for 24 h. Culture supernatants were then harvested, and centrifuged at 1500 rpm for 10 min to remove cell debris. The supernatants were concentrated 30 times using Amicon®Ultra-15 3 K Centrifugal Filter Devices (Merck, Darmstadt, Germany) [13]. Concentrates were exposed to ultraviolet irradiation at an intensity of 0.15 mW/cm2 for 30 min in order to inactivate the infectivity of the virus, filtered through a 0.20-µm filter, and named Med24. Mock-infected cells were also incubated in serum-free medium, supernatants were harvested 24 h later, treated as described for RH2-infected cells, and named MedC.

4.3. Proteomic Analysis

Three samples were used each in the proteomic analysis of Med24 and MedC [45]. Protein concentrations in Med24 and MedC were assessed, and 4 volumes of cold acetone per 100 µg were added to each sample. They were kept at −20 °C overnight, and centrifuged at 15,000 rpm for 10 min to collect precipitates. Proteins were dried, and denatured by resuspending in 20 µL of lysis buffer (50 mM Tris-HCl, pH 9.0, 6 M uric acid, and 5% sodium deoxycholate), and reduced with 2 µL of reduction buffer (10 mM dithiothreitol) at 37 °C for 1 h. In order to block cysteine, 1 µL of cysteine-blocking buffer (55 mM iodoacetamide) was added, and proteins were alkylated in the dark at 25 °C for 30 min. After a 10-fold dilution of samples with 50 mM Tris-HCl (pH 9.0), proteins were disintegrated with trypsin at 37 °C for 16 h. An equal volume of ethyl acetate was added and acidified to a final concentration of 0.5% trifluoroacetic acid. The aqueous solution after centrifugation at 14,000 rpm for 2 min was harvested. Samples were desalted on C18-Stage Tips and labeled with the iTRAQ tag dissolved in 70 µL ethanol at room temperature for 1 h.
In the LC-MS/MS analysis, the Ultimate 3000 Nano LC system (Thermo Fisher Scientific, Waltham, MA, USA) was used for ultra performance liquid chromatography (UPLC) and connected to the Q-Exactive hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific) with a nano-electrospray ionization source. Labeled samples were injected into UPLC and concentrated in a C18 reverse phase trap column (100 µm I.D. × 5 mm length, Thermo Fisher Scientific) at a flow rate of 4 µL/min. Samples were then separated using a C18 reversed phase column (75 μm I.D. × 150 mm long, Nikkiso Tecnos, Tokyo) at a flow rate of 300 nL/min. A fixed mobile phase condition to Solvent A was water containing 0.1% formic acid and to Solvent B was acetonitrile containing 0.1% formic acid. Peptides were ionized by the positive mode of nano-electrospray ionization, which was a 1.8-kV capillary voltage.
Protein data were analyzed using Mascot Distiller v2.4 (Matrix Science, London, UK) http://www.matrixscience.com/mascot_support_v2_4.html, and a list of peaks was generated based on the recorded fragmentation spectra. In the identification of this protein, the UniProt amino acid sequence database (released on 4 March 2015: http://www.uniprot.org/) with biological species limited to the mouse (NCBI Taxonomy ID: 10090) and herpesviridae (ID: 10292) was used. The identified proteins were checked using the UniProt-GOA database (v140, http://www.ebi.ac.uk/GOA), and the annotation of Gene Ontology was added. Qualitative data and quantitative values were assessed using Mascot v2.4. In protein identification, the criteria of false rates of detection were less than 1%. The mass error was 10 ppm in Precursor Mass, and 0.01 Da in Fragment Mass. Due to the semi-quantitative nature of this approach, proteins were classified into three categories based on the values of iTRAQ quantitation (<5, low abundance; 5 to 10, medium abundance; and >10, high abundance) [46].

4.4. Statistical Analysis

Statistical analyses were performed using the Student’s t-test with Microsoft Excel (Microsoft, Redmond, WA, USA). Results were expressed as the mean ± SD. Differences were considered to be significant at p < 0.05.

5. Conclusions

Our results indicate that a HSV-1 RH2 infection increases the release of danger signal proteins and viral gene products, but decreases the release of ECM components. These changes may alter the tumor microenvironment and promote anti-tumor immunity against SCC.

Acknowledgments

We would like to thank Kazuaki Takafujie of the Center for Research Education, Osaka University Graduate School of Medicine for his assistance with proteomic analysis.

