Chemokines and cytokines in the tumor microenvironment influence immune cell infiltration and activation. To elucidate their role in immune cell recruitment during oral cancer development, we generated a mouse tongue cancer model using the carcinogen 4-nitroquinoline 1-oxide (4NQO) and investigated the carcinogenetic process and chemokine/cytokine gene expression kinetics in the mouse tongue. C57/BL6 mice were administered 4NQO in drinking water, after which tongues were dissected at 16 and 28 weeks and subjected to analysis using the RT2 Profiler PCR Array, qRT-PCR, and pathologic and immunohistochemical analyses. We found that Th1-associated chemokine/cytokine (Cxcl9, Cxcl10, Ccl5, and Ifng) and Treg-associated chemokine/cytokine (Ccl17, Ccl22, and Il10) mRNA levels were simultaneously increased in premalignant lesions of 4NQO-treated mice at 16 weeks. Additionally, although levels of Gata3, a Th2 marker, were not upregulated, those of Cxcr3, Ccr4, and Foxp3 were upregulated in the tongue tissue. Furthermore, immunohistochemical analysis confirmed the infiltration of CD4+, CD8+, and Foxp3+ cells in the tongue tissue of 4NQO-treated mice, as well as significant correlations between Th1- or Treg-associated chemokine/cytokine mRNA expression and T cell infiltration. These results indicate that CD4+, CD8+, and Foxp3+ cells were simultaneously recruited through the expression of Th1- and Treg-associated chemokines in premalignant lesions of 4NQO-induced mouse tongue tissue.

Combined treatment of human oral squamous cell carcinoma (OSCCs) with DNA methyltransferase inhibitors (DNMTis), histone methyltransferase inhibitors (HMTis), and histone deacetylase inhibitors (HDACis), and the molecular mechanisms underlying their anticancer effects, have not been fully elucidated. Herein, we investigated the cytotoxic effects of combined DNMTis (5-Aza-deoxycytidine: 5-Aza-dC, RG108), HMTis (3-deazaneplanocin A: DZNep), and HDACis (trichostatin A: TSA) treatment on human OSCC cells and explored their molecular mechanisms. Combined 5-Aza-dC, or RG108, and TSA treatment significantly decreased HSC-2 and Ca9-22 cell viability. Combinatorial DZNep and TSA treatment also decreased Ca9-22 cell viability. Although caspase 3/7 activation was not observed in HSC-2 cells following combined treatment, caspase activity was significantly increased in Ca9-22 cells treated with DZNep and TSA. Moreover, combined treatment with 5-Aza-dC, RG108, and TSA increased the proportion of HSC-2 and Ca9-22 cells in the S and G2/M phases. Meanwhile, increased phosphorylation of the histone variant H2A.X, a marker of double-stranded DNA breaks, was observed in both cells after combination treatment. Hence, the decreased viability induced by combined treatment with epigenomic inhibitors results from apoptosis and cell cycle arrest in S and G2/M phases. Thus, epigenomic therapy comprising combined low concentrations of DNMTi, HMTi, and HDACi is effective against OSCC.

Chemokines are a group of cytokines involved in the mobilization of leukocytes, which play a role in host defense and a variety of pathological conditions, including cancer. Interferon (IFN)-inducible chemokines C-X-C motif ligand 9 (CXCL), CXCL10, and CXCL11 are anti-tumor chemokines; however, the differential anti-tumor effects of IFN-inducible chemokines are not completely understood. In this study, we investigated the anti-tumor effects of IFN-inducible chemokines by transferring chemokine expression vectors into a mouse squamous cell carcinoma cell line, SCCVII, to generate a cell line stably expressing chemokines and transplanted it into nude mice. The results showed that CXCL9- and CXCL11-expressing cells markedly inhibited tumor growth, whereas CXCL10-expressing cells did not inhibit growth. The NH2-terminal amino acid sequence of mouse CXCL10 contains a cleavage sequence by dipeptidyl peptidase 4 (DPP4), an enzyme that cleaves the peptide chain of chemokines. IHC staining indicated DPP4 expression in the stromal tissue, suggesting CXCL10 inactivation. These results suggest that the anti-tumor effects of IFN-inducible chemokines are affected by the expression of chemokine-cleaving enzymes in tumor tissues.