Identification of signal transduction that induces anticancer drug resistance for clinical application

诱导抗癌耐药性的信号转导的鉴定用于临床应用

• 批准号: 18590656
• 负责人: NAKANO Shuji
• 金额: $ 2.52万
• 依托单位: Nakamura Gakuen College (2007)Kyushu University (2006)
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2006
• 资助国家: 日本
• 起止时间: 2006 至 2007
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-18590656/
• 关键词: Anticancer drug resistance; signal transduction; molecular target; EGFR; Bax; Tyrosine kinase

Although gefitinib, a selective inhibitor of epidermal growth factor receptor (EGFR) tyrosine kinase, has been clinically demonstrated to be effective for certain cancer cell types, the molecular mechanisms of the anti-tumor activity have not been fully elucidated. In this study, we investigated the mechanism of gefitinib-induced growth inhibition and apoptosis in HAG-1 human gallbladder adenocarcinoma cells. Treatment of gefitinib at a dose of 1 mM resulted in a significant growth inhibition, and the cell number irreversibly declined after 72-h incubation, with a progressive expansion of apoptotic cell population over 120-h. Following 2-h treatment, gefitinib significantly inhibited EGFR autophosphorylation and subsequent downstream signaling pathway through Erk and Akt, and induced accumulation of cells in the G0/G1 phase of the cell cycle at 24-h, accompanied by a concomitant increase in p21 transcript and increased expression of p27. Gefitinib did not affect the amount of total and … More phosphorylated p53 at serine 15, but upregulated the expression of total Bax, with subsequent increase in p18 Bax, an active form of Bax. The expression of Bcl-2 and Bad was unchanged. An increase in gefitinib-induced expression of total Bax might be due to the decreased degradation of Bax, because the level of Bax mRNA has not been altered by gefitinib treatment. Gefitinib promoted the cleavage of full-length p21 Bax into p18 Bax in mitochondrial-enriched fraction, a characteristic feature of Bax activation toward apoptosis. Moreover, blockade of Bax by using anti-Bax small interfering double stranded RNA (siRNA) significantly reduced gefitinib-induced apoptosis. Taken together, these data suggest a critical role of p18 Bax in gefitinib-induced apoptosis.Next, we investigated the mechanistic role of Src and Ras, major oncogene products implicated in the pathogenesis of many human cancers in gefitinib sensitivity. Using parental and v-src- or c-H-ras-transfected HAG-1 human gallbladder adenocarcinoma cell lines, effects of gefitinib on cytotoxicity, cell cycle purtubation and apoptosis, and tyrosine phosphorylation of EGFR, Akt, and Erk were determined by WST-1 assay, flow cytometry, and Western blots, respectively Activated Ras and Src conferred a strong resistance to gefitinib by nearly 30-fold and 200-fold, respectively. Geftinib induced accumulation of cells in the G0/G1 phase of the cell cycle at 24-h, with progressive expansion of apoptotic cell population in parental HAG-1 cells, but these effects were completely abolished in v-src- or c-H-ras-transfected cell line. Upon gefitinib treatment, EGFR activation and subsequent downstream activation through Erk and Akt were significantly inhibited in HAG-1 cells. By contrast, gefinitib failed to inhibit the activation of both Akt and Erk in v-src-transfected cells and Erk, but not Akt in c-H-ras-transfected cells, despite the blockade of EGFR activation in these respective cell lines. Treatment of v-sre-transfected cells with herbimycin A, a Src tyrosine kinase inhibitor, partially revearsed the gefinitib resustance, with concomitant inhibition of Akt and Erk. Our results suggest that activated Ras and Src could induce gefinitib resistance by activating either or both of Akt and Erk signaling pathways, thus providing a strategic rationate for assessment of these specific signaling molecules downstream of EGFR to customize treatment. Less

