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Protease-activated receptor 1 (PAR1) is known as a thrombin receptor. Recent studies have reported PAR1 expression in various malignancies; however, its role in oral squamous cell carcinoma (OSCC) requires clarification. A previous study showed that down-regulation of ΔNp63, a homolog of p53, augments PAR1 expression in OSCC. In the present study, the association of PAR1 expression with clinicopathological findings in OSCC was examined retrospectively. Expression of PAR1, thrombin, and ΔNp63 was examined immunohistochemically in OSCC specimens. Patients were divided into three groups based on the expression pattern of PAR1 at the invasive front: group A, PAR1-negative in both cancer and stromal cells; group B, positive in stromal cells but negative in cancer cells; group C, positive in both cancer and stromal cells. Histologically high-grade tumours were significantly more common in group C. Patients in group C had the highest incidence rate of nodal metastasis (P

Objective Cytokeratin (CK) 19 is a member of the acidic type I CK family. Recently, CK19 expression has been found in various tumor tissues; however, the significance of this remains unknown. The purpose of this study is to clarify the roles of CK19 in the progression of oral squamous cell carcinoma (OSCC). Methods A total of 100 patients who had been diagnosed with OSCC at our department between January 2011 and December 2016 was included. The patients were divided into three groups based on an optimal cut-off points (5% and 77%) of the labeling index (LI) as follows: group A; LI Results Histologically high-grade tumors were significantly more common in group C than in groups A and B. Furthermore, the incidence of nodal metastasis was significantly higher in group C than in other groups. Intense CK19 immunoreactivity was detected in metastatic lymph nodes of groups B and C, but not from group A. Moreover, patients with advanced pN stage and extranodal extension were more common in groups B and C than group A. Disease-specific survival curves revealed poorer prognoses in group C. Conclusions These results suggest that CK19 is involved in OSCC invasion and metastasis and could be a novel biomarker of highly invasive OSCC with metastatic potential.

Gestational nutrition is widely recognized to affect an offspring’s future risk of lifestyle-related diseases, suggesting the involvement of epigenetic mechanisms. As folic acid (FA) is a nutrient essential for modulating DNA methylation, we sought to determine how maternal FA intake during early pregnancy might influence tumor sensitivity in an offspring. Dams were maintained on a FA-depleted (FA(−)) or normal (2 mg FA/kg; FA(+)) diet from 2 to 3 days before mating to 7 days post-conception, and their offspring were challenged with chemical tumorigenesis using 7,12-dimethylbenz[a)anthracene and phorbol 12-myristate 13-acetate for skin and 4-nitroquinoline N-oxide for tongue. In both squamous tissues, tumorigenesis was more progressive in the offspring from FA(−) than FA(+) dams. Notably, in the skin of FA(−) offspring, the expression and activity of cylindromatosis (Cyld) were decreased due to the altered DNA methylation status in its promoter region, which contributed to increased tumorigenesis coupled with inflammation in the FA(−) offspring. Thus, we conclude that maternal FA insufficiency during early pregnancy is able to promote neoplasm progression in the offspring through modulating DNA methylation, such as Cyld. Moreover, we propose, for the first time, “innate” utero nutrition as the third cause of tumorigenesis besides the known causes—hereditary predisposition and acquired environmental factors.

Collagen type XVII (COL17) is expressed in various tissues and its aberrant expression is associated with tumour progression. In this study, we investigated the regulation of COL17 expression in oral squamous cell carcinoma (OSCC) using the cell lines NA, SAS, Ca9-22, and Sa3. COL17 was induced upon p53 activation by cisplatin in SAS; however, this effect was more limited in NA and hardly in Ca9-22 and Sa3, with mutated p53. Moreover, COL17 was found to be regulated by miR203a-3p in all cell lines. Our data suggest that COL17 expression in OSCC cell lines is regulated by p53 and miR203a-3p.

