Role of autophagy in oncolytic herpes simplex virus type 1-induced cell death in squamous cell carcinoma cells.

Herpes simplex virus type 1 (HSV-1) is one of the most widely studied viruses for oncolytic virotherapy. In squamous cell carcinoma (SCC) cells, the role of autophagy induced by neurovirulence gene-deficient HSV-1s in programmed cell death has not yet been elucidated. The oncolytic HSV-1 strain RH2, which lacks the γ34.5 gene and induces the fusion of human SCC cells, was used. RH2 replicated and induced cell death in SCC cells. RH2 infection was accompanied by the aggregation of microtubule-associated protein 1 light chain 3 (LC3) in the cytoplasm, the conversion of LC3-I to LC3-II and the formation of double-membrane vacuoles containing cell contents. No significant changes were observed in the expression of Bcl-2 or Bax, while a slight decrease was observed in that of Beclin 1. The autophagy inhibitors, 3-methyladenine (3-MA) and bafilomycin A1, did not affect viral replication, but significantly inhibited the cytotoxicity of RH2. The caspase-3 inhibitor z-DEVD-fmk and caspase-1 inhibitor z-YVAD-fmk also reduced the cytotoxicity of RH2. These results demonstrated that γ34.5 gene-deficient HSV-1 RH2 induced autophagic cell death in SCC cells as well as pyroptosis and apoptosis.

Immunogenic cell death by oncolytic herpes simplex virus type 1 in squamous cell carcinoma cells.

Molecules essential for the induction of immunogenic cell death (ICD) are called damage-associated molecular patterns (DAMPs). The effects of oncolytic herpes simplex virus type 1 (HSV-1) on the production of DAMPs were examined in squamous cell carcinoma (SCC) cells. The cytopathic effects of HSV-1 RH2 were observed in mouse SCCVII cells infected at a high multiplicity of infection (MOI), and the amounts of viable cells were decreased. After being infected with RH2, ATP and high mobility group box 1 (HMGB1) were released extracellulary, while calreticulin (CRT) translocated to the cell membrane. A flow-cytometric analysis revealed an increase in the number of annexin-V and propidium iodide (PI)-stained cells; and the amount of cleaved poly (ADP-ribose) polymerase (PARP) was increased. The killing effect of RH2 was reduced by pan-caspase inhibitor z-VAD-fmk and the caspase-1 inhibitor z-YVAD-fmk, suggesting the involvement of apoptosis and pyroptosis. In C3H mice bearing synergic SCCVII tumors, the growth of tumors injected with the supernatant of RH2-infected cells was less than that of tumors injected with phosphate-buffered saline (PBS). These results indicate that oncolytic HSV-1 RH2 produces DAMPs from SCC cells to induce cell death. This may contribute to the enhancement of tumor immunity by oncolytic HSV-1.

Ultrasound as a method to enhance antitumor ability of oncolytic herpes simplex virus for head and neck cancer.

Low-intensity ultrasound is a useful method to enhance the delivery of drugs to target cells via a range of mechanisms including the transient formation of micropores in the cell membrane, a process known as sonoporation. The effect of ultrasound on oncolytic herpes simplex virus type-1 (HSV-1) infection in oral squamous cell carcinoma (SCC) was examined. Human SCC cell line SAS and oncolytic HSV-1 RH2, which was deficient in the neurovirulent γ134.5 gene and exhibited cell fusion actions, were used. Cells grown in multi-well plates were infected with HSV-1 and exposed to ultrasound in the presence or absence of microbubbles after an adsorption period. The number of plaques was significantly greater than that of the untreated control. SAS cells were inoculated subcutaneously into nude mice and tumors were produced. Tumors were injected with HSV-1 RH2 with or without microbubbles and then exposed to ultrasound through the covering skin. The amount of the virus in tumor tissues 3 days after the injection was higher in tumors treated with HSV-1 RH2 and ultrasound than in tumors treated with RH2 only. The expression of the HSV-1 antigen was also increased by ultrasound and microbubbles. Tumor growth was suppressed with HSV-1 RH2 in combination with ultrasound, especially with microbubbles. These results indicated that ultrasound increased the efficiency of the HSV-1 infection in SAS cells and nude mouse tumors. This method can potentially be useful to enhance the antitumor effects of oncolytic HSV-1 on head and neck cancer treatment.