Author Contributions

Shinya Tada and Masakazu Hamada performed the experiments. Shinya Tada and Yoshiaki Yura designed the study and wrote the manuscript.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. A model of the augmentation of antitumor immunity by an oncolytic virus. (A) Immunological abnormalities associated with TME inhibit the priming of antitumor adaptive immunity or tolerize tumor-specific CD4+ and CD8+ T cells; (B) Oncolytic viruses induce ICD with the release of secretomes containing DAMPs, PAMPs, proinflammatory cytokines, and TAA. This triggers DC maturation and cytotoxic CD8+ T cell proliferation. The reduced secretion of stromal components due to viral infection also overcomes TME-associated immunosuppression. TME, tumor microenvironment; ECM, extracellular matrix; TAA, tumor-associated antigen; ICD, immunogenic cell death; DC, dendritic cell; PAMPs, pathogen-associated molecular patterns; DAMPs, damage-associated molecular patterns.
Figure 1. A model of the augmentation of antitumor immunity by an oncolytic virus. (A) Immunological abnormalities associated with TME inhibit the priming of antitumor adaptive immunity or tolerize tumor-specific CD4+ and CD8+ T cells; (B) Oncolytic viruses induce ICD with the release of secretomes containing DAMPs, PAMPs, proinflammatory cytokines, and TAA. This triggers DC maturation and cytotoxic CD8+ T cell proliferation. The reduced secretion of stromal components due to viral infection also overcomes TME-associated immunosuppression. TME, tumor microenvironment; ECM, extracellular matrix; TAA, tumor-associated antigen; ICD, immunogenic cell death; DC, dendritic cell; PAMPs, pathogen-associated molecular patterns; DAMPs, damage-associated molecular patterns.
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Table 1. Up-regulated proteins in the abundance group of secretomes a. a Proteins up-regulated by more than 1.4 fold by the strain RH2 infection are listed. b Mean of three assessments. * p < 0.05.
Table 1. Up-regulated proteins in the abundance group of secretomes a. a Proteins up-regulated by more than 1.4 fold by the strain RH2 infection are listed. b Mean of three assessments. * p < 0.05.
Accession No.Protein NameMW (kDa)iTRAQ Quantitation bFold Changep-Value
Med24MedC
Q9QXS1Plectin53479.7541.480.057
B7FAU9 Filamin, alpha 28073.749.71.480.004 *
P68372 Tubulin beta-4B 5054.7371.480.0027 *
Q9JKF1 Ras GTPase-activating-like protein IQGAP1 18947.733.71.420.09
Q02053 Ubiquitin-like modifier-activating enzyme 1 11840281.430.058
Q5SXR6 Clathrin heavy chain 1924026.31.510.023 *
P80314 T-complex protein 1 subunit beta573923.71.650.012 *
P63038 60-kDa heat shock protein, mitochondrial 6132.420.31.590.22
P42932 T- complex protein 1 subunit theta 6031.320.31.540.0013 *
Q9CWJ9 Bifunctional purine biosynthesis 6427.7171.630.021 *
protein PURH
P11983 T-complex protein 1 subunit alpha 6025.716.71.540.0088 *
P56480 ATP synthase subunit beta, mitochondrial 5624171.410.42
Q64737 Trifunctional purine biosynthetic protein 10822.7161.420.12
adenosine-3
Q9JHU4 Cytoplasmic dynein 1 heavy chain 1 53226.7122.220.16
D3YW87 Filamin-C 28933.320.71.610.033 *
P80318 T-complex protein 1 subunit gamma 6122.713.31.70.0078 *
P80315 T-complex protein 1 subunit delta 5819.313.31.450.016 *
Q7TPV4 Myb-binding protein 1A 15221.311.31.560.064
Q8CGC7 Bifunctional glutamate/proline-tRNA ligase17019.711.31.740.033 *
Q03265 ATP synthase subunit alpha, mitochondrial 6019.310.31.870.17
P23116 Eukaryotic translation initiation factor 31621711.31.50.078
subunit A
Q78PY7 Staphylococcal nuclease domain-containing10216.7111.510.12
protein1
B1AX58 Plastin-3 721611.31.410.00015 *
Q9D8N0 Elongation factor 1-gamma 5016.710.31.610.18