We previously revealed that epithelial-to-mesenchymal transition (EMT) was mediated by ΔNp63β, a splicing variant of ΔNp63, in oral squamous cell carcinoma (OSCC). Recent studies have highlighted the involvement of microRNA (miRNA) in EMT of cancer cells, though the mechanism remains unclear. To identify miRNAs responsible for ΔNp63β-mediated EMT, miRNA microarray analyses were performed by ΔNp63β-overexpression in OSCC cells; SQUU-B, which lacks ΔNp63 expression and displays EMT phenotypes. miRNAs microarray analyses revealed miR-205 was the most up-regulated following ΔNp63β-overexpression. In OSCC cells, miR-205 expression was positively associated with ΔNp63 and negatively with zinc-finger E-box binding homeobox (ZEB) 1 and ZEB2, potential targets of miR-205. miR-205 overexpression by miR-205 mimic transfection into SQUU-B cells led to decreasing ZEB1, ZEB2, and mesenchymal markers, increasing epithelial markers, and reducing cell motilities, suggesting inhibition of EMT phenotype. Interestingly, the results opposite to this phenomenon were obtained by transfection of miR-205 inhibitor into OSCC cells, which express ΔNp63 and miR-205. Furthermore, target protector analyses revealed direct regulation by miR-205 of ZEB1 and ZEB2 expression. These results showed tumor-suppressive roles of ΔNp63β and miR-205 by inhibiting EMT thorough modulating ZEB1 and ZEB2 expression in OSCC.

We previously reported that epithelial-to-mesenchymal transition (EMT) was mediated by ΔNp63β in oral squamous cell carcinoma (OSCC). In this study, DNA microarray analyses were performed using ΔNp63β-overexpressing OSCC cells to identify genes associated with ΔNp63β-mediated EMT. Thereby, we focused on kallikrein-related peptidase (KLK) 6, most up-regulated following ΔNp63β-overexpression, that activates protease-activated receptors (PARs). In RT-PCR analyses, ΔNp63 was positively associated with KLK6 and PAR2 and negatively with PAR1 in OSCC cells. By ΔNp63 knockdown, KLK6 and PAR2 expression was decreased and PAR1 was increased. Furthermore, KLK6 knockdown led to enhancing migration and invasion, and inhibiting proliferation, suggesting EMT-phenotypes. Although, in the KLK6 or PAR2 knockdown cells, phosphorylation of ERK was reduced, it was restored in the KLK6 knockdown OSCC cells treated with recombinant KLK6 proteins. Immunohistochemistry showed ΔNp63, KLK6, and PAR2 were more strongly expressed in the epithelial dysplasia and central region of OSCC than normal oral epithelium, whereas PAR1 expression was undetectable. Interestingly, at the invasive front of OSCC, ΔNp63, KLK6, and PAR2 were reduced, but PAR1 was elevated. In addition, the OSCC patients with decreasing KLK6 expression at the invasive front had more unfavourable prognosis. These results suggested differential roles of KLK6 in malignant transformation and EMT; high ΔNp63β expression up-regulates KLK6-PAR2 and down-regulates PAR1, inducing malignant transformation in oral epithelium with stimulating proliferation through ERK signal activation. Moreover, KLK6-PAR2 expression is down-regulated and PAR1 is up-regulated when ΔNp63β expression is decreased, leading to EMT with enhancing migration and invasion through ERK signal reduction at the invasive front.

Oral squamous cell carcinoma (OSCC) constitutes over 90% of all cancers in the oral cavity. The prognosis for patients with invasive OSCC is poor; therefore, it is important to understand the molecular mechanisms of invasion and subsequent metastasis not only to prevent cancer progression but also to detect new therapeutic targets against OSCC. Recently, extracellular vesicles-particularly exosomes-have been recognized as intercellular communicators in the tumor microenvironment. As exosomic cargo, deregulated microRNAs (miRNAs) can shape the surrounding microenvironment in a cancer-dependent manner. Previous studies have shown inconsistent results regarding miR-200c-3p expression levels in OSCC cell lines, tissues, or serum-likely because of the heterogeneous characters of the specimen materials. For this reason, single-cell clone analyses are necessary to effectively assess the role of exosome-derived miRNAs on cells within the tumor microenvironment. The present study utilized integrated microarray profiling to compare exosome-derived miRNA and exosome-treated cell-derived mRNA expression. Data were acquired from noninvasive SQUU-A and highly invasive SQUU-B tongue cancer cell clones derived from a single patient to determine candidate miRNAs that promote OSCC invasion. Matrigel invasion assays confirmed that hsa-miR-200c-3p was a key pro-invasion factor among six miRNA candidates. Consistently, silencing of the miR-200c-3p targets, CHD9 and WRN, significantly accelerated the invasive potential of SQUU-A cells. Thus, our data indicate that miR-200c-3p in exosomes derived from a highly invasive OSCC line can induce a similar phenotype in non-invasive counterparts.

