Combining the use of a chemotherapeutic agent with oncolytic virotherapy is a useful way to increase the efficiency of the treatment of cancer. The effect of the histone diacetylase (HDAC) inhibitor trichostatin A (TSA) on the antitumor activity of a herpes simplex virus type-1 (HSV-1) mutant was examined in oral squamous cell carcinoma (SCC) cells. Immunoblotting analysis and immunoflourescence staining revealed that a cytoplasmic nuclear factor-κB (NF-κB) component, p65, translocated into the nucleus after infection with γ134.5 gene-deficient HSV-1 R849, indicating that R849 activated NF-κB. TSA induced acetylation of p65 and increased the amount of p65 in the nucleus of oral SCC cells. Treatment of R849-infected cells with TSA also increased the amount of nuclear p65 and binding of NF-κB to its DNA-binding site and an NF-κB inhibitor SN50 diminished the increase in nuclear p65. In the presence of TSA, the production of virus and the expression of LacZ integrated into R849 and glycoprotein D, but not ICP0, ICP6 and thymidine kinase, were increased. The viability of cells treated with a combination of R849 and TSA was lower than that of those treated with R849 only. After treatment with TSA, expression of the cell cycle kinase inhibitor p21 was upregulated and the cell cycle was arrested at G1. These results indicate that TSA enhanced the replication of the HSV-1 mutant through the activation of NF-κB and induced cell cycle arrest at G1 to inhibit cell growth. TSA can be used as an enhancing agent for oncolytic virotherapy for oral SCC with γ134.5 gene-deficient HSV-1.
The usage of replication-competent herpes simplex virus type 1 (HSV-1) mutants is an approach to cancer therapy because the replication of viruses within cancer cells can result in their destruction.1 As the γ134.5 gene is responsible for the neurovirulence of HSV-1, γ134.5 gene-deficient mutants are considered to be useful as vectors for oncolytic therapy.2, 3 However, it was reported that they exerted only limited antitumor activity on their own. This is in part because of the reduction of the replication potential of the virus within targeted tumors because of the complete elimination of the γ134.5 gene. Thus, recent studies have focused on the effect of combining oncolytic virotherapy with chemotherapy or construction of viruses encoding therapeutic transgenes.4, 5, 6, 7, 8, 9
The ubiquitous nuclear factor NF-κB is a critical regulator of the expression of numerous genes implicated in immune and inflammatory responses, cellular proliferation and differentiation and cell survival.10, 11 It is activated by a broad variety of stimuli, such as growth factors, cytokines, ionizing radiation, ultraviolet light, chemotherapeutic drugs and bacterial and viral infections.10, 12, 13 This transcription factor is composed of homo- or heterodimers with various combinations of five subunits: p50/p105, p52/p100, p65 (RelA), RelB and c-Rel. The most abundant form of NF-κB is a heterodimer of p50 and p65.13 In unstimulated cells, NF-κB is sequestered in the cytoplasm in an inactive form through its association with a member of an inhibitory family, the most characterized member of which is IκBα. Upon stimulation, inhibitor kappa B (IκB) is rapidly phosphorylated on Ser32 and Ser 36 by the cytoplasmic IκB kinase (IKK) complex, which triggers its polyubiquitination and subsequent degradation.14 The released NF-κB translocates into the nucleus to regulate the expression of multiple target genes, including those coding for its own inhibitor, IκB. This negative feedback ensures that NF-κB is removed from DNA-binding sites and transported back to the cytoplasm, thereby terminating NF-κB-dependent transactivation.