Enhancement of systemic tumor immunity for squamous cell carcinoma cells by an oncolytic herpes simplex virus.

RH2 is a neurovirulent γ134.5 gene-deficient herpes simplex virus type 1 (HSV-1) with a lytic ability in human squamous cell carcinoma (SCC) cells; it is related to spontaneously occurring HSV-1 mutant HF10. The effect of RH2 on SCC was examined using a syngeneic C3H mouse model. After infection of mouse SCCVII cells with RH2, cell viability was decreased at first, but recovered by prolonged culture, indicating the limited replication of RH2. The antitumor ability of RH2 was examined using a bilateral SCCVII tumor model. The growth of the RH2-injected tumors was suppressed compared with that of phosphate-buffered saline-injected tumors. Moreover, the growth of contralateral tumor of RH2-treated mice was also suppressed significantly. The splenocytes of C3H mice treated with RH2 lysed more SCCVII cells than NFSaY83 cells and YAC-1 cells. The cytotoxicity of the splenocytes on SCCVII cells was significantly greater than that of splenocytes from tumor-bearing mice. Removal of CD8(+) T cells from splenocytes decreased their cell killing activity remarkably. The antitumor effect of RH2 on SCCVII xenografts in nude mice was not demonstrated. These results indicate that RH2 exhibited a suppressive effect on mouse SCC, even if the replication of RH2 was limited. This is ascribed to the ability of RH2 to enhance existing tumor-specific cytotoxic T lymphocyte activity.

The effects of trichostatin A on the oncolytic ability of herpes simplex virus for oral squamous cell carcinoma cells.

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-kappaB (NF-kappaB) component, p65, translocated into the nucleus after infection with gamma(1)34.5 gene-deficient HSV-1 R849, indicating that R849 activated NF-kappaB. 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-kappaB to its DNA-binding site and an NF-kappaB 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-kappaB 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 gamma(1)34.5 gene-deficient HSV-1.

ESE-1 inhibits the invasion of oral squamous cell carcinoma in conjunction with MMP-9 suppression.

OBJECTIVES: Matrix metalloproteinases (MMPs) regulated by ets transcription factors facilitate carcinoma cell invasion. An ets family member, ESE-1, is expressed specifically in epithelial tissues, but its association with MMPs is obscure. In this study, we investigated whether ESE-1 regulates invasion of oral squamous cell carcinoma (SCC) via transcriptional activity of MMP-9. METHODS: HSC-3 and KB were used as human oral SCC lines. The expression of ESE-1 and MMP-9 was detected by in situ hybridization and immunohistochemistry. Invasion assay, gelatin zymography and Northern blotting were used to detect the invasion activity, the gelatinolytic activity and the expression of MMP-9 in the ESE-1 transfectants. Luciferase assays and mutation analysis were used for the transcriptional analysis of MMP-9 promoter region by ESE-1. RESULTS: ESE-1 was expressed in the intermediate layer but not in the invasive area, in which MMP-9 was expressed, in the oral SCC tissues. ESE-1 suppressed invasion activity and 92 kDa gelatinolytic activity in HSC-3 as a result of transfection. ESE-1 regulates MMP-9 expression in a negative manner and the ets binding site on the MMP-9 promoter contributed to suppression by ESE-1. CONCLUSIONS: These findings indicate that ESE-1 negatively regulates the invasion of oral SCC via transcriptional suppression of MMP-9.

Enhancement of antitumor activity of herpes simplex virus gamma(1)34.5-deficient mutant for oral squamous cell carcinoma cells by hexamethylene bisacetamide.