15-Deoxyspergualin (DSG) is an immunosuppressive agent being clinically used. Unlike tacrolimus and cyclosporine A, it does not inhibit the calcineurin pathway, and its mechanism of action and target molecule have not been elucidated. Therefore, we previously prepared biotinylated derivative of DSG (BDSG) to fish up the target protein. In the present research, we identified poly(rC) binding protein 2 (PCBP2) as a DSG-binding protein using this probe. DSG was confirmed to bind to PCBP2 by pull-down assay. Intracellular localization of PCBP2 was changed from the nucleus to the cytoplasm by DSG treatment. DSG inhibited the cell growth, and over-expression of PCBP2 reduced the anti-proliferative activity of DSG. PCBP2 is known to regulate various proteins including STAT1/2. Thus, we found PCBP2 as the first target protein of DSG that can explain the immunosuppressive activity.

Emerging evidence indicates that cancer-derived exosomes increase the tumorigenic potential of tumor cells by reprogramming the cells associated with the tumor microenvironment. Our aim was to examine the cross talk via exosomes between two oral squamous cell carcinoma (OSCC) clones from the same patient. Our data showed that exosomes derived from highly metastatic SQUU-B cells conferred metastatic ability to nonmetastatic SQUU-A cells and subsequently reduced mRNA expression of cytokeratin 13, which is strongly linked to malignant transformation of OSCCs. The results suggest that multiple cell clones secrete their unique exosomes within the malignant tumor mass, which mediate paracrine interactions with other cell clones and affect clinical prognosis.

Extracellular matrix (ECM) components are critical for all aspects of cell proliferation, adhesion, and morphological alteration. Recent progress has yielded multiple molecular drugs that specifically target gene products which are expressed at high levels in tumor cells. We investigated whether the sensitivity of tumor cells to molecular target drugs could be altered when cells were cultured on surfaces with various coating conditions such as lysine, laminin, Matrigel, collagen type I, and human fibronectin (HFN). This study evaluates the IC50 values of imatinib in oral squamous cell carcinoma (OSCC) cell lines when cells are cultured on plates coated with ECM components such as collagen type I and HFN. Four OSCC cell lines—SQUU-A, SQUU-B, SAS, and NA— are used. Cell proliferation was assessed using WST-8 reagent. Collagen type I and HFN significantly enhanced OSCC cell proliferation compared with control. Imatinib cytotoxicity was demonstrated following culture of OSCCs in culture plates coated with collagen type I or HFN. However, there were no significant changes in imatinib IC50 values between collagen type I and HFN. These results indicate that some molecular target drugs exhibit cancer cell cytotoxicity without being influenced by cell environment factors such as the ECM. These results may aid in the search for molecular target drugs to apply in the clinical chemotherapy of OSCC.

A new class of a dual inhibitor of Aurora kinase and tubulin polymerization was created by introducing various substituted phenoxyethylamino or pyridyloxyethylamino groups to the 2-position of 3-cyano-4-methyl-6-(5-methyl-3-pyrazoloamino)-pyridine. Compound 3g exhibited Aurora kinase inhibition, excellent protein kinase selectivity to Aurora kinase in comparison with 66 other kinases, inhibition of phosphorylation of Ser10 of histone H3 as an Aurora kinase inhibitor, inhibition of tubulin polymerization in vitro, good cell membrane permeability, and a good PK profile. Therefore compound 3g was effective in some antitumor mouse models at a dose of 30 mg/kg po qd.

