ベスナリノン ニヨル ヒト ダエキセンガン サイボウ ノ ゾウショク ヨクセイ キコウ ノ カイセキ

唾液腺癌は化学療法、放射線療法に抵抗性で局所再発、遠隔転移の多い予後不良の悪性腫瘍である。ベスナリノン は強心剤として開発されたが、その副作用である無顆粒球症発症の原因究明の過程で多くの悪性腫瘍細胞に対して分 化・アポトーシス誘導活性を有すること明らかにされ、唾液腺癌を含む多くの悪性腫瘍に対して分化誘導療法が試 みられている。本研究ではベスナリノン処理によりG1 arrestが誘導されるヒト唾液腺癌細胞(TYS)を用いてベスナ リノンによる細胞増殖抑制作用の分子機構の解明を試みた。 解析は(1)ベスナリノン処理TYS細胞より調製したmRNAを用いanti-sense oriented expression cDNA library を構築し、そのlibraryをTYS細胞にトランスフェクション後、ベスナリノン耐性株を選択し、挿入されている cDNA断片を回収するanti-sense expression cloning 法と、(2)上記libraryをランダムにシークェンスし分化、増殖 に関与すると考えられる遺伝子のペスナリノンによる発現誘導を検索し、up-regulationがあれば、その分子を中心に そのシグナルを上流、下流へと追っていくcandidate gene searching 法にて行った。 その結果、anti-sense expression cloning 法にてribosomal protein L21 cDNAを得た。ribosomal protein L21 anti-sense cDNAを強制発現させた細胞はベスナリノンに対する感受性が低下していたが、その作用はおそらくある 種のタンパク合成の阻害によるものであり、ribosomal protein L21タンパクの特異的な発現抑制がベスナリノン抵抗 性の原因とは考え難かった。一方、candidate gene searching 法でマウスあるいはラットの細胞においてtransforming growth factor-β1 (TGF-β1)あるいはfollicle-stimulating hormone (FSH)により誘導される遺伝子として報告されて いるTGF-β1-stimulated clone 22 (TSC-22) cDNA断片を得た。TSC-22遺伝子はTYS細胞においてベスナリノン 処理にて明らかな発現誘導が認められたため、ヒトTSC-22cDNAの全長のクローニング、構造解析、機背繍析を試 みた。5'-RACE変法でopen reading frameを含むほほ完全長1.6kbのヒトTSC-22cDNAを得た。全塩基配列を決 定したところ予想されるアミノ酸配列はマウス、ラットに98.6%の相同性が認められた。またロイシンジッパー構 造が認められたが、その近傍にDNA結合領域あるいは核移行シグナルが存在しないことより、bZIP転写因子と結 合しその転写活性を抑制するdominant negative 転写制卸因子である可能性が示唆された。TSC-22遺伝子はTYS 細胞において細胞密度に依存しその発現が増加し、ベスナリノンはその発現を更に増強した。タンパク合成阻害剤シ クロヘキシミドを用いた実験によりベスナリノンによるTSC-22遺伝子の発現増強は間接作用であることが示唆され た。次にTSC-22の上流、下流のシグナルを解析する目的でTGF-β1、p21WAF1の発現を検索した。ベスナリノン はTYS細胞においてTGF-β1をわずかに誘導し、p21WAF1を著明に誘導した。またp21WAF1の誘導は直接作用 であることが明らかとなった。更にTGF-β1はp21WAF1、TSC-22の発現を誘導した。次にTSC-22アンチセン スオリゴヌクレオチドのTYS細胞に対する影響を検索すると、対数増殖期には全く影響を与えないが、細胞密度が 上がり増殖を止めるべき時期になると細胞増殖促進作用が認められた。またTSC-22アンチセンスオリゴヌクレオチ ドはベスナリノンによるTYS細胞の増殖抑制作用を阻害した。 以上の結果より、ベスナリノンはTYS細胞においてTGF-β1あるいは他のタンパク質を介したTSC-22遺伝子の 誘導、更に直接作用あるいはTGF-β1を介したp21WAF1の誘導により細胞周期をG1期に止め、細胞増殖抑制作 用を示すことが示唆された

Carrier cell-mediated cell lysis of squamous cell carcinoma by squamous cell carcinoma antigen 1 promoter-driven oncolytic adenovirus

