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结直肠癌(CRC)是最常见的消化道恶性肿瘤,在中国的发病率仅次于肺癌,且呈现逐年升高的趋势[1]。结直肠癌的发病机制具有多因素、多阶段、多基因突变等特点[2,3]。结直肠癌早期发现的概率低,多数患者出现明显症状后才被确诊,此时手术通常已无法完全控制肿瘤进展,需化疗控制病情[4]。结直肠癌患者最常用的化疗药物为奥沙利铂和五氟尿嘧啶(5-FU),分别是DNA合成抑制剂和胸苷酸合成酶抑制剂,影响细胞DNA复制和转录,最终导致细胞死亡[5-7]。近年来,复发/难治性结直肠癌患者逐渐升高,对多数化疗药无应答,其5年生存率低于10%[8-9]。结直肠癌患者耐药情况越来越严重,解决耐药问题的首要条件为阐明结直肠癌患者化疗耐药的机制。
随着人们对肠道微生物研究的深入,已明确表明其与多种肠道疾病相关,其中具核梭杆菌(Fn)与结直肠癌发生发展的关系研究最透彻[10-13]。在口腔、胃肠道、呼吸道等环境内均明确发现Fn,其是一种专性厌氧性、G−菌,可黏附、侵入细胞,获得适合生存的环境[14-16]。Fn可以产生毒力因子、提升白介素17(IL-17)和肿瘤坏死因子(TNF)等促炎因子水平、抑制自然杀伤细胞的细胞毒活性、招募肿瘤浸润性骨髓细胞等,促进肿瘤发生发展和耐药机制的产生[11-13, 17-19]。例如,Fn促进了CRC的进展,并使奥沙利铂和5-FU化疗耐药[20],因此,解决Fn高富集后促进CRC进展并产生化疗耐药的问题,有望提升结直肠癌患者化疗的有效率。
本研究首先在类器官层面验证Fn促结直肠癌增殖作用,其次比较考察前期筛选获得的抗Fn化合物在Fn与结肠癌HCT116细胞共孵育条件下对其体外抗癌活性的影响,最后优选高活性化合物开展其对Fn灌胃干预下裸鼠结肠癌HCT116移植瘤抗癌药效评价,从而为新型抗结直肠癌药物研发提供先导化合物。
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连续4 d对PBS和不同浓度Fn干预下的固定位置直肠癌类器官生长情况的拍照观察结果显示,PBS阴性对照组的直肠癌类器官随时间呈缓慢生长;但是,1×104 CFU/ml浓度的Fn干预下的直肠癌类器官随时间呈急速生长,且呈浓度依赖(图1)。
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用CCK-8法分别比较测定了前期筛选获得的9个MIC达2.0~32 μg/ml的抗Fn活性化合物对人结直肠癌HCT116细胞及在最优MOI条件下与Fn共孵育的人结直肠癌HCT116细胞的体外IC50,结果显示,9个受试化合物均能显著提升Fn与结肠癌细胞共孵育条件下的体外抗肿瘤活性,其中,甲氨蝶呤提升幅度最大,达143倍(表1)。
表 1 Fn对抗Fn化合物(MIC)的体外抗癌活性(IC50)的影响
名称 MIC
(μg/ml)HCT116
(IC50,μmol/L)HCT116 + Fn
(IC50,μmol/L)新鱼腥草素钠 32 0.48 0.12 对甲氧基苯甲醛 32 4.22 1.77 PA-824 32 9.62 5.62 甲氨蝶呤 32 4.30 0.03 吲哚-3-甲酸 16 8.86 2.72 己烯雌酚 32 23.10 9.35 2'-脱氧胞嘧啶核苷 8 0.78 0.07 5-硝基-8-羟基喹啉 2 22.85 3.93 特地唑胺 2 4.29 1.42 通过实时无标记肿瘤细胞分析技术检测了优选抗Fn活性化合物甲氨蝶呤在Fn与HCT116细胞共孵育条件下对肿瘤细胞生长的影响,实时检测结果显示,甲氨蝶呤也能显著抑制Fn的促肿瘤细胞生长作用,且呈浓度依赖性(图2)。
综上所述,优选甲氨蝶呤开展后续Fn灌胃干预下裸鼠人结肠癌HCT116移植瘤的体内抗癌药效评价。
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在Fn灌胃干预下,抗Fn优选化合物甲氨蝶呤(0.5 mg/kg)以及PD-1(5.0 mg/kg)单独用药的抑瘤率分别为11.39%、53.95%;当甲氨蝶呤(0.5 mg/kg)与PD-1(5.0 mg/kg)联用后,其抑瘤率可显著提升至67.46%(表2和图3B)。给药组荷瘤裸鼠的体重无显著变化,与对照组相似(图3A)。
表 2 甲氨蝶呤单用及联合PD-1对Fn干预下裸鼠移植瘤的抑瘤率
组别 PD-1 甲氨蝶呤 单独用药 53.95% 11.39% 联合PD-1 − 67.46% $ {\chi ^2} $ − 65.82 P − <0.01
Screening of small molecule inhibitors against Fusobacteriumnucleatum and study of their anti-colorectal activity
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摘要:
目的 基于市售化合物库的表型筛选获得抗具核梭杆菌(Fn)活性化合物,评价其Fn干预下的抗结直肠癌活性,为新型抗结直肠癌药物研发提供先导化合物。 方法 首先验证Fn促结直肠癌类器官增殖效应;其次,比较考察抗Fn化合物在Fn与结肠癌HCT116细胞共孵育下对其体外抗癌活性的影响;最后,评价高活性化合物对Fn灌胃干预下裸鼠结肠癌HCT116移植瘤的体内抗癌药效。 结果 Fn可显著促进直肠癌类器官增殖;9个抗Fn化合物均能显著提升Fn与HTC116细胞共孵育下的体外抗癌活性,其中,甲氨蝶呤抗癌活性最强,IC50达0.03 μmol/L;甲氨蝶呤(0.5 mg/kg)与PD-1(5.0 mg/kg)联用能显著抑制Fn灌胃干预下裸鼠人结肠癌HCT116移植瘤的生长,抑瘤率达67.46%,抑瘤效果优于单药。 结论 Fn小分子抑制剂甲氨蝶呤对有Fn干预的结直肠癌细胞和裸鼠移植瘤具有良好的体内外抗癌活性;这为后续结构优化打下基础,并有望拓展甲氨蝶呤的新适应证。 Abstract:Objective To screen small molecule inhibitors against Fusobacterium nucleatum(Fn) based on commercially available compound libraries, followed to investigate their anti-colorectal cancer activities under Fn intervention in order to obtain novel anti-colorectal cancer lead compounds. Methods Firstly, to validate the promotion of colorectal cancer proliferation on organoid by Fn. Secondly, comparative investigation for the effects of anti-Fn compounds on their in vitro anticancer activity under Fn’s co-incubation with colorectal cancer HCT116 cells. Finally, evaluation for in vivo anticancer efficacy of highly active compounds on nude mouse colon cancer HCT116 transplanted tumor under the intervention of Fn by gavage. Results Fn could significantly promote the proliferation of rectal cancer organoids. 9 ant-Fn active compounds could significantly enhance their in vitro anticancer activity under Fn’s co-incubation with HCT116 cells. Methotrexate had the strongest anti-cancer activity with IC50 as 0.03 μmol/L. The combined use of methotrexate(0.5 mg/kg)and PD-1(5.0 mg/kg)had a stronger anti-tumor effect than their standalone use. Conclusion As new small molecule inhibitor against Fn, methotrexate exhibits good in vitro and in vivo anti-colorectal cancer activity against HCT116 cells and nude mouse xenografts under Fn intervention. This lays the foundation for subsequent structural optimization, and is expected to expand the new indications for methotrexate. -
Key words:
- Fusobacterium nucleatum /
- phenotypic screening /
- anti-crc /
- organoids /
- methotrexate
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表 1 Fn对抗Fn化合物(MIC)的体外抗癌活性(IC50)的影响
名称 MIC
(μg/ml)HCT116
(IC50,μmol/L)HCT116 + Fn
(IC50,μmol/L)新鱼腥草素钠 32 0.48 0.12 对甲氧基苯甲醛 32 4.22 1.77 PA-824 32 9.62 5.62 甲氨蝶呤 32 4.30 0.03 吲哚-3-甲酸 16 8.86 2.72 己烯雌酚 32 23.10 9.35 2'-脱氧胞嘧啶核苷 8 0.78 0.07 5-硝基-8-羟基喹啉 2 22.85 3.93 特地唑胺 2 4.29 1.42 表 2 甲氨蝶呤单用及联合PD-1对Fn干预下裸鼠移植瘤的抑瘤率
组别 PD-1 甲氨蝶呤 单独用药 53.95% 11.39% 联合PD-1 − 67.46% $ {\chi ^2} $ − 65.82 P − <0.01 -
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