Histone acetylation is tightly controlled by histone acetyltransferases and histone deacetylases (HDACs).15, 16 Changes in histone acetyltransferase or HDAC activity occur in cancers and have prompted the search for pharmacological agents capable of inhibiting these enzymes.16 Indeed, HDAC inhibitors (HDACi’s) have been shown to exhibit antitumor activity in a number of cancers.15, 17, 18 HDACs function not only to deacetylate core histones leading to repressive changes in chromatin structure, but also to deacetylate various host transcription factors, altering their transcriptional activity. NF-κB is subjected to reversible acetylation, and HDACs play important roles in its deacetylation.19, 20 Trichostatin A (TSA) is a natural HDACi that promotes histone hyperacetylation and strongly induces apoptosis by altering the expression of some apoptotic genes.21, 22, 23 However, it was also reported to show relatively modest antitumor activity in cases of head and neck squamous cell carcinoma (SCC) cells. The limited effect of TSA was associated with NF-κB’s activation by this HDACi.18
With regard to HSV infections, NF-κB is activated early during an HSV-1 infection, translocates to the nucleus24, 25 and prevents virus-induced early apoptosis of infected cells to complete its replication cycle.25, 26, 27 Whether an HDACi that activates NF-κB could affect the replication of oncolytic HSV-1 mutants has not been investigated. In this study, we examined the effects of TSA on the NF-κB activity of oral SCC cells and antitumor activity of γ134.5 gene-deficient mutants. The results indicate that TSA can be used as an enhancing agent for oncolytic virotherapy in cases of oral SCC.
Materials and methods
Cells, virus and reagents
The human oral SCC cell line SAS, Ca9-22 and HSC were obtained from the Japanese Collection of Research Bioresources (Tokyo, Japan). Cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U ml−1 penicillin and 100 μg ml−1 streptomycin and grown in an incubator at 37 °C in a humidified atmosphere with 5% CO2. The HSV-1 mutant R84928 was grown in semiconfluent Vero cell monolayers. The infectivity of HSV-1 was determined by the formation of plaques on Vero cell monolayers covered with 0.3% methylcellulose. TSA was obtained from Sigma (St Louis, MO) and dissolved in dimethyl sulfoxide. SN50 was obtained from Calbiochem (Darmstadt, Germany).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay
Cells grown in 96-well culture dishes were infected with HSV-1 at a multiplicity of infection (MOI) of 2, whereas controls were mock-infected. After incubation for various intervals, 10 μl of a 5 mg ml−1 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide solution was added to each well with 100 μl of medium. Cells were incubated for 4 h at 37 °C, and then 100 μl of 0.04 N HCl in isopropanol was added. They were mixed thoroughly to dissolve the dark blue crystals. After letting stand overnight at room temperature, the plates were read on a Benchmark Plus microplate spectrophotometer (Bio-Rad Laboratories, Hercules, CA) with a reference wavelength of 630 nm and a test wavelength of 570 nm. Background absorbance at 630 nm was subtracted from the 570-nm reading. Changes from room air controls were calculated.
Preparation of nuclear and cytoplasmic extracts
Nuclear and cytoplasmic extracts were prepared according to the method reported by Taddeo et al.27 Cells were collected and rinsed once with cold PBS, transferred to a microtube and lysed by gentle inversion in hypotonic lysis buffer containing 10 mM HEPES (pH 7.5), 10 mM KCl, 3 mM MgCl2, 0.05% Nonidet P-40, 1 mM EDTA, 1 mM dithiothreitol, 10 mM NaF, 10 mM β-glycerophosphate, 0.1 mM sodium orthovanadate and protease inhibitor mixture. Samples were incubated on ice for 30 min before centrifugation at 500 g for 5 min at 4 °C. The supernatant fluids were transferred into fresh tubes and used as cytoplasmic extract. The nuclear pellets were rinsed twice in a hypotonic lysis buffer containing 0.1% Nonidet P-40 and lysed with buffer containing 50 mM HEPES (pH 7.9), 250 mM KCl, 1% Nonidet P-40, 5% glycerol, 0.1 mM EDTA, 1 mM dithiothreitol, 10 mM NaF, 10 mM glycerophosphate, 0.1 mM sodium orthovanadate and protease inhibitor mixture. The samples were frozen and thawed three times and incubated on ice for 30 min. Insoluble material was pelleted in a microtube at 16 000 g for 10 min at 4 °C and the supernatant fluid was used as nuclear extract.