Q8BGQ7 Alanine--tRNA ligase, cytoplasmic 10715.710.31.510.21
Q61553 Fascin 551510.31.450.049 *
Q8CIE6 Coatomer subunit alpha 13814.7101.470.025 *
P21550 Beta-enolase 473825.31.50.002 *
Q922F4 Tubulin beta-6 chain 5027.717.71.570.011 *
Table 2. Down-regulate proteins in the abundance group of secretomes a. a Proteins down-regulated by less than 0.5 fold by the RH2 infection are listed. b Mean of three assessments.
Table 2. Down-regulate proteins in the abundance group of secretomes a. a Proteins down-regulated by less than 0.5 fold by the RH2 infection are listed. b Mean of three assessments.
Accession No.Protein Name MW (kDa)iTRAQ Quantitation b Fold Changep-Value
Med24MedC
A0A087WR50Fibronectin26333.799.00.340.00032
P35441Thrombospondin-113015.748.30.320.00045
E9PZ16Basement membrane-specific heparan4709.350.00.190.0011
sulfate proteoglycan core protein
P97298Pigment epithelium-derived factor 4616.742.70.390.0011
A2AQ53Fibrillin-131210.041.70.240.0000830
Q3UQ28Peroxidasin homolog1656.736.30.180.0019
P3343472-kDa type IV collagenase 7411.0 31.30.350.0074
E9PZ00 Prosaposin 619.0 28.30.320.0016
D3Z598 Latent-transforming growth factor beta-binding protein 41676.0 29.70.20.00022
Q07797 Galectin-3-binding protein 647.3 25.70.290.00022
P28653 Biglycan 424.7 27.70.170.0011
P10605 Cathepsin B 3710.0 21.30.470.023
Q00493 Carboxypeptidase E 5310.3 21.00.490.011
Q8VHY0 Chondroitin sulfate proteoglycan 42526.3 25.00.250.0015
Q06890 Clusterin 526.3 23.30.270.00044
Q9R118 Serine protease HTRA1 517.0 22.30.310.00078
P07214 SPARC 346.3 22.70.280.00029
Q61398 Procollagen C 504.7 24.30.190.0000940
Q9QZF2 Glypican-1 617.3 19.70.370.0078
Q640N1 Adipocyte enhancer-binding protein 11285.7 20.70.270.0004
Q8BND5 Sulfhydryl oxidase 1 835.3 21.00.250.0018
Q02819 Nucleobindin-1 537.7 17.30.440.016
Q9R0E2 Procollagen-lysine, 2-oxoglutarate845.7 17.70.320.0037
5-dioxygenase 1
Q61468 Mesothelin 696.3 16.70.380.00039
M0QWP1 Agrin 2172.7 18.30.150.00000480
Q61508 Extracellular matrix protein 1 633.0 17.70.170.0014
O08665 Semaphorin-3A 895.3 15.00.360.0054
Q9WVH9 Fibulin-5 502.3 18.00.130.00071
Q60963 Platelet-activating factor acetylhydrolase 492.3 17.70.130.00037
O88325 Alpha-N-acetylglucosaminidase 833.0 15.70.190.00046
P21460 Cystatin-C 165.3 13.30.340.0058
P55065 Phospholipid transfer protein 540.7 18.00.040.00048
P25785 Metalloproteinase inhibitor 2 245.7 12.70.450.012
Q8QZR4Out at first protein homolog324.7 13.70.340.0049
G3XA35 MCG116562, isoform CRA_a 2634.7 13.30.350.007
Q8CE08 Prostatic acid phosphatase 442.7 15.30.17 0.0000700
P41245 Matrixmetalloproteinase -9 813.0 14.70.20.0013
P12023 Amyloid beta A4 protein 873.3 13.30.250.0042
P18242 Cathepsin D 455.0 11.70.430.034
Q9WV54 Acid ceramidase 452.7 14.00.190.00071
P08121 Collagen alpha-I (III) chain 1392.0 13.30.150.011
G3UWC2 N-acetylated alpha-linked acidic dipeptidase 2, isoform CRA_a871.7 11.70.140.0013
Q62165 Dystroglycan 972.3 10.30.230.00043
Q62356 Follistatin-related protein 1 352.3 10.00.230.0019
P11087 Collagen alpha-1(I) chain 1382.0 10.30.190.011
Q9EPL2 Calsyntenin-1 1090.7 11.30.060.0015
O88207 Collagen alpha-1(V) chain 1840 12.000.00048
Q04857 Collagen alpha-1 (VI) chain 1080 12.000.0000310
O09159 Lysosomal alpha-mannosidase 1150 10.000.0000652
P47880 Insulin-like growth factor-biding protein 6 250 10.000.00098
Table 3. DAMPs and DAMPs-related proteins in secretomes. a Mean of three assessments. * p < 0.05.
Table 3. DAMPs and DAMPs-related proteins in secretomes. a Mean of three assessments. * p < 0.05.
Accession No.Protein Name MW (kDa)iTRAQ Quantitation aFold Changep-Value
Med24 MedC
DAMPs
P63017 Heat shock cognate 71-kDa protein 7168.063.71.070.41
P11499Heat shock protein HSP 90-beta 8366.756.71.180.046 *
