ヒト胃癌細胞株におけるp53遺伝子の構造異常解析

取得学位 : 博士（医学）, 学位授与番号 : 医博甲第1050号, 学位授与年月日：平成4年3月25日,学位授与年：199

胃癌におけるCOX-2発現の意義とCOX-2阻害剤による転移抑制に関する研究

金沢大学附属病院a)臨床検体をもちいた胃癌組織におけるCOX-2の発現切除胃癌80例をもちい、抗COX-2モノクローナル抗体にて免疫組織学的に検討したところ、72.5%の胃癌原発巣にCOX-2の発現を認めたが非癌部の粘膜上皮にはその発現を認めなかった。ただし、COX-2発現の有無と臨床病理学的因子との相関は認めなかった。b)COX-2阻害剤による血管新生抑制効果受精鶏卵の漿尿膜上にCOX-2阻害剤を吸着させたディスクをのせ孵卵したのち、その血管新生抑制効果を検討した。COX-2阻害剤としてNSAIDsの1つであるIndomethacinおよび特異的阻害剤JTE-522をもちいたところ、血管新生を濃度依存性に抑制したが、本来血管新生抑制剤であるTNP-470に比べ、高濃度の条件を必要とした。c)胃癌培養細胞株におけるCOX-2の発現とCOX-2阻害剤による増殖抑制効果ウェスタンブロット法にてCOX-2の発現を検討し、高発現株としてMKN45、低発現株としてMKN28を同定した。これら2株をもちい、IndomethacinおよびJTE-522による増殖抑制効果を検討した。in vitroとしてMTT assay法をもちいたところ、IndomethacinおよびJTE-522は濃度依存性に増殖を抑制したが、その抑制効果は高発現株であるMKN45において顕著であった。また、in vivoとしてヌードマウスの皮下に両細胞株を移植した後、COX-2阻害剤を投与したところ、Indomethacinでは高発現株であるMKN45のみ増殖を抑制したが、JTE-522では増殖抑制効果は明らかでなかった。以上より、胃癌においてCOX-2は高率に発現しているものの、臨床病理学的にはその働きは明らかでなかった。わずかながら血管新生に関与している可能性があるが、特異的阻害剤では増殖抑制効果は少なく、Indomethacinによる効果はCOX-2を阻害する以外のメカニズムが関与しているものと考えられた。研究課題/領域番号:11770691, 研究期間(年度):1999-2000出典：「胃癌におけるCOX-2発現の意義とCOX-2阻害剤による転移抑制に関する研究」研究成果報告書　課題番号11770691（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （ https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-11770691/）を加工して作

