[临床试验] 研究发现绿原酸可调节巨噬细胞极化治疗胶质瘤 II期临床试验即将启动
临床试验信息平台
第3期
除了临床试验之外,绿原酸深层机制的研究上也取得了重要发现,中国医学科学院药物研究所陈晓光研究员团队与首都医科大学附属北京世纪坛医院(现工作单位为首都医科大学附属北京天坛医院)李文斌教授团队合作发现绿原酸可以通过调节巨噬细胞极化来抑制胶质瘤生长,相关研究成果在线发表在nature旗下期刊《Scientific Reports》
胶质瘤是最常见的颅内恶性肿瘤,占所有原发性中枢神经系统肿瘤的32%,占所有恶性脑肿瘤的81%,具有发病率高、复发率高、死亡率高和治愈率低等“三高一低”的特点,并伴有年轻化发病的趋势。
到目前为止,替莫唑胺是临床治疗胶质瘤唯一的一线药物。然而,其临床有效率不足45%,有一部分患者使用替莫唑胺近期疗效较好但长期疗效不佳,并且替莫唑胺临床易产生耐药。因此,迫切需要研发新型的药物用于临床上胶质瘤的治疗。
巨噬细胞和小胶质细胞作为重要的一类免疫细胞,约占整个胶质瘤组织的30%,与肿瘤的增殖和侵袭密切相关[1]。巨噬细胞被广泛认为可分为两类:促炎的M1型和抗炎的M2型巨噬细胞。M1型巨噬细胞可增强Th1免疫反应,通过其促炎活性杀死微生物、促进肿瘤裂解;M2型巨噬细胞可通过促进血管生成因子及免疫抑制型细胞因子的分泌促进肿瘤生长。这提示我们,调节胶质瘤中巨噬细胞的表型可以达到治疗胶质瘤的目的。
绿原酸(chlorogenic acid, CHA),是一种低分子量(MW:354.30)的多酚类化合物,具有广泛的药理作用,如抗菌、消炎、解毒、利胆、降压、增强机体免疫和抗肿瘤等。四川九章生物科技有限公司研发的注射用绿原酸是我国具有自主知识产权的1.1类抗癌新药,目前已经完成复发性胶质瘤治疗的Ⅰ期临床试验。
近期,中国医学科学院药物研究所陈晓光研究员团队与首都医科大学附属北京世纪坛医院(现工作单位为首都医科大学附属北京天坛医院)李文斌教授团队合作发现绿原酸可以通过调节巨噬细胞极化来抑制胶质瘤生长。
本项研究的论文《Chlorogenic acid inhibits glioblastoma growth through repolarizating macrophage from M2 to M1 phenotype》于2017年1月在线发表在nature旗下期刊《Scientific Reports》。研究者:薛妮娜(并列一作)、周秦(并列一作)、季鸣、金晶、来芳芳、陈菊、张梦甜、贾静、杨华蓉、张洁、李文斌(通讯作者)、蒋建东(通讯作者)、陈晓光(通讯作者)。
研究内容简介如下
Figure 1. Effect of CHA on macrophage marker expression induced by lipopolysaccharide (LPS) and interferon (IFN)-γ. Macrophages were treated with LPS (10 ng/ml) and IFNγ (20 ng/ml) with or without different concentrations of CHA for 24 h or 48 h. The mRNA levels of M1-marker gene iNOS and M2-marker gene Arg in Ana-1 (A) and RAW264.7 cells (B) were measured by real-time RT-PCR. The expression of mRNAs was normalized to GAPDH. The expressions of CD11c and MHC Class II IA-IE in RAW264.7 cells for 24 h (C) or 48 h (D) were evaluated by flow cytometry. The histogram bars represent three independent experiments. The data are presented as the mean ± SD. *p-value < 0.05, **p-value < 0.01 vs. LPS/IFNγ .