For the detection of proteins other than NF-κB p65, cells were washed in PBS and lysed in a lysis buffer containing 20 mM Tris-HCl (pH 7.4), 0.1% SDS, 1% Triton X-100, 1% sodium deoxycholate and protease inhibitor mixture. After sonication on ice and subsequent centrifugation at 15 000 g for 10 min at 4 °C, the supernatant was collected and the protein concentration was determined using a Protein Assay Kit (Bio-Rad). Sample protein (20 μg) was electrophoresed through a polyacrylamide gel and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA) by electroblotting. The membrane was probed with antibodies, and antibody-binding was detected using an enhanced chemiluminescence kit (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's instructions. Rabbit polyclonal antibodies against NF-κB p65 and poly (ADP-ribose) polymerase (PARP) (Cell Signaling Technology, Beverly, MA), and mouse monoclonal antibodies against p21 (Cell Signaling), glycoprotein D (gD) (Chemicon International, Temecula, CA) and β-actin (Sigma) were used. The β-actin expression was assessed to ensure equal protein loading. Antibodies conjugated with horseradish peroxidase were used as secondary antibody.
One milliliter of nuclear extract was immunoprecipitated by incubation with 2 μg of rabbit polyclonal antibody against p65 for 2 h at 4 °C, then 2 mg of protein G-Sepharose (Amersham Biosciences) was added, and the samples were further incubated for 2 h. Immunoprecipitates were washed three times with lysis buffer, boiled for 5 min after 2 × SDS sampling buffer was added and subjected to immunoblotting using pan-Acetyl (Santa Cruz Biotechnology, Santa Cruz, CA), a goat polyclonal antibody against an acetylated polypeptide, overnight a 4 °C, followed by a donkey anti-goat IgG conjugated with horseradish peroxidase (Cell Signaling), and protein bands were detected by enhanced chemiluminescence.
Electrophoretic mobility shift assay
Electrophoretic mobility shift assays were carried out using a digoxigenin gel sift kit (Boehringer Mannheim Biochemica, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, the oligonucleotide 5′-IndexTermAGTTGAGGGGACTTTCCCAGGC-3′ containing a kB-binding site (Sigma) was digoxigenin-labeled using a 3′-end labeling kit, and the DNA probe was incubated with 10 μg of the nuclear extract at room temperature for 10 min. Thereafter, protein–DNA complexes were separated on a 7.5% nondenaturing polyacrylamide gel, and electrically transferred to a nylon membrane (Amersham Biosciences) for chemiluminescence detection. The intensity of each band was measured with the Image J program.
Reverse-transcription PCR and PCR analyses
Total RNA was extracted from cells using ISOGEN (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. RT-PCR was performed using a Takara RNA PCR kit (Takara, Tokyo, Japan). One microgram of RNA was reverse-transcribed using avian myeloblastosis virus (AMV) reverse transcriptase, and cDNAs encoding ICP0, ICP6, thymidine kinase (TK), gD, LacZ and β-actin gene sequences were amplified by PCR using specific primers. The sequences of the primers used were as follows:
ICP0 forward, 5′-IndexTermGACAGCAAAAATCCCCTGAG-3′; ICP 0 reverse, 5′-IndexTermACGAGGGAAAACAATAAGGG-3′; ICP6 forward, 5′-IndexTermGACAGCCATATCCTGAGC-3′; ICP6 reverse, 5′-IndexTermACTCACAGATCGTTGACGACCG-3′; TK forward, 5′-IndexTermATACCGACGATATGCGACCT-3′; TK reverse, 5′-IndexTermTTATTGCCGTCATAGCGCGG-3′; gD forward, 5′-IndexTermATGGGAGGCAACTGTGCTAT-3′; gD reverse, 5′-IndexTermCTCGGTGCTCCAGGATAAAC-3′; LacZ forward, 5′-IndexTermGCGTTACCCAACTTAATCG-3′; and LacZ reverse, 5′-IndexTermTGTGAGCGAGTAACAACC-3′; β-actin forward, 5′-IndexTermGTGGGCCGCTCTAGGCACCAA-3′; and β-actin reverse, 5′-IndexTermCTCTTTGATGTCACGCACGATTTC-3′.9 To confirm the integrity of each RNA sample, a PCR analysis of the β-actin gene was performed. The PCR amplification of cDNAs was carried out in volumes of 50 μl for 30 cycles at a denaturing temperature of 94 °C for 30 s, an annealing temperature of 60 °C for 30 s and an extension temperature of 72 °C for 2 min using a Gene Amp PCR system 9700 (Applied Biosystems, Foster City, CA). PCR products were subsequently size-fractionated on 1.5% agarose gels, stained with ethidium bromide and photographed under transmitted UV light.