胃癌腹膜転移における診断法の開発とアンギオテンシン系を介した線維化機構とその制御

(1)臨床例における腹膜転移(再発)の早期診断腹膜播種が確定している症例において血清IV型コラーゲン値は全例高値であったが、腹膜再発や再燃の過程において画像診断や臨床症状、既存の血清腫瘍マーカーと比較し、早期診断に有用かどうかを検討した。現在、腹膜転移を有する13症例を追跡しているが、いずれの症例においても症状の進行とともに血清IV型コラーゲン値は上昇している。また、画像診断や臨床症状、腫瘍マーカーの上昇が明確でない腹膜再発高危険症群のなかに血清IV型コラーゲン値の上昇してきた症例が5例存在しており、うち3例は腹腔鏡検査において腹膜再発と診断された。残り2例はcut off値付近を推移しており経過観察中である。(2)胃癌細胞株における検討胃癌におけるアンギオテンシン1型受容体についての検討In vitroにおいてヒト胃癌細胞株5株中4株にアンギオテンシン1型受容体(AT1)が発現しており、アンギオテンシンIIの添加によりその増殖率は亢進した。また、この増殖率亢進はAT1の拮抗剤であるcandesartanにより抑制された。このAT1を介した増殖率亢進の機序は、ERKのリン酸化に伴う増殖シグナルおよびNF-kBの活性化に伴うsurvivinの誘導による抗アポトーシス効果であることを証明した。ヌードマウスに高度腹膜転移株OCUM-2MD3を1x107個腹腔内投与したところ、移植4週目には癌性腹水を伴う腹膜播種が形成されたが、組織学的には腫瘍の線維化はそれほど顕著ではなかった。そこで、AT1を介した臓器の線維化についての検討は断念し、AT1を介した腫瘍増殖に対する分子標的治療の可能性についてin vivoで検討した。移植1週間後からcandesartan 10mg/kgを連日経口投与した群はコントロール群と比較し、有意に生存率の延長を認めた。以上より、胃癌腹膜転移の早期診断に血清IV型コラーゲン値測定が有用である可能性が示された。また、胃癌の増殖および線維化にはAT1を介する経路が存在し、その拮抗剤であるcandesartan(ブロプレス:降圧剤)をもちいた分子標的治療の可能性が示された。Evaluation of the serum type IV collagen for early diagnosis of peritoneal metastases (recurrence)The purpose of this study is to determine whether the type IV collagen, which is marker of hepatic fibrosis, could be useful biomarker for peritoneal metastasis. Type IV collagen was measured in 112 patients who were diagnosed wit gastric cancer : early cancer 55 (Group A), advanced cancer 43 (Group B), patients with peritoneal metastasis 27 (Group C). And they were compared with conventional biomarker (CEA, CA19-9, CAl25). The median type IV collagen levels in Group C was significantly higher than Group A and B (p<0.0001). In the clinical course of 13 patients with peritoneal metastasis, all of the serum type IV collagen levels were increasing according to their tumor progression. Furthermore, 3 of 5 patients, who were in the high risk group for peritoneal recurrence, were found peritoneal recurrence by laparoscopy without clinical, radiological, biochemical symptoms. These findings sugge st that the serum type IV collagen is a useful marker for early diagnosis of peritoneal metastasis with gastric cancer.4Angiotensin II activates MAP kinase and NF-kB through angiotensin II type I receptor in gastric cancer cellsFour of 5 gastric cancer cell lines were found angiotensis II type 1 (AT1) receptor expression using western blot analysis. Angiotensin II stimulated growth of AT1 positive gastric cancer cell and this proliferative response was inhibited by candesartan, a specific AT1 receptor antagonist. Angiotensin II plays a role in the growth of AT1 positive gastric cancer cells through MAP kinase activation by ERK1/2 phosphorylation and surviving induction as antiapoptotic molecule by NF-kB activation.One of the AT1 receptor expressing cell lines, OCUM2MD3, which had a very high peritoneal metastatic potential, was used as in vivo experiment. OCUM2MD3 made peritoneal metastasis including massive ascites and metastatic nodules 4weeks after intraperitoneal injection of 1×107 cells in nude mice. To elucidate whether over-expression of AT1 correlate to tumor progression, AT1 receptor antagonist, candesartan, has been treated everyday from 1 week after tumor injection. The candesartan group showed a significant longer survival than control group. These finding suggest that AT1 activation correlates tumor progression and its receptor antagonist may be a candidate for the molecule targeting therapy.研究課題/領域番号:17591383, 研究期間(年度):2005-2006出典：「胃癌腹膜転移における診断法の開発とアンギオテンシン系を介した線維化機構とその制御」研究成果報告書　課題番号17591383 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

Effects of valproic acid on the cell cycle and apoptosis through acetylation of histone and tubulin in a scirrhous gastric cancer cell line