Current oncolytic viruses exert only limited antitumor activity on their own. There is a need to increase their oncolytic capability. We evaluated the effect of a differentiating reagent, hexamethylene bisacetamide (HMBA), on the antitumor activity of a gamma(1)34.5-deficient herpes simplex virus type 1 (HSV-1) R849 for human oral squamous cell carcinoma (SCC) cells. Hexamethylene bisacetamide increased the viral yield, especially at a low input multiplicity of infection (MOI), and the transcription of immediate early genes of HSV-1. Hexamethylene bisacetamide treatment promoted the cytopathic effect of R849 and increased the proportion of dead cells. Hexamethylene bisacetamide produced more apoptotic cells in R849-infected cells as compared with parental HSV-1(F)-infected cells. The growth of oral SCC xenografts in nude mice was markedly suppressed by treatment with R849 in combination with HMBA, and the survival of the co-treated animals was significantly prolonged as compared with that of animals treated with R849 only. Herpes simplex virus type 1 mRNA was expressed in tumors and trigeminal neurons, but not in brain, lung, liver, and kidney. These results indicate that HMBA enhances the antitumor activity of R849 through the expression of immediate early genes without increasing its toxicity. Hexamethylene bisacetamide can be used as an enhancing agent for oncolytic therapy with HSV-1 mutants.

A modified repositioning system for segmental resection of the mandible.

Mandibular reconstruction is required after segmental resection of the mandible. Several techniques have been proposed but have several drawbacks. A modified system (based on Leibinger's titanium-positioning system) that can reposition the residual mandible easily and accurately without interfering with the reconstructive procedure was developed. This system has been used successfully in more than 10 patients, with no complications.

Induction of endonuclease G-mediated apopotosis in human oral squamous cell carcinoma cells by protein kinase C inhibitor safingol.

PKC inhibitor safingol suppressed the growth of human oral squamous cell carcinoma (SCC) cells significantly at concentrations that inhibit PKC isoforms. Safingol inhibited the translocation of PKC following treatment with 12-o-tetradecanoylphorbol 13-acetate (TPA) in PKC alpha-EGFP-transfected cells, but not in PKC beta-EGFP- transfected cells, indicating selective inhibition for PKC alpha in oral SCC cells. Flow cytometric analysis and DNA analysis by agarose gel electrophoresis revealed an increase in the proportion of sub-G(1) cells and DNA fragmentation in safingol-treated cells. Mitochondrial membrane potential was decreased, and cytochrome c was released from mitochondria. However, the safingol-induced cell death was not accompanied by activation of caspase 3 and cleavage of poly (ADP-ribose) polymerase (PARP). The broad-spectrum caspase inhibitor BD-fmk failed to prevent safingol-induced cell death. Another apoptogenic factor endonuclease G, but not apoptosis-inducing factor (AIF), was also released from mitochondria and translocated to the nucleus. These results suggest that PKC alpha inhibitor safingol induces an endonuclease G- mediated apoptosis in a caspase-independent manner.

Phase II study of a novel oral formation of 5-fluorouracil in combination with low-dose cisplatin as preoperative chemotherapy of oral squamous cell carcinoma.

TS-1 is a novel oral 5-fluorouracil containing tegaful (prodrug of 5-FU) and two biochemical modulators. These modulators feature effect-enhancing and adverse reaction-reducing activity. We investigated the histological response and toxicities of combination chemotherapy with TS- 1 and low-dose cisplatin and evaluated its usefulness as preoperative chemotherapy Forty-four newly diagnosed patients with stage Il-IV oral squamous cell carcinoma were enrolled in this study from February 2002 to April 2004. Patients were administered TS-1 80 mg/m2/day (days 1-14) and cisplatin 5 mg/m2/day (days 1-5 and 8-12) followed by radical surgery within 2 weeks. The histopathological effect of chemotherapy, which was a surrogate endpoint of this trial, was evaluated with surgical or biopsy specimens. The rate of histological antitumor effect was as follows: complete response (CR) 36.4%, partial response (PR) 25.0%, minor response (MR) 18.1% and no change (NC) 20.5%. The rate of histological response (CR + PR) was 61.4%. The CR rate of effective cases was 59.3%. The main toxicities occurred in bone marrow and the digestive tract. The incidence of severe toxicity such as grade 3 or 4 was 4.5% in anemia, 9% in leukocytopenia, 11.4% in neutropenia, 4.5% in thrombocytopenia and 2.3% in anorexia, diarrhea and urticaria. Most patients showed no toxicity or mild toxicities. TS- 1 with low-dose cisplatin has highly effective antitumor activity and mild toxicities. In particular, the CR rate was very high. It is suggested that this regimen is suitable for neoadjuvant chemotherapy. We expect that this chemotherapy will contribute to avoidance of surgery for small tumors (stages I and II) and will enable function-preserving surgery for advanced tumors.