Flow cytometric analysis
Cells were fixed in cold 80% ethanol for 1 h at 4 °C. The fixed cells were collected by centrifugation, and the pellet was resuspended in propidium iodide (PI) staining reagent composed of 0.1% Triton X-100, 0.1 mM EDTA, 0.05 mg ml−1 RNase A (50 U mg−1) and 50 μg ml−1 PI, and then stored in the dark at room temperature until analyzed. Cell cycle analysis was performed with a fluorescence-activated cell sorter (FACSort; Becton Dickinson, Mountain View, CA) and interpreted with the aid of Mod Fit LT software (Varity Software House, Topsham, ME).
Confocal laser microscopic analysis
For the immunofluorescence analysis of p65, cells were fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton X-100 in PBS for 3 min at 4 °C and incubated with rabbit polyclonal antibodies against p65 for 1 h at room temperature. After washing, the cells were incubated with rhodamine-conjugated goat anti-rabbit IgG (Leinco Technologies, St Louis, MO). After a wash, the cells were stained with 4, 6-diamidino-2-phenylindole (Roche, Indianapolis, IN), and coverslips were mounted onto microscope slides using ProLong antifade mounting reagent (Molecular Probes, Eugene, OR). The slides were analyzed by a confocal laser-scanning microscope LMS 410 (Carl Zeiss Microimaging, Thomwood, NY).
Mean values were compared using the unpaired t-test. P<0.05 indicated a significant difference among groups.
Nuclear accumulation of NF-κB by infection with HSV-1 mutant in oral SCC cells
In response to stimuli that activate NF-κB, NF-κB components, p50 and p65, translocate into the nucleus after degradation of the NF-κB inhibitor IκB by IKK.14 To determine whether the HSV-1 mutant could activate NF-κB, SAS cells were infected with R849 at an MOI of 2, and cytoplasmic and nuclear extracts were subjected to an immunoblot analysis using antibodies against p65. As PARP was expressed in the nucleus,26 the blotted membranes were also reacted with anti-PARP antibody. The expression of p65 was increased in the nucleus from 6 h after infection and became remarkable at 12 h (Figure 1a).
To further examine the localization of p65 in R849-infected cells, immunofluorescent staining was performed. In uninfected cells, p65 was observed mainly in the cytoplasm. Twelve hours after infection, cells became rounded and the nucleus was stained with anti-p65 antibody strongly as compared with uninfected cells (Figure 1b). In the following experiments, infection with R849 was performed at an MOI of 2.
Acetylation and nuclear accumulation of p65 by TSA
In the nucleus, p65 is acetylated by histone acetyltransferase and binds to its DNA-binding site. Acetylated p65 was constantly deacetylated by HDAC. HDACi has been shown to inhibit deacetylation and increases the level of nuclear p65.18 SAS cells were cultured in the presence or absence of 0.3 μM TSA, and the acetylated p65 was detected in combination with anti-p65 and anti-acetylated polypeptide antibodies. Acetylated p65 increased in a time-dependent manner from 6 h after the treatment (Figure 2a). When the expression of p65 was examined by immunoblot analysis, it was found that nuclear p65 increased after treatment with TSA (Figure 2b).