The squamous cell carcinoma antigen (SCCA) serves as a serological marker for squamous cell carcinomas. Molecular cloning of the SCCA genomic region has revealed the presence of two tandemly arrayed genes, SCCA1 and SCCA2. We examined the promoter activity of the 5'-flanking proximal region of the SCCA1 gene. Deletion analysis of SCCA1 promoter identified a 175-bp core promoter region and an enhancer region at -525 to -475 bp upstream of the transcription start site. The transcriptional activity of the SCCA1 promoter was up-regulated in squamous cell carcinoma cells, compared with normal keratinocyte, normal non-keratinocyte and adenocarcinoma cells. Five tandem repeats of enhancer increased SCCA1 promoter activity by 4-fold. Oncolytic adenovirus driven by the SCCA1 promoter with 5 tandem repeats of enhancer specifically killed squamous cell carcinoma cells in vitro and in vivo. A549 carrier cells infected with the oncolytic adenovirus induced complete regression of tumor by overcoming immunogenicity and adenovirus-mGM-CSF augmented the antitumor effect of carrier cells. These findings suggest that SCCA1 promoter is a potential target of gene therapy for squamous cell carcinoma

CXCR3+ macrophage in Sjögren's syndrome

Background: Mechanisms underlying immune cells' recruitment and activation into the inflammatory lesions of lip salivary glands (LSGs) from primary Sjögren's syndrome (pSS) patients are incompletely understood. Chemokines play pivotal roles in these processes, so we investigated the clinical significance of chemokine receptor CXCR3 and its ligands in the autoimmune lesions of pSS. Methods: We histologically determined the grade of LSG samples from 22 pSS patients and subjected the samples to immunofluorescence analysis to determine the expressions of CXCR3 and its ligands: CXCL9, CXCL10, and CXCL11. To identify the immune cells expressing CXCR3 in the LSGs, we performed double immunofluorescence analysis using antibodies against CD3 (pan-T cells), CD80 (M1 macrophages), CD163 (M2 macrophage), and CD123 (plasmacytoid dendritic cells: pDCs). The relationship between the grade of lymphocytic infiltration and the number of positively stained cells was analyzed by Spearman's rank correlation test. Results: The expressions of CXCL9 and CXCL10 showed particularly intense staining in the LSG samples' ductal cells. The CXCR3 expression was detected mainly in CD80+ and CD163+ macrophages. The number of CXCR3+CD163+ macrophages inversely correlated with the LSG inflammatory lesions' severity (rs= −0.777, p<0.001). Conclusions: Our results suggest that the enhanced production of CXCL9 and CXCL10 from ductal cells results in the CXCR3+ macrophages' migration. There was an inverse correlation between these two parameters: i.e., the number of CXCR3+CD163+ macrophages decreased as the lymphocytic infiltration grade increased. Although CXCR3 is expressed in all of the innate immune cells, CXCR3+CD163+ M2 macrophages may contribute to the anti-inflammatory functions in pSS lesions

Distinct Regulation of CXCL10 Production in Salivary Gland Cells

CXCL10, a CXC chemokine induced by interferon-gamma [IFN-γ], has been observed in a wide variety of chronic inflammatory disorders and autoimmune conditions. Although CXCL10 is known to be overexpressed in the salivary glands of individuals with primary Sjögren's syndrome (pSS), it is unclear which cells produce CXCL10 under what types of stimulations. Here we investigated the precise molecular mechanisms by which CXCL10 was produced in human salivary gland ductal (NS-SV-DC) and acinar (NS-SV-AC) cell lines. Our results demonstrated that NS-SV-DC cells produced higher levels of CXCL10 compared to NS-SV-AC cells. In addition, our findings demonstrated that the regulator of the enhancement of CXCL10 was different between NS-SV-DC and NS-SV-AC cells; i.e., interferon-gamma (IFN-γ) had more potential than interferon-alpha (IFN-α), tumor necrosis factor (TNF)-α, and interleukin (IL)1-β in the induction of CXCL10 production in NS-SV-DC cells, whereas TNF-α had potential to induce CXCL10 production in NS-SV-AC cells. A Western blot analysis demonstrated that IFN-γ enhanced the production of CXCL10 via both the JAK/STAT1 pathway and the NF-κB pathway in NS-SV-DC cells, whereas TNF-α enhanced the production of CXCL10 via the NF-κB pathway in NS-SV-AC cells. The results of study suggest that the CXCL10 overexpression in the salivary glands is caused mainly by IFN-γ-stimulated salivary gland ductal cells. The enhanced production of CXCL10 by IFN-γ from ductal cells may result in the inflammation of pSS lesions