Background. Management of peritoneal dissemination is the most critical problem in gastric cancer. This study was performed to investigate the inhibitory effects of valproic acid (VPA) on a highly peritoneal-seeding cell line of human scirrhous gastric cancer, OCUM-2MD3, and to explore the mechanism and the potential of VPA. Methods. The effects of VPA on the growth of OCUM-2MD3 cells were assessed by MTT assay. In addition, paclitaxel (PTX) was combined with VPA to evaluate their synergistic effects. HDAC1 and HDAC2 expression were evaluated by western blotting in OCUM-2MD3 cells and other gastric cancer cell lines (TMK-1, MKN-28). The acetylation status of histone H3 and α-tubulin after exposure to VPA were analyzed by western blotting. The activities of cell cycle regulatory proteins and apoptosis-modulating proteins were also examined by western blotting. The effects of VPA in vivo were evaluated in a xenograft model, and apoptotic activity was assessed by TUNEL assay. Results. OCUM-2MD3 cells showed high levels of HDAC1 and HDAC2 expression compared with TMK-1 and MKN-28. The concentration of VPA required for significant inhibition of cell viability (P < 0.05) was 5 mM at 24 h and 0.5 mM at 48 h and 72 h. The inhibition of VPA with PTX showed dose-dependent and combinatorial effects. VPA increased acetyl-histone H3, acetyl α-tubulin, and p21WAF1 levels accompanied by upregulation of p27, caspase 3, and caspase 9, and downregulation of bcl-2, cyclin D1, and survivin. In the xenograft model experiment, the mean tumor volume of the VPA-treated group was significantly reduced by 36.4%, compared with that of the control group at 4 weeks after treatment (P < 0.01). The apoptotic index was significantly higher in the VPA-treated group (42.3% ± 3.5%) than in the control group (7.7% ± 2.5%) (P < 0.001). Conclusions. VPA induced dynamic modulation of histone H3 and α-tubulin acetylation in relation with the anticancer effect and the enhancement of PTX in the OCUM-2MD3 cell line. Therefore, VPA in combination with PTX is expected to be a promising therapy for peritoneal dissemination of scirrhous gastric cancer. © 2010 Yagi et al; licensee BioMed Central Ltd

Gene amplification of CCNE1, CCND1, and CDK6 in gastric cancers detected by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization

New and effective treatments for advanced gastric cancer are urgently needed. Cyclins E and D1 form a complex with cyclin-dependent kinase 2, 4, or 6 to regulate G1-S transition. The G1-S regulatory genes encoding cyclin E (CCNE1), cyclin D1 (CCND1), and CDK6 (CDK6) are frequently amplified in gastric cancer and may therefore influence molecularly targeted therapies against ERBB2 or EGFR when coamplified. A total of 179 formalin-fixed and paraffin-embedded gastric cancer specimens were examined for these gene amplifications by multiplex ligation-dependent probe amplification and fluorescence in situ hybridization. Amplification of at least 1 G1-S regulatory gene was found in 35 tumors (CCNE1 amplification, 15% of samples; CCND1, 6%; CDK6, 1%). In 13 of the 35 tumors, dual-color fluorescence in situ hybridization identified coamplification of the G1-S regulatory genes with ERBB2, EGFR, and/or KRAS in single cancer nuclei. The observation that cells with G1-S regulatory gene amplification contained clonal subpopulations with coamplification of ERBB2, EGFR, or KRAS in 5 early and 3 advanced cancers suggests that amplification of the G1-S regulatory genes represents an early event, which precedes ERBB2, EGFR, or KRAS amplification. Amplified CCNE1, CCND1, and CDK6 in advanced gastric cancer may be potentially useful as direct targets for molecular therapy or for combination therapy with ERBB2 or EGFR inhibitors. Multiplex ligation-dependent probe amplification could be a useful tool for identification of patients who would benefit from such therapies. © 2016 Elsevier Inc.Embargo Period 12 month

Semi-comprehensive analysis of gene amplification in gastric cancers using multiplex ligation-dependent probe amplification and fluorescence in situ hybridization