Modification of nuclear localization of p65 and NF-κB binding to its DNA-binding site in HSV-1 mutant-infected oral SCC cells by TSA
The effect of TSA on the localization of NF-κB p65 in HSV-1 mutant-infected cells was examined by immunoblot analysis. SAS cells were infected with R849. After adsorption for 1 h, cell monolayers were washed with PBS twice and cultured in the presence or absence of TSA. At 12 h after infection, an increase of p65 in the nucleus was observed. SN50 is an inhibitor of NF-κB that prevents the translocation of cytoplasmic NF-κB into the nucleus.29 In the presence of 9 μM SN50, the nuclear NF-κB was markedly decreased in R849-infected cells (Figure 3). When infected cells were cultured in the presence of TSA and SN50, the increase of nuclear p65 by TSA was diminished (Figure 3).
Whether R849 and TSA could increase the binding of NF-κB to its DNA-binding site was also examined by electrophoretic mobility shift assay. SAS cells were infected with R489 and cultured in the presence or absence of TSA. The nuclear extracts were reacted with the κB site of DNA and the complexes were analyzed by undenatured polyacrylamide gel electrophoresis. R849 increased NF-κB binding, and the addition of TSA further increased the binding (Figure 4). Measurement of the intensity of each band with the Image J program revealed that NF-κB binding was increased 8- and 12-fold by treatment with R849 and a combination of R849 and TSA, respectively.
Increase of virus production by TSA
SAS cells were infected with R849 and cultured in the presence or absence of TSA, and the yield of virus was measured 12, 24 and 36 h after infection. TSA increased viral yield 10-fold at 24 h after infection, but there was no increase 36 h after infection (Figure 5a). When infected cells were cultured in the presence of SN50 for 24 h, the yield was decreased to 62% of the untreated control. After the treatment with a combination of TSA and SN50 for 24 h, the enhancement by TSA was diminished and the increase in viral yield was 3.3-fold (Figure 5b).
Modification of viral gene expression by TSA
R849-infected cells were cultured in the presence or absence of TSA for 12 h, and the expressions of the immediate early gene ICP0, early genes ICP6 and TK and late genes LacZ inserted into γ134.5 and gD were examined by reverse-transcription PCR. There was an increase of LacZ and gD, but not of ICP0, ICP6 and TK after treatment with TSA (Figure 6a). Increased expression of gD on treatment with TSA was observed by immunoblot analysis (Figure 6b).
Enhancement of suppression of cell viability by TSA
To determine the effect of HSV-1 infection and TSA on the growth of oral SCC cells, SAS cells were infected with R849 and/or cultured in the presence or absence of TSA and cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Forty-eight hours after infection, the viability of infected cells decreased to 68% of the uninfected control. On incubation of uninfected cells in the presence of 0.1 μM TSA for 48 h, the cell viability was maintained at the initial level. However, when SAS cells were infected with R849 and cultured in the presence of TSA, cell viability was decreased to 50% of the untreated control, suggesting that TSA enhanced, although insignificantly (P>0.05), the suppressive effect of R849 on cell growth (Figure 7a). At a concentration of 0.3 μM, TSA itself suppressed cell growth, and a combination of R849 and TSA decreased cell viability more efficiently than R849 or TSA only (Figure 7b). The differences between the combined treatment and R849 only at 48 and 72 h after infection were significant (P<0.05).
Induction of p21 expression and cell cycle arrest by TSA
The growth inhibition mediated by HDACi is postulated to be due to the induction of a cell cycle kinase inhibitor, p21.30, 31 R849-infected SAS cells were treated with TSA. Immunoblot analysis revealed that p21 became detectable in cells treated with TSA. Induction of p21 expression by TSA was also observed in R849-infected cells, although the level was lower than that in uninfected cells (Figure 8a). After treatment with TSA for 12 h, the proportion of cells in G0/G1 was increased to 75%, whereas that in the untreated control was 42%. The G0/G1 peak was not increased by R849 infection only, but was increased to 72% after treatment with TSA (Figure 8b).