Effect of Ca2+-dependent cell death on the release of herpes simplex virus.

The effect of a variety of cell death-inducing reagents on the release of herpes simplex virus type 1 (HSV-1) was examined. Ionomycin was found to increase the release of HSV-1, whereas no significant increase was observed by the treatment with TNF-alpha, anti-Fas antibody, C2-ceramide, sphingosine, H-7, tyrphostin and camptothecin. Ionomycin induced an immediate early peak and a subsequent long-lasting elevation of intracellular Ca(2+) concentration ([Ca(2+)]i). In the absence of extracellular Ca(2+), ionomycin neither elevated [Ca(2+)]i nor increased the release of HSV-1 from the infected cells, indicating that Ca(2+) influx play an important role in the release of HSV-1. Studies with trypan blue and annexin V staining revealed that the ionomycin-induced alteration of [Ca(2+)]i was accompanied by cell death of the infected cells. Disintegration of cell membrane, cytoplasmic vacuole formation and the leakage of virus particles from the cell surface were observed by electron microscopy. These results indicate that Ca(2+)-dependent cell death showing necrotic alteration is responsible for the enhanced release of HSV-1. The data also give some initial insights into the functional importance of cell death during the late stages of HSV-1 infection.

Combination effect of adenovirus-mediated pro-apoptotic bax gene transfer with cisplatin or paclitaxel treatment in ovarian cancer cell lines.

To develop a novel therapeutic strategy for ovarian cancer, we constructed a recombinant adenovirus which highly expresses pro-apoptotic Bax protein and examined its therapeutic effect on a series of ovarian cancer cell lines: A2780, A2780/cDDP, OVCAR-3 and SK-OV-3. A recombinant adenovirus carrying the Bax-alpha gene (AxCALNKYbax) induced high expression of the Bax-alpha protein in all the cell lines. The cytotoxic effect of Bax was observed in three ovarian cancer cell lines: the per cent reduction in the number of cells was 40.0% for cisplatin-sensitive A2780, 50.0% for cisplatin-resistant A2780/cDDP, and 64.8% for marginally cisplatin-resistant OVCAR-3. In contrast, it was only 12.3% for cisplatin-resistant SK-OV-3. Cisplatin-resistant A2780/cDDP had a p53 mutation and exhibited attenuated Bax induction after cisplatin treatment, which may explain why supplementation of Bax was effective in this chemoresistant ovarian cancer. Combination with cisplatin or paclitaxel enhanced the cytotoxic effect of Bax induction in all but one cell line including cisplatin-resistant A2780/cDDP. It appears that adenovirus-mediated Bax induction, with or without combination with conventional chemotherapy, useful strategy for the treatment of ovarian cancer.

Enhancing effects of fibroblast growth factor on the proliferation of salivary gland carcinoma cells and salivary gland carcinogenesis.