吉非替尼是一种表皮生长因子受体（EGFR）酪氨酸激酶的选择性抑制剂，临床上已证实对某些癌细胞有效，但其抗肿瘤活性的分子机制尚未完全阐明。本研究旨在探讨吉非替尼诱导人胆囊腺癌细胞HAG-1生长抑制和凋亡的机制。吉非替尼以1 mM的剂量处理导致显著的生长抑制，并且在72 h孵育后细胞数量不可逆地下降，在120 h内凋亡细胞群逐渐扩增。处理2小时后，吉非替尼显着抑制EGFR自磷酸化以及随后通过Erk和Akt的下游信号通路，并诱导24小时细胞聚集在细胞周期的G 0/G1期，同时伴随p21转录增加和p27表达增加。吉非替尼不影响总的量， ...更多信息 磷酸化p53的丝氨酸15，但上调总Bax的表达，随后增加p18 Bax，Bax的活性形式。Bcl-2和Bad的表达无变化。吉非替尼诱导的总Bax表达增加可能是由于Bax降解减少，因为吉非替尼处理未改变Bax mRNA水平。吉非替尼促进了富含细胞的部分中全长p21 Bax裂解为p18 Bax，这是Bax活化朝向凋亡的特征。此外，通过使用抗Bax小干扰双链RNA（siRNA）阻断Bax显著减少吉非替尼诱导的细胞凋亡。两者合计，这些数据表明，p18 Bax在吉非替尼诱导apoptosis.Next中的关键作用，我们研究了Src和Ras的机制作用，这两种主要癌基因产物在吉非替尼敏感性中与许多人类癌症的发病机制有关。采用WST-1法、流式细胞术和Western印迹法分别检测了吉非替尼对HAG-1人胆囊癌细胞株的细胞毒性、细胞周期调控和凋亡以及EGFR、Akt和Erk酪氨酸磷酸化的影响。活化的Ras和Src分别使吉非替尼产生了近30倍和200倍的强耐药性。Geftinib在24 h时诱导细胞聚集在细胞周期的G 0/G1期，在亲代HAG-1细胞中凋亡细胞群进行性扩增，但这些作用在v-src或c-H-ras转染的细胞系中完全消除。在吉非替尼处理后，HAG-1细胞中EGFR活化和随后通过Erk和Akt的下游活化被显著抑制。相比之下，吉非替布未能抑制v-src转染细胞中Akt和Erk的激活，以及c-H-ras转染细胞中Erk的激活，但不能抑制Akt的激活，尽管在这些相应的细胞系中EGFR激活被阻断。用一种Src酪氨酸激酶抑制剂除莠霉素A处理v-sre转染的细胞，部分逆转了吉非替尼的耐药性，同时抑制了Akt和Erk。我们的研究结果表明，激活的Ras和Src可以通过激活Akt和Erk信号通路中的一个或两个来诱导吉非替尼耐药，从而为评估EGFR下游的这些特异性信号分子以定制治疗提供了战略依据。少

项目成果

Activated Src and Ras induce gefitinib resistance by activation of signaling pathways downstream of epidernial growth factor receptor in human gallbladder adenocarcinoma cells.

激活的 Src 和 Ras 通过激活人胆囊腺癌细胞中表皮生长因子受体下游的信号通路来诱导吉非替尼耐药。

• 发表时间: 2006
• 期刊: Cancer Chemother Pharmacol. 58
• 作者: Qin B;Ariyama H;Baba E;Tanaka R;Kusaba H;Harada M;Nakano S.
• 通讯作者: Nakano S.

In-Vitro differential metabolism and activity of 5-FU between short-term, high dose and long-term low dose treatments

短期、高剂量和长期低剂量治疗之间 5-FU 的体外代谢和活性差异

• 发表时间: 2006
• 期刊: Anticancer Drugs 17/4
• 作者: Yamaguchi K;Shimamura T;Hyodo I;Koizumi W;Doi T;Narahara H;Komatsu Y;Kato T;Saitoh S;Akiya T;Munakata M;Miyata Y;Maeda Y;Takiuchi H;Nakano S;Esaki T;Kinjo F;and Sakata Y.;Qin B;Qin B
• 通讯作者: Qin B

Nanog pseudogene 8 expression in gastrointestinal cancer cells.

Nanog 假基因 8 在胃肠道癌细胞中的表达。

• 发表时间: 2007
• 作者: Uchino K;Hirano G;Shirakawa T;Isobe T;Makiyama A;Arita S;Shibata Y;Kusaba H;Baba E;Nakano S.
• 通讯作者: Nakano S.

Feasibility study of ambulatory continuous infusion of 5-fluorouracil followed by cisplatin through hepatic artery for metastatic colorectal cancer.

经肝动脉门诊持续输注5-氟尿嘧啶随后顺铂治疗转移性结直肠癌的可行性研究。

• 发表时间: 2006
• 期刊: Cancer Chemother Pharmacol. 57(1)
• 作者: Qin B;Kato K;Mitsugi K;Nakamura M;Tanaka R;Baba E;Ariyama H;Kuroiwa T;Harada M;Nakano S.
• 通讯作者: Nakano S.

Regulation of exosomal HLA production of B cells by NF-kB and MAP kinases

NF-kB 和 MAP 激酶对 B 细胞外泌体 HLA 产生的调节

• 发表时间: 2007
• 作者: Yamaguchi K;Shimamura T;Hyodo I;Koizumi W;Doi T;Narahara H;Komatsu Y;Kato T;Saitoh S;Akiya T;Munakata M;Miyata Y;Maeda Y;Takiuchi H;Nakano S;Esaki T;Kinjo F;and Sakata Y.;Qin B;Qin B;Qin B;Ariyama H;Qin B;有田 修二
• 通讯作者: 有田 修二