The prognosis of patients with gastric carcinomas at an advanced stage still remains dismal, and therefore novel therapeutic modalities are urgently needed. Since the successful targeting of amplified ERBB2 with a humanized monoclonal antibody, the amplified genes of other receptor tyrosine kinases such as EGFR, FGFR2, and MET, as well as those of other cell regulator genes, are being considered as candidate targets of molecular therapy. The aim of the present study was to determine the amplification status of 26 genes, which are frequently amplified in solid cancers, in advanced gastric cancers. A total of 93 formalin-fixed and paraffin-embedded advanced gastric cancer tissues were examined by multiple ligation-dependent probe amplification, and 32 cases with ‘gain’ or ‘amplified’ status of 16 genes were further examined for the respective gene amplification by fluorescence in situ hybridization (FISH) and for the respective protein overexpression by immunohistochemistry. The frequencies of gene amplifications in advanced gastric cancers were as follows: ERBB2 (13 cases, 14%), FGFR2 (7 cases, 8%), MYC (7 cases, 8%), TOP2A (7 cases, 8%), MET (4 cases, 4%), MDM2 (4 cases, 4%), CCND1 (3 cases, 3%), FGF10 (2 cases, 3%), and EGFR (1 case, 1%). Amplification of the receptor tyrosine kinases genes occurred in a mutually exclusive manner except for one tumor in which ERBB2 and FGFR2 were both amplified but in different cancer cells. Co-amplification of ERBB2 and MYC, and EGFR and CCND1, in single nuclei but on different amplicons, was confirmed in one case each. Attempts at correlating the FISH status with the immunohistochemical staining pattern showed variable results from complete concordance to no correlation. In conclusion, combination of multiple ligation-dependent probe amplification and FISH analysis is a feasible approach for obtaining the semi-comprehensive genetic information that is necessary for personalized molecular targeted therapy.Modern Pathology advance online publication, 6 March 2015; doi:10.1038/modpathol.2015.33

Potential of extravasated platelet aggregation as a surrogate marker for overall survival in patients with advanced gastric cancer treated with preoperative docetaxel, cisplatin and S-1: a retrospective observational study

Background: The theory of extravasated platelet aggregation in cancer lesions was recently introduced. We investigated the association of platelet aggregation in gastric cancer stroma with clinicopathological features, chemotherapeutic response, pathological response, and survival. Methods: The study comprised 78 patients with advanced gastric cancer who had undergone gastrectomy with or without combination of docetaxel, cisplatin and S-1 (DCS) as preoperative chemotherapy between 2005 and 2014. The patients were divided into two groups: patients who had received preoperative DCS therapy forming the p-DCS group and patients who had not received preoperative DCS therapy forming the control group. The 39 patients in the control group had received gastrectomy and postoperative chemotherapy of S-1 alone. Platelet aggregation in biopsy specimens before preoperative DCS therapy in the p-DCS group and at the time of diagnosis in the control group were evaluated using CD42b immunohistochemical staining. Results: Twenty-four patients in the p-DCS group and 19 in the control group were found to have platelet aggregation in their cancer stroma. Patients with histologically confirmed platelet aggregation had significantly higher rates of chemoresistance (58.3%) than those without platelet aggregation (20.0%) (P = 0.019). According to multivariate analysis, CD42b expression (odds ratio: 5.102, 95% confidence interval: 1.039-25.00, P = 0.045) was correlated with chemoresistance. CD42b expression and histological non-responder status were both significantly correlated with poor overall survival (OS) (P = 0.012, P = 0.016); however, RECIST was not correlated with OS. In the control group, CD42b expression was also significantly correlated with poor overall survival (OS) (P = 0.033). In the p-DCS group, according to multivariate analysis, male sex (hazard ratio: 0.281, 95% confidence interval: 0.093-0.846, P = 0.024) was correlated with good prognosis and CD42b expression (hazard ratio: 4.406, 95% confidence interval: 1.325-14.65, P = 0.016) with poor prognosis. Conclusions: This study suggests that platelets in gastric cancer stroma may create a favorable microenvironment for chemoresistance. CD42b immunohistochemical staining of biopsy specimens is a promising candidate for being a prognostic marker in patients with gastric cancer. © 2017 The Author(s)