The proliferation of mouse submandibular gland carcinoma YT-12 cells was stimulated by endothelial cell growth factor (ECGF)/bovine brain-derived acidic fibroblast growth factor (aFGF) and recombinant human aFGF. To determine whether aFGF was capable of modifying salivary gland carcinogenesis, the effect of brain-derived aFGF was examined in vivo. Mice in Groups 1 and 2 were injected with 9,10-dimethyl-1,2-benzanthracene (DMBA) into the left submandibular gland, and then Group 1 mice received bovine brain-derived aFGF and Group 2 mice received vehicle subcutaneously for 10 weeks. Group 3 and 4 mice received either bovine brain-derived aFGF or vehicle only. Sixteen weeks after the start of the experiment, the incidence of submandibular gland carcinomas in Group 1 was significantly greater than that in Group 2. Immunohistochemical study indicated that ducts in the normal submandibular glands and carcinomas showed positive staining with anti-aFGF antibody. Immunoblot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed the expression of aFGF in these tissues. FGF receptor (FGFR)-1 and FGFR-4 were detectable in the mouse submandibular glands and carcinomas. These findings suggest that bovine brain-derived aFGF stimulates the proliferation of submandibular gland carcinoma cells and promotes mouse submandibular gland carcinogenesis.

Enhancement of herpes simplex virus-induced polykaryocyte formation by 12-O-tetradecanoyl phorbol 13-acetate: association with the reorganization of actin filaments and cell motility.

A morphological change induced by syn- herpes simplex virus type 1 (HSV-1), polykaryocyte formation, was enhanced by treatment with 12-O-tetradecanoyl phorbol 13-acetate (TPA) in A431 cells. TPA treatment decreased the number of stress fibers, but led to the development of spike-like filopodia and actin-containing long projections. Similar reorganization of actin filaments was observed in HSV-1-induced polykaryocytes. The actin filament-disrupting drug cytochalasin D, but not the microtubule-disrupting drug nocodazole, inhibited the effect of TPA on polykaryocyte formation, indicating that the actin microfilament system plays a key role in this event. HSV-1 glycoprotein D (gD) was present in the cytoplasm of HSV-1-infected cells and gD gene-transfected cells; its expression became prominent at long cell projections in the presence of TPA. These findings suggest that the reorganization of actin filaments and cell motility are associated with the enhancing effect of TPA on HSV-1-induced polykaryocyte formation.

Mental nerve neuropathy as a result of primary herpes simplex virus infection in the oral cavity. A case report.

We describe a 25-year-old woman who had mental nerve neuropathy. The symptom was attributed to herpes simplex virus infection, which appeared as herpetic gingivostomatitis 4 days after the extraction of the lower third molar. This case suggests that herpes simplex virus can infect the inferior alveolar nerve through an extraction wound and can induce mental nerve neuropathy.

[Orofacial alpha herpesvirus infection].

The mouth is the most common site of primary and recurrent herpes simplex virus (HSV) infection. The clinical appearance of recurrent intraoral HSV infection is similar to that of recurrent aphthous stomatitis. Reactivation of HSV occurs in bone marrow transplantation and is more frequent in patients conditioned with total body irradiation than in patients conditioned without total body irradiation. Although the effect of oral acyclovir to prevent recurrent herpes labialis is not confirmed, recurrent HSV lesions can be treated with the ointment formulation successfully. A therapeutic approach using replication-competent HSV may be useful in the treatment of tumors of epithelial origin, such as carcinoma of the upper aerodigestive tract.

Effect of epidermal growth factor administration on the development of mouse salivary gland carcinomas.

This study investigated whether epidermal growth factor (EGF) administration was capable of modifying salivary gland carcinogenesis. Two groups of mice were given 1 mg of 9,10-dimethyl-1,2-benzanthracene (DMBA) into the left submandibular gland, and then Group 1 mice received 2 microg of EGF and Group 2 mice received vehicle subcutaneously for 8 weeks. Mice in two other groups, 3 and 4, received either EGF or vehicle alone. Twelve weeks after the start of the experiment, the incidences of submandibular gland carcinomas in Groups 1 and 2 were 39% and 58%, respectively, although this difference was not statistically significant. Duct- and cyst-like structures and carcinomas in the left submandibular glands were weakly stained by anti-EGF receptor (EGFR) antibody. Immunoblot and reverse transcriptase polymerase chain reaction (RT-PCR) analyses revealed the expression of EGFR in the submandibular glands and carcinomas. However, EGFR was undetectable in YT cells that were derived from a submandibular gland undifferentiated carcinoma of a Group 2 mouse. These findings indicate that EGF does not promote tumor induction in mouse salivary gland carcinogenesis. This may be ascribed in part to the low expression level of EGFR in tumor cells.

