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Objective Accumulating evidence has demonstrated the protumor role of estrogen receptor (ER)–mediated signaling in multiple cancer types, which is distinct from this signaling in sex steroid–dependent organs. However, its role in oral squamous cell carcinoma (OSCC) remains unclear. Study Design We assessed the expression of ERα and ERβ in human OSCC tissues by immunohistochemistry and evaluated the expression of both receptors in OSCC cell lines by immunoblotting and flow cytometry. To further assess the contribution of ER-mediated signals to oral cancer progression, proliferation, invasion, and chemosensitivity, cell lines were stimulated with the ER agonist β-estradiol. Results Immunohistochemical analysis of OSCC tissues showed that ERβ was present in the cytoplasm and nuclei of OSCC cells. In contrast, ERα was not detected in any of the cases analyzed. Additionally, the proliferation and invasiveness of OSCC cells were significantly elevated following stimulation with β-estradiol. Chemotherapeutic agent–induced apoptosis of cancer cells was attenuated by pretreatment with β-estradiol. Conclusions ER-mediated signaling plays a crucial role in oral cancer progression by facilitating the proliferation, invasion, and chemoresistance of OSCC cells, indicating its potential for developing novel targeted therapies for this type of cancer.

The repurposing of existing FDA-approved non-cancer drugs is a potential source of new treatment options for cancer patients. An anti-inflammatory drug, 5-aminosalicylic acid (5-ASA), has been clinically used to treat inflammatory bowel disease. Hyperthermia (HT) is widely applicable addendum therapy with the existing cancer treatment modalities. Here, we addressed how 5-ASA combined with HT induces lethal effects in human oral squamous cell carcinoma (OSCC) HSC-3 cells. We found that 5-ASA/HT combination significantly inhibited the viability of HSC-3 cells, while cytotoxic effects in primary human dermal fibroblast cells were minor. Apoptotic endpoints were significantly increased by the 5-ASA/HT combined treatment, as evidenced by presence of Annexin V-FITC/PI positive cells, loss of MMP, Bcl-2/Bax ratio alteration, and increased Fas, cleaved Bid, and caspase expression. Interestingly, the enhancement of apoptosis was reversed in the presence of ON/ONOO− scavengers. These findings indicate that the combination treatment enhances apoptosis via ON/ONOO− mediated ER stress-Ca2+-mitochondria signaling and caspase-dependent apoptotic pathways. Our findings provide novel evidence that the combination of 5-ASA and HT is a promising approach for the enhancement of apoptosis; it may serve as an effective strategy for treating human OSCC.

Objectives Aging has been suggested to be associated with immune dysregulation. An understanding of alterations in the host immunity with advancing age is, therefore, important for designing immune therapy for elderly cancer patients. In this context, not much is known about age-associated alterations in the immune system in oral cancer. Methods To evaluate age-associated alterations in the immune system, which might affect anti-tumor immune responses in oral cancer, we performed a comparative analysis of the proportion of different immune cells, the proliferative capacity of T cell compartment, and the response against immune therapies targeting immune check point molecules between young and aged oral cancer-bearing mice. Results The proportion of immune regulatory cells, such as regulatory T cells and myeloid derived suppressor cells, was significantly increased in aged mice compared to that in young mice. Moreover, the expression of PD-1 and CTLA-4 on both CD4+ and CD8+ T cells was elevated in aged mice compared to that in young mice, and the proliferative abilities of CD4+ and CD8+ T cells derived from aged mice were significantly reduced following stimulation of T-cell receptors. Moreover, tumor growth was significantly enhanced in aged mice compared to that in young mice. However, immunotherapies targeting PD-1, CTLA-4, and PD-L1 resulted in faster tumor regression in aged mice than in young mice. Conclusions Together, our results indicate that age-associated alterations in the immune system are directly associated with the impairment of anti-tumor immunity in aged mice bearing oral cancer, and might facilitate the progression of the tumor.

Objective Recent studies have demonstrated the pro-tumour role of CD36 in multiple cancer types. However, its role has not been well elucidated in oral squamous cell carcinoma (OSCC). Here, we aimed to evaluate the role of CD36 in proliferation and migration of OSCC cells. Methods Human OSCC cell lines HSC-2, HSC-3, HSC-4 and Ca9-22 were assessed for proliferation by staining with the cell proliferation marker Ki-67. We also assessed migration activity, and the expression of cell adhesion molecules such as E-cadherin and β-catenin and platelet-derived growth factor receptors (PDGFRs) of CD36-positive cells. Results CD36-positive cells showed increased expression of Ki-67 and migration activity compared with CD36-negative cells. Moreover, CD36-positive cells showed reduced expression of E-cadherin and β-catenin, whereas the expression of PDGFRs increased compared with that in CD36-negative cells. Conclusions Our results strongly suggest that CD36 has an important role in facilitating the proliferation and migration activity of OSCC cells, indicating its usefulness in the diagnosis of high-grade tumour and targeted therapy of oral cancer.

