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.

Head and neck cancer has been treated by a combination of surgery, radiation, and chemotherapy. In recent years, the development of immune checkpoint inhibitors (ICIs) has made immunotherapy a new treatment method. Oncolytic virus (OV) therapy selectively infects tumor cells with a low-pathogenic virus, lyses tumor cells by the cytopathic effects of the virus, and induces anti-tumor immunity to destroy tumors by the action of immune cells. In OV therapy for head and neck squamous cell carcinoma (HNSCC), viruses, such as herpes simplex virus type 1 (HSV-1), vaccinia virus, adenovirus, reovirus, measles virus, and vesicular stomatitis virus (VSV), are mainly used. As the combined use of mutant HSV-1 and ICI was successful for the treatment of melanoma, studies are underway to combine OV therapy with radiation, chemotherapy, and other types of immunotherapy. In such therapy, it is important for the virus to selectively replicate in tumor cells, and to express the viral gene and the introduced foreign gene in the tumor cells. In OV therapy for HNSCC, it may be useful to combine systemic and local treatments that improve the delivery and replication of the inoculated oncolytic virus in the tumor cells.

Safingol, L- threo-dihydrosphingosine, induces cell death in human oral squamous cell carcinoma (SCC) cells through an endonuclease G (endoG) -mediated pathway. We herein determined whether safingol induced apoptosis and autophagy in oral SCC cells. Safingol induced apoptotic cell death in oral SCC cells in a dose-dependent manner. In safingol-treated cells, microtubule-associated protein 1 light chain 3 (LC3)-I was changed to LC3-II and the cytoplasmic expression of LC3, amount of acidic vesicular organelles (AVOs) stained by acridine orange and autophagic vacuoles were increased, indicating the occurrence of autophagy. An inhibitor of autophagy, 3-methyladenine (3-MA), enhanced the suppressive effects of safingol on cell viability, and this was accompanied by an increase in the number of apoptotic cells and extent of nuclear fragmentation. The nuclear translocation of endoG was minimal at a low concentration of safingol, but markedly increased when combined with 3-MA. The suppressive effects of safingol and 3-MA on cell viability were reduced in endoG siRNA- transfected cells. The scavenging of reactive oxygen species (ROS) prevented cell death induced by the combinational treatment, whereas a pretreatment with a pan-caspase inhibitor z-VAD-fmk did not. These results indicated that safingol induced apoptosis and autophagy in SCC cells and that the suppression of autophagy by 3-MA enhanced apoptosis. Autophagy supports cell survival, but not cell death in the SCC cell system in which apoptosis occurs in an endoG-mediated manner.

Sphingosine kinase 1 (SphK1) overexpressed in head and neck squamous cell carcinoma (SCC) regulates tumor growth. The effects of PF-543, a specific SphK1 inhibitor, on human SCC cells were examined. The proportion of viable cells after PF-543 treatment decreased in a time- and dose-dependent manner, and cell death occurred in SphK1-expressing SCC cells. Flow cytometry analysis revealed that PF-543 induced both necrosis and apoptosis. PF-543 also induced granular accumulation of LC3 and conversion from LC3-I to LC3-II, which was blocked by autophagy inhibitors, wortmannin, 3-methyladenine (3-MA), and bafilomycin A1. Treatment of head and neck SCC cells with autophagy inhibitors and PF-543 increased the proportion of cells with necrosis and apoptosis, indicating that autophagy acts to promote cell survival. Reactive oxygen species (ROS) scavenger reduced the cytotoxicity of PF-543. These results demonstrated that PF-543 induces apoptosis, necrosis, and autophagy in human head and neck SCC cells, and that autophagy antagonizes either necrosis or apoptosis.

Conventional chemotherapy and targeted therapies have limited efficacy against advanced head and neck squamous cell carcinoma (HNSCC). The immune checkpoint inhibitors (ICIs) such as antibodies against CTLA-4, PD-1, and PD-L1 interrupt the co-inhibitory pathway of T cells and enhance the ability of CD8⁺ T cells to destroy tumors. Even in advanced HNSCC patients with recurrent diseases and distant metastasis, ICI therapy shows efficiency and become an effective alternative to conventional chemotherapy. However, as this therapy releases the immune tolerance state, cytotoxic CD8⁺ T cells can also attack organs and tissues expressing self-antigens that cross-react with tumor antigens and induce immune-related adverse events (irAEs). When patients with HNSCC are treated with ICIs, autoimmune diseases occur in multiple organs including the skin, digestive tract, endocrine system, liver, and respiratory tract. Treatment of various malignancies, including HNSCC, with ICIs may result in the appearance of oral irAEs. In the oral cavity, an oral lichenoid reaction (OLR) and pemphigoid develop. Sicca syndrome also occurs in association with ICIs, affecting the salivary glands to induce xerostomia. It is necessary to elucidate the pathogenic mechanisms of these intractable diseases that are not seen with conventional therapy. Early diagnosis and appropriate approaches to irAEs are needed for efficient treatment of advanced HNSCC by ICIs.