Expression of abnormal transcripts of the FHIT (fragile histidine triad) gene in ovarian carcinoma.

To elucidate the role of the FHIT (fragile histidine triad) gene in ovarian carcinogenesis, the expression of the gene was analysed by reverse transcription-polymerase chain reaction (RT-PCR) in 51 cases of ovarian carcinoma, 6 cases of borderline tumour and 4 cases of benign ovarian tumour. The concomitant expressions of normal and abnormal FHIT transcripts were detected in 39% of carcinomas and in 83% of borderline tumours, while benign tumours and normal ovarian tissues expressed only normal transcript. In addition, there were 4 (8%) cases of carcinoma lacking expression of normal FHIT transcript, all of which were in advanced stages (stage III-IV) and poorly differentiated. These results suggest that the expression of abnormal transcripts of the FHIT gene is a feature of ovarian malignant/borderline tumours and that the complete loss of normal FHIT expression is related to the progression of ovarian carcinoma in a subset of the cases. However, abnormal FHIT transcripts themselves were not associated with any clinicopathological parameters, such as clinical stage, histological subtype of tumour, grade of differentiation or outcome of the patient. Additionally, abnormal FHIT expression was not associated with the presence of loss of heterozygosity (LOH) at this locus, suggesting that abnormal FHIT transcripts are not derived from genetic alteration or that genetic alteration at this locus is complicated.

Human chorionic gonadotropin (hCG) inhibits cisplatin-induced apoptosis in ovarian cancer cells: possible role of up-regulation of insulin-like growth factor-1 by hCG.

Gonadotropins have been suggested to play a role in the development or progression of ovarian cancer, and we have previously reported the expression of luteinizing hormone/ human chorionic gonadotropin (LH/hCG) receptor in 40% of epithelial ovarian carcinomas. To examine the biological effect of LH/hCG on ovarian cancer cells, apoptosis induced by cisplatin with or without hCG treatment was investigated in 2 ovarian cancer cell lines, OVCAR-3 and SK-OV-3. Stimulation of cell proliferation by hCG was also studied. In addition, to analyze further the mechanism of hCG signaling involved in apoptosis-inhibition, we examined the expression of LH/hCG receptors and the regulation by hCG for apoptosis-inhibitory pathways, such as the bcl-2/bax system and the insulin-like growth factor-1 (IGF-1)/IGF-1 receptor (IGFR) system. hCG did not increase cell proliferation in either cell line. However, hCG treatment suppressed cisplatin-induced apoptosis by 58% in the OVCAR-3 cells, as shown by immunofluorescent staining and quantitation of DNA fragmentation. LH/hCG receptor mRNA was expressed only in OVCAR-3, and no apoptosis-inhibitory effect of hCG was observed in the SK-OV-3 cells that did not express the receptor. In the OVCAR-3 cells, hCG significantly increased mRNA expression of IGF-1, but did not change mRNA levels of bcl-2/bax. Our findings suggest that LH/hCG influences the chemosensitivity of ovarian cancer cells through an apoptosis-inhibitory signal possibly via up-regulation of IGF-1 expression.

Early invasive adenocarcinoma of the fallopian tube: a case report and review of the literature.

We present an early invasive adenocarcinoma of the fallopian tube, which was incidentally found in a 45-year-old woman undergoing a laparotomy for uterine myoma. Histological examination of the hydropic tubes revealed widespread endosalpingeal hyperplasia without atypia in both tubes. In addition, the left tube contained 3 scattered lesions of carcinoma in situ, one of which was accompanied by a microfocus of definite stromal invasion confined within the endosalpingeal mucosa. Such a case seems extremely rare, and it might represent the histological appearance of an early invasive feature of tubal carcinoma. We reviewed previously reported cases of in situ and/or early invasive carcinomas of the fallopian tube with respect to the pathological diagnosis and histogenesis of primary tubal adenocarcinomas.