KRT13 and UPK1B for differential diagnosis between metastatic lung carcinoma from oral squamous cell carcinoma and lung squamous cell carcinoma

Abstract Oral squamous cell carcinomas unusually show distant metastasis to the lung after primary treatment, which can be difficult to differentiate from primary squamous cell carcinoma of the lung. While the location and number of tumor nodules is helpful in diagnosing cases, differential diagnosis may be difficult even with histopathological examination. Therefore, we attempted to identify molecules that can facilitate accurate differential diagnosis. First, we performed a comprehensive gene expression analysis using microarray data for OSCC-LM and LSCC, and searched for genes showing significantly different expression levels. We then identified KRT13, UPK1B, and nuclear receptor subfamily 0, group B, member 1 (NR0B1) as genes that were significantly upregulated in LSCC and quantified the expression levels of these genes by real-time quantitative RT-PCR. The expression of KRT13 and UPK1B proteins were then examined by immunohistochemical staining. While OSCC-LM showed no KRT13 and UPK1B expression, some tumor cells of LSCC showed KRT13 and UPK1B expression in 10 of 12 cases (83.3%). All LSCC cases were positive for at least one of these markers. Thus, KRT13 and UPK1B might contribute in differentiating OSCC-LM from LSCC

Prognostic significance of interleukin-8 and CD163-positive cell-infiltration in tumor tissues in patients with oral squamous cell carcinoma.

PURPOSE:We investigated whether serum interleukin (IL)-8 reflects the tumor microenvironment and has prognostic value in patients with oral squamous cell carcinoma (OSCC). EXPERIMENTAL DESIGN:Fifty OSCC patients who received radical resection of their tumor(s) were enrolled. Preoperative sera were measured for IL-8 by ELISA. Expression of IL-8 and the infiltration of immune cells in tumor tissues were analyzed by an immunohistochemical staining of surgical specimens. RESULTS:We found that disease-free survival (DFS) was significantly longer in the Stage I/II OSCC patients with low serum IL-8 levels compared to those with high levels (p = 0.001). The tumor expression of IL-8, i.e., IL-8(T) and the density of CD163-positive cells in the tumor invasive front, i.e., CD163(IF) were correlated with the serum IL-8 level (p = 0.033 and p = 0.038, respectively), and they were associated with poor clinical outcome (p = 0.007 and p = 0.002, respectively, in DFS) in all patients. A multivariate analysis revealed that N status, IL-8(T) and CD163(IF) significantly affected the DFS of the patients. Further analysis suggested that combination of N status with serum IL-8, IL-8(T) or CD163(IF) may be a new criterion for discriminating between OSCC patients at high and low risk for tumor relapse. Interestingly, the in vitro experiments demonstrated that IL-8 enhanced generation of CD163-positive M2 macrophages from peripheral blood monocytes, and that the cells produced IL-10. CONCLUSIONS:These findings indicate that IL-8 may be involved in poor clinical outcomes via generation of CD163-positive M2 macrophages, and that these factors in addition to N status may have prognostic value in patients with resectable OSCSS

Paclitaxel Potentiates the Anticancer Effect of Cetuximab by Enhancing Antibody-Dependent Cellular Cytotoxicity on Oral Squamous Cell Carcinoma Cells In Vitro

Administration of cetuximab (C-mab) in combination with paclitaxel (PTX) has been used for patients with head and neck squamous cell carcinoma (SCC) clinically. In this study, we attempted to clarify the molecular mechanisms of the enhancing anticancer effect of C-mab combined with PTX on oral SCC cells in vitro. We used two oral SCC cells (HSC4, OSC19) and A431 cells. PTX alone inhibited cell growth in all cells in a concentration-dependent manner. C-mab alone inhibited the growth of A431 and OSC19 cells at low concentrations, but inhibited the growth of HSC4 cells very weakly, even at high concentrations. A combined effect of the two drugs was moderate on A431 cells, but slight on HSC4 and OSC19 cells. A low concentration of PTX enhanced the antibody-dependent cellular cytotoxicity (ADCC) induced by C-mab in all of the cells tested. PTX slightly enhanced the anticancer effect of C-mab in this ADCC model on A431 and HSC4 cells, and markedly enhanced the anticancer effect of C-mab on OSC19 cells. These results indicated that PTX potentiated the anticancer effect of C-mab through enhancing the ADCC in oral SCC cells