文章首先确认了CHA(≤10μM)对原代神经元细胞、胶质细胞系、巨噬细胞系和一些肿瘤细胞系没有直接的细胞毒作用。
巨噬细胞具有很强的可塑性,在LPS/IFNγ诱导下可活化为M1表型,在IL-4或IL-13诱导下,可活化为M2表型。我们采用PCR和FCS技术,在小鼠单核/巨噬细胞系ANA-1和RAW264.7细胞中给予LPS/IFNγ或IL4刺激与CHA联用,发现CHA可进一步增加M1表型Marker(iNOS,MHCⅡ-IA/IE和CD11c)的表达,降低M2型Marker(ARG1和CD206)的表达。这提示CHA不仅可以促进巨噬细胞进一步极化成M 1表型,还可以抑制极化的M2表型的巨噬细胞。
Figure 2. Effect of CHA on macrophage marker expression induced by interleukin (IL)-4. Ana-1 and RAW264.7 cells were treated with interleukin (IL)-4 (20 ng/ml) in the presence of DMSO or different concentrations of CHA for 24 h. The mRNA levels of M1-marker gene iNOS and M2-marker gene Arg in Ana-1 (A) and RAW264.7 cells (B) were measured by real-time RT-PCR. The expression of mRNAs was normalized to GAPDH. The expressions of CD206 in Ana-1 (C) and RAW264.7 cells (D) were evaluated by flow cytometry.The histogram bars represent three independent experiments. The data are presented as the mean ± SD. *p-value < 0.05, **p-value < 0.01 vs.IL-4.
接下来,我们探索了CHA发挥极化作用的具体分子机制。STAT1和STAT6信号通路分别介导巨噬细胞M1和M2型极化。Western Blot实验结果显示,CHA可进一步激活与M1表型极化相关的磷酸化STAT1水平,而在巨噬细胞极化成M2表型的基础上,CHA降低与M2表型相关的磷酸化STAT6水平。
为了进一步确认CHA是通过调节巨噬细胞极化而影响肿瘤的增殖。我们在巨噬细胞和肿瘤细胞共培养模型上发现CHA可以促进M1型巨噬细胞诱导凋亡样肿瘤细胞的产生,并抑制M2型巨噬细胞诱导肿瘤细胞增殖的作用。
Figure 3. Effect of CHA on tumor cells growth in co-culture of tumor cells with macrophages. U87-RFP-Luc or MFC-GFP tumor cells (1×103) were mono-cultured in lipopolysaccharide (LPS) (10 ng/ml) and interferon (IFN)-γ (20 ng/ml) (M1 stimulator) or interleukin (IL)-4 (20 ng/ml) (M2 stimulator) alone, or in combination with indicated concentration of CHA for 48 h, or were co-cultured with M1 stimulator or M2 stimulator-treated macrophages in the presence of CHA for 48 h. The tumor cellular morphology was visualized by fluorescence microscopy. The proliferation was represented as fluorescent area or overall photon counts of cells by using software of ImageProPlus and EnSpire Multimode Plate Reader, respectively. The data are presented as the mean ± SD. *p-value < 0.05, **p-value < 0.01; as evaluated using Student’s t-test.
在小鼠胶质瘤G422皮下移植瘤模型中,CHA显示出一定的抑制胶质瘤生长的作用。与体外作用机制一致,FCS和IHC的结果显示:CHA可增加荷瘤鼠肿瘤组织中CD11c阳性的M1型巨噬细胞,并降低CD206阳性的M2型巨噬细胞。
Figure 4. Effect of CHA on glioblastoma progression in G422 xenograft model. ICR mice were injected subcutaneously in the flank with G422 glioma cells in and were treated with CHA (20 mg/kg or 40 mg/kg per day) for 14 days or cisplatinum (CPT) (6 mg/kg per week).
小结
CHA一方面通过促进M1型巨噬细胞极化,另一方面通过抑制M2型巨噬细胞极化,从而发挥抗胶质瘤生长的作用。该研究为CHA应用于胶质瘤的临床治疗提供重要的理论依据。
Figure 5. Schematic representation of proposed mechanism for CHA-mediated anti-tumor activity in glioblastoma.
参考文献:[1] Grauwet K, Chiocca EA. Glioma and microglia, a double entendre [J]. Nat Immunol, 2016,
17:1240-1242.
论文原文下载(扫描二维码)
[临床试验招募]天津环湖医院高压氧联合放化疗治疗胶质母细胞瘤临床研究招募患者
[公告] 神经肿瘤(胶质瘤)临床试验信息平台开通 | 李文斌点评
注册临床试验平台会员,请点击本文左下角阅读原文;或扫描下面二维码
“神经肿瘤(胶质瘤)临床试验信息平台”由神外前沿新媒体和中国医师协会脑胶质瘤专业委员会药物与生物治疗学组共同发起,定期发布中枢神经系统肿瘤的临床试验信息、报道临床试验进展;审稿人为首都医科大学附属北京天坛医院李文斌教授。联系方式:微信号53880941;邮件vip@vipyiyi.com;