Nuclear factor-κB-activating signals activate IKK to phosphorylate IκB, which is followed by the degradation of IκB by the proteosome. During the entry of HSV-1, a first wave of IKK activity is observed. IKK’s activation at this time is rapid, transient and independent of viral replication. The induction is followed by the degradation of IκB and triggering of NF-κB DNA-binding activity, which lasts for 2 h. After the onset of viral DNA synthesis at approximately 3 h after infection, the infected cells begin to accumulate large amounts of complementary viral RNAs, sequences derived from at least 50% of the viral DNA. These RNAs activate protein kinase R, which is responsible for phosphorylation of IκB by IKK.25, 27, 32 In this way, HSV-1 infection induces a second wave of IKK activity, which persists at increased levels for several hours after infection, leads to the complete degradation of IκB and induces a strong and persistent nuclear translocation and increase in p50/65-dependent DNA-binding activity.
In normal cells infected with HSV-1, the subunit of eukaryotic translation initiation factor (eIF-2α) is phosphorylated by protein kinase R and all protein synthesis is prevented, followed by the termination of viral replication. The γ134.5 protein of HSV-1 blocks the effect of activated protein kinase R by recruiting the protein phosphatase 1α to dephosphorylate eIF-2α. Thus, in the absence of this gene, the HSV-1 mutant cannot replicate in normal cells.33 However, as the activation of mitogen-activated protein kinase (MAPK) kinase (MEK), a critical downstream RAS effector kinase, suppresses the induction of protein kinase R, γ134.5-deficient HSV-1 is allowed to replicate in cancer cells with high MEK activity.32 Previously, we indicated that γ134.5-deficient HSV-1 could replicate in oral SCC cells.9 In this study, we examined the effect of infection by a γ134.5-deficient HSV-1 mutant on the distribution of p65 in oral SCC cells and found that NF-κB components were increased in the nucleus after infection. The increase in NF-κB was confirmed by immunofluorescent staining and a similar translocation of NF-κB was observed in other oral SCC cell lines, such as Ca9-22 and HSC cells (data not shown). Furthermore, the NF-κB inhibitor SN50, which inhibits the translocation of NF-κB into the nucleus, suppressed the increase in nuclear p65. Thus, we concluded that infection by a γ134.5-gene deficient mutant could activate NF-κB in oral SCC cells.
Trichostatin A is considered to be a potent antitumor drug that inhibits HDAC. However, Duan et al.18 reported that TSA promoted the acetylation of p65 and increased the NF-κB activity in head and neck cancer cells, which was responsible for the modest antitumor effect of TSA on these cancer cells. In this study, TSA enhanced the acetylation of p65 and accumulation of p65 in the nucleus of oral SCC cells. As measured by electrophoretic mobility shift assay, more NF-κB bound to the DNA-binding site after treatment with TSA. These findings suggest that TSA enhances the activation of NF-κB in HSV-1 mutant-infected oral SCC cells. The increase in nuclear NF-κB caused by TSA was partly suppressed in the presence of the NF-κB inhibitor SN50, so TSA may affect the translocation of NF-κB. However, Chen et al.20 reported that TSA prolonged both TNF-induced DNA-binding activity and the presence of NF-κB in the nucleus, delaying the export to the cytoplasm after binding with IκB. Thus, it is more likely that TSA acetylates nuclear p65 and prolongs the retention of NF-κB in the nucleus. In any case, it can be stated that TSA promotes the nuclear localization of NF-κB in the HSV-1 mutant-infected cells and acetylated p65 would fully activate transcription of target genes of NF-κB.
Goodkin et al.26 proposed that NF-κB was required as an antiapoptotic factor in prolonging functional cell survival, and thus efficient viral replication. Gregory et al.34 examined the replication of HSV-1 in cell lines with deletions of IKK1 or IKK2 and found an 86–94% loss of viral yield compared with normal cells. In cells where NF-κB was blocked by dominant-negative IκB expression, HSV failed to suppress apoptosis.34 We found that the NF-κB inhibitor SN50 had a weak inhibitory effect on viral yield, and suppressed the TSA-induced enhancement of the yield, indicating an important role for NF-κB in HSV-1 replication. It is also indicated that NF-κB activity is required to promote the expression of genes of HSV-1.25 During productive infection of HSV-1, viral genes were expressed in a tightly regulated temporal cascade, consisting of immediate-early, delayed-early and late genes.35 NF-κB promotes the expression of immediate early genes of HSV-1 because of the binding sites of the promoter regions of these genes. Amici et al.25 reported that NF-κB is recruited to the promotor of immediate-early gene ICP0 in HSV-1-infected keratinocytes, enhancing ICP0 gene transcription. We found upregulation of late genes gD- and γ134.5-delivered LacZ genes, although no particular change in the expression of immediate- and delayed-early genes was observed. As the expression of late genes depends on that of immediate- and late-early genes, these early genes may also be affected by TSA. Together, it was concluded that TSA activated NF-κB and promoted the replication of the HSV-1 mutant R849 in oral SCC cells, by upregulating the transcription of viral genes.