P60710Actin, cytoplasmic 1 42 49.036.01.360.027 *
Q3U2G2Heat shock 70 kDa protein 4 94 36.030.71.170.071
P07901Heat shock protein HSP 90-alpha 85 57.749.71.160.052
P10107Annexin A1 39 30.029.01.030.75
P10853Histone H2B type 1-F/J/L 14 27.329.30.930.71
P6303860-kDa heat shock protein, mitochondrial61 32.320.31.590.22
P16045Galectin-1 15 22.319 .01.180.067
P07356 Annexin A2 3915.314.31.070.72
Q61171Peroxiredoxin -2 2215.013.71.100.33
O08709Peroxiredoxin -6 2511.79.71.210.18
Q9JMH6Thioredoxin reductase 1, cytoplasmic6710.78.31.280.28
Q61699Heat shock protein 105 kDa 9612.38.01.540.25
P16110 Galectin-3 2810.05.71.670.031 *
P99029 Peroxiredoxin-5, mitochondrial 229.06.31.420.091
P63158 High mobility group protein B1 255.74.71.220.66
P15864 Histone H1.2 213.07.00.430.0081 *
P10639Thioredoxin 125.73.71.550.10
P14211Calreticulin 483.73.31.120.90
DAMPs-related proteins
Q3V117 ATP-citrate synthase 12128.021.01.330.025 *
P56480ATP synthase subunit beta, mitochondrial 5624.017.01.410.42
Q03265 ATP synthase subunit alpha, mitochondrial 6019.310.31.870.17
Q9DB20 ATP synthase subunit O, mitochondrial 234.74.01.170.80
Table 4. HSV-1 proteins in secretomes. a Mean of three assessments. UL, unique sequence of the L component; US, unique sequence of the S component; ICP, infected cell protein; VP, virion polypeptide.
Table 4. HSV-1 proteins in secretomes. a Mean of three assessments. UL, unique sequence of the L component; US, unique sequence of the S component; ICP, infected cell protein; VP, virion polypeptide.
Accession No.Protein Name MW (kDa)GeneiTRAQ Quantitation a
Med24
F8RG70 Ribonucleoside-diphosphate reductase large subunit, ICP6124UL3933.7
D3YP93 Single-stranded DNA-binding protein, ICP8 128UL29 32.7
L0N5H2 DNA polymerase processivity subunit 51UL42 19.3
B9VQC5 Transcriptional regulator ICP4 133α4 7.7
D3YP82 Major capsid protein, VP5 149UL19 14.3
A0A0B5E4C5 UL12, Exonuclease 67UL12 13.0
D3YPB9 Multifunctional expression regulator, ICP27 55UL54 13.0
A0A0F7GRC1 Deoxyuridine triphosphatase 39UL50 10.0
F8RCG6 Tegument protein UL37 121UL37 10.0
A0A0B5E4D5 Thymidine kinase 41UL23 8.3
A0A0B5E4F4 VP13/VP14 74UL47 7.7
D0V7M2 Capsid protein VP23 34UL18 7.7
A2I996 Glycoprotein D 43US6 6.7
L0N6C7 Envelope glycoprotein E 59US8 5.3
P10224 Ribonucleoside-diphosphate reductase small subunit 38UL40 6.0
F8RCR4 Tegument protein VP11/12 78UL46 5.3
F8RDC2 DNA packaging tegument protein UL25 63UL25 5.0
L0N3F0 Tegument protein VP22 32UL49 4.3
D3YPB2 Transactivating tegument protein VP16 54UL48 3.7
A0A0F7CY40 Nuclear egress membrane protein 30UL34 4.7
A0A0F7GTB2 Capsid maturation protease 67UL26 1.7
A0A089YQM2 Glycoprotein B 100UL27 1.7
A0A089YQK4 UL2, Uracil-DNA glycosylase 36UL2 1.3
F8RFA5 Tegument protein UL21 58UL21 1.3
A0A0B5EE83 Glycoprotein I 41US7 1.0
D3YP85 Envelope glycoprotein H 90UL22 0.7
L0N3E3 Envelope glycoprotein C 55UL44 0.7
L0N6J6 DNA polymerase 137UL30 0.7
A0A089WZ14 Type II membrane protein 18UL45 0.7

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Tada, S.; Hamada, M.; Yura, Y. Proteomic Analysis of Secretomes of Oncolytic Herpes Simplex Virus-Infected Squamous Cell Carcinoma Cells. Cancers 2018, 10, 28. https://doi.org/10.3390/cancers10020028

AMA Style

Tada S, Hamada M, Yura Y. Proteomic Analysis of Secretomes of Oncolytic Herpes Simplex Virus-Infected Squamous Cell Carcinoma Cells. Cancers. 2018; 10(2):28. https://doi.org/10.3390/cancers10020028

Chicago/Turabian Style

Tada, Shinya, Masakazu Hamada, and Yoshiaki Yura. 2018. "Proteomic Analysis of Secretomes of Oncolytic Herpes Simplex Virus-Infected Squamous Cell Carcinoma Cells" Cancers 10, no. 2: 28. https://doi.org/10.3390/cancers10020028

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