Oral squamous cell carcinoma (OSCC) is often associated with lymphatic rather than hematogenous metastasis; however, a determinant factor for this process has not been elucidated. This study examined the effect of OSCC-derived exosomes on angiogenesis and lymphangiogenesis, closely related with hematogenous and lymphatic metastasis, respectively. Our data demonstrated that OSCC-derived exosomes stimulated the expression of VEGFs and their receptors, essential regulators of angiogenesis and lymphangiogenesis, in human lymphatic endothelial cells but not in human vein endothelial cells. These results suggest that specific exosomes have differential tropism toward a certain cell type, defining the modality of metastasis.

Bone deficiency causes osteoporosis and often decreases quality of life in patients with rheumatoid arthritis. Estrogens are known to protect elderly women from bone loss. Synthesis of new estradiol–bisphosphonate conjugates (E 2 –BPs) was accomplished and their in vivo activity as bone-specific estrogens were examined. Among them, MCC-565 showed selective estrogenic activity in bones; but it showed little estrogenic activity in the uterus. We also found that the linker moiety in E 2 –BPs was essential for the absorption and specificity of the conjugates.

Design and synthesis of 15-deoxyspergualin–biotin conjugates as novel binding probes for target protein screening

A new class of Aurora A kinase inhibitor was created by transforming 4-(5-methyl-3-pyrazoloamino)pyrimidine moiety of VX-680 to 3-cyano-6-(5-methyl-3pyrazoloamino)pyridine. Compound 6 exhibited a potent Aurora A kinase inhibitory activity, excellent selectivity to Aurora B kinase and other 60 kinases, good cell permeability and good PK profile. Therefore compound 6 was effective in antitumor mice model at a dose of 30 mg/kg po qd without decrease of body weight.

3-Deoxy-1,2-O-isopropylidene-α-D-glycero-D-erythro-pentofuranose (11) was stereoselectively converted into the Witting salt 8 corresponding to the C-1–C-6 portion of amphotericin B aglycon (1) in 57% overall yield in 8 steps. The aldehydic segment 9 or 10 corresponding to the C-7–C-12 portion of 1 was also derived in 6 steps from 11 in 53 or 38% overall yield, respectively. Wittig condensation of 8 with 9 or 10 followed by four-step conversion afforded the C-1–C-12 segment, (3S,5R,8R,9R,11S)-1-O-t-butyldimethylsilyl-12-iodo-3,5:8,9-di-O-isopropylidene-11-O-[(2-methoxyethoxy)methyl]-1,3,5,8,9,11-dodecanehexol(4)or(2S,4R,5R,8R,10S)-1,2-anhydro-12-O-t-butyldimethylsilyl-4,5:8,10-di-O-isopropylidene-1,2,4,5,8,10,12-dodecaneheptol (5) in 26 or 15% overall yield from 11, respectively.

The title compound, (3S,4S,5S,9S,11R,12R,15R,17S)-4-[(t-butyldimethylsilyloxy)methyl]-1,3,5,9,11,12,15,17,19-nonahydroxy-3,5:11,12:15,17-tri-O-isopropylidene-19-O-(4-methoxybenzyl)-9-O-[(2-methoxyethoxy)methyl]-7-nonadecanone (1) was effectively synthesized through the coupling of (3S,5R,8R,9R,11S)-12-iodo-3,5:8,9-di-O-isopropylidene-1-O-(4-methoxybenzyl)-11-O-[(2-methoxyethoxy)methyl]-1,3,5,8,9,11-dodecanehexol (2) and (3S,4R,5S)-4-[(t-butyldimethylsilyloxy)methyl]-3,5-O-isopropylidene-1,1-bis(methylthio)-1-trimethylstannyl-6-heptene-3,5-diol (36). The C-13–C-19 segment 36 was enantiospecifically synthesized from 1,2:5,6-di-O-isopropylidene-α-D-thero-D-glycero-3-hexofuranosulose (7) via (2S,4R,5S)-5,6-epoxy-3-[(t-butyldimethylsilyloxy)methyl]-1-hexen-3-ol (6a) in 11.6% overall yield.