Studies exploring chemotherapy–oncolytic virus combinations or viruses encoding therapeutic transgenes have demonstrated that synergistic tumor killing can either be replication-dependent, with enhanced replication resulting in enhanced potency,4, 5, 6, 9 or replication-independent, with enhanced potency being caused by mechanisms other than enhanced viral replication.7, 8 We found that TSA had a suppressive effect on the growth of oral SCC cells at 0.3 μM, but its effect was insufficient at 0.1 μM. When TSA was combined with R849, cell viability was decreased more strongly than with R849 only, indicating that TSA promoted the antitumor activity of the γ134.5 gene-deficient HSV-1 mutant. In a recent clinical study, when two patients with oral SCC had a single intratumoral injection of HSV1716 at a low dose, HSV1716 showed no evidence of viral replication and little evidence of biological activity.36 This indicates that a dose of 5 × 105 PFU is too low to produce a therapeutic effect and that viral replication in oral SCC must be enhanced. As TSA increased the expression of viral genes and the production of progeny virus in oral SCC cells (Figure 6), it may be used as an agent to promote the replication of oncolytic HSV-1s in oral SCC tissues.
On the other hand, HDACi including TSA elicits a range of biological responses that affect tumor growth and survival, including inhibition of cell cycle progression, induction of tumor cell-selective apoptosis, suppression of angiogenesis and modulation of immune responses.17 With regard to the cell cycle, the growth inhibition mediated by HDACi in human tumor cells is primarily due to an induction of p21 expression, with a subsequent inhibition of cyclin/cyclin-dependent kinase (CDK) activity.30, 31 We found that TSA induced the expression of p21 and arrested the cell cycle at G1 in oral SCC cells. This must contribute to the enhanced suppression of cell viability in cultures treated with a combination of R849 and TSA. Cyclin/CDK activity is known to be required for HSV-1 replication,37 which may abolish the enhancing effect of TSA on the yield of virus after an incubation for 36 h (Figure 5a). Recently, Liu et al.38 reported that combination therapy of HSV-1G47 delta with TSA enhanced antitumor efficacy and antiangiogenesis. They identified cyclin D1 blockade and vascular endothelial growth factor inhibition as the main mechanisms for antitumor and antiangiogeneic activities. This indicates that synergistic tumor killing by combination treatment with TSA is replication independent.
We have not determined the antitumor efficiency of combined oncolytic therapy with R849 and TSA in vivo, but concurrent systemic TSA and intratumoral G47delta administration was reported to result in enhanced antitumor efficacy and antiangiogenesis in animal models.38 A similar antitumor effect may be expected because induction of CDK inhibitor p21, as the downregulation of cyclin D1, can affect the growth of tumors.
In conclusion, we showed that TSA increased NF-κB activity early on in the infection by R849 and promoted the replication of HSV-1. TSA also acted on histone and induced the expression of p21 and cell cycle arrest at G1. It can be stated that TSA enhances antitumor activity of oncolytic HSV-1 R849 for oral SCC in replication-dependent and replication-independent manners.
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This investigation was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan.
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Katsura, T., Iwai, S., Ota, Y. et al. The effects of trichostatin A on the oncolytic ability of herpes simplex virus for oral squamous cell carcinoma cells. Cancer Gene Ther 16, 237–245 (2009). https://doi.org/10.1038/cgt.2008.81
- herpes simplex virus mutant
- oncolytic virotherapy
- deacetylase inhibitor
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