Cancer is often associated with dysregulation of both the humoral and cellular immune response, which in some instances is believed to result from changes in immune cell populations. For example, immunosuppressive CD11b(+)Gr-1(+) myeloid-derived suppressor cells have been shown to proliferate in the tumor microenvironment and surrounding tissues, highlighting the relationship between tumor growth and impairment of the immune response. However, the role of myeloid-derived suppressor cells in cancer progression has not been fully characterized because these cells are heterogeneous with properties influenced by the type and location of the tumor. Here, we show that CD11b(+)Gr-1(+) cells are elevated in the peripheral blood, spleen, and tumor of mice with oral squamous cell carcinoma. The phenotype and function of these cells varied depending on the tissue of origin. In particular, CD11b(+)Gr-1(+) cells in tumors expressed PD-L1 more abundantly than those in other tissues. Accordingly, CD11b(+)Gr-1(+) cells from tumors, but not from the spleen, suppressed T cell proliferation in vitro. The results suggest that tumor-derived or immune factors result in the accumulation of phenotypically and functionally diverse populations of CD11b(+)Gr-1(+) cells in mice with oral squamous cell carcinoma. The data also indicate that PD-L1 expression in CD11b(+)Gr-1(+) cells contributes to immune suppression, implying that targeting both myeloid-derived suppressor cells and PD-L1 would be an effective immunotherapeutic strategy against oral cancer.

Background: Although caspase-3 is a key molecule for apoptosis induction, recent evidence has suggested its protumoral role in various human malignancies. The aim of the present study was to investigate the expression of cleaved caspase-3 (the active form of caspase-3) in both clinical samples and cell lines from oral squamous cell carcinomas (OSCCs) and elaborate on its contribution to the protumor role in oral cancer. Methods: The expression of cleaved caspase-3 was immunohistochemically evaluated in samples from 30 patients with OSCCs. The samples were either from biopsies or surgically-resected specimens with a mix of clinical stages and tumor site origins. The expression of cleaved caspase-3 was further examined in three OSCC cell lines. Results: In addition to apoptotic cancer cells, all the cases of OSCCs demonstrated a surprisingly positive expression of cleaved caspase-3. A diffuse, cytoplasmic pattern was particularly prominent in in situ carcinoma cells, invasive carcinoma cells, and metastatic cancer cells that lacked apoptotic morphology. On the other hand, non-neoplastic, normal epithelial cells were completely negative for cleaved caspase-3. In all the OSCC cell lines studied, cleaved caspase-3 was expressed in the cytoplasm and nucleus of cancer cells. Flow cytometric analysis also confirmed that the activation level of caspase-3 in non-apoptotic cancer cells was relatively lower than that in apoptotic cancer cells. Moreover, caspase-3 inhibition by caspase-3 specific inhibitor decreased the proliferation of OSCC cells. Conclusions: Because cleaved caspase-3 is selectively expressed in non-apoptotic OSCC cells and is associated with cell proliferation, these findings implicate caspase-3 signaling in promoting the progression of oral cancer.

Summary Objectives Gemcitabine (GEM) is a pyrimidine nucleoside analogue that is a new chemotherapeutic agent used for treating various cancers. Because accumulating evidence indicates that GEM may activate host immune responses, its potential as an immune modulator in cancer chemotherapy has generated considerable interest. Materials and methods In the present study, we investigated the antitumor effects of GEM using a mouse oral cancer model using immunological analyses. We examined apoptotic cell death of tumor cells with GEM treatment both in vitro and in vivo. We also investigated whether in vivo administration of GEM affected the distributions of immune cells, tumor-cell surface expression levels of immune accessory molecules and T cell immune responses in tumor-bearing mice. Results GEM induced significant oral cancer-cell apoptosis in vitro, and in vivo GEM administration markedly attenuated established mouse tumor growth. In vivo GEM administration decreased the numbers of both myeloid-derived suppressor cells (MDSCs) and B cells in tumor-bearing mice and enhanced dendritic cell maturation. Moreover, GEM treatment upregulated tumor-cell surface expressions of several immune accessory molecules and adhesion molecules, including CD80, CD86, CD40, ICAM-1, VCAM-1, and P-selectin. Remarkably, these tumor cells augmented tumor specific T-cell responses. Conclusion These results suggest that GEM can induce host antitumor immune responses, which would facilitate antitumor effects in the treatment of oral cancer.