目的·检测Fas相关死亡结构域蛋白(Fas-associated protein with death domain,FADD)在头颈部鳞状细胞癌(head and neck squamous cell carcinoma,HNSCC)中的表达水平,并探究FADD促进HNSCC细胞增殖的分子机制。方法·利用GEPIA ...目的·检测Fas相关死亡结构域蛋白(Fas-associated protein with death domain,FADD)在头颈部鳞状细胞癌(head and neck squamous cell carcinoma,HNSCC)中的表达水平,并探究FADD促进HNSCC细胞增殖的分子机制。方法·利用GEPIA 2数据库分析肿瘤组织中FADD表达水平及其与预后的关系;通过对HNSCC组织进行免疫组织化学染色(immunohistochemistry staining,IHC),探究FADD在正常、不典型增生和肿瘤组织中的表达水平变化;构建稳定低表达FADD的人HNSCC Fadu、HSC3细胞株,并通过蛋白印迹实验和实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)方法进行验证;使用LiveCyte活细胞追踪系统、克隆形成、细胞活力检测等方法探究FADD对HNSCC细胞增殖水平的调控作用;使用免疫共沉淀串联质谱(co-immunoprecipitation mass spectrum,Co-IP/MS)鉴定与FADD发生相互作用的蛋白,并应用CRISPR/Cas9技术、LiveCyte活细胞追踪系统、蛋白印迹实验等方法对与FADD相互作用的蛋白进行进一步机制研究。结果·数据库分析显示FADD在头颈鳞癌中显著高表达,并与患者不良预后相关。免疫组化染色表明FADD在HNSCC患者正常组织、不典型增生及肿瘤组织中的表达水平呈现递增趋势。在HNSCC细胞中敲低FADD后,与对照组相比,细胞的增殖能力显著降低,形成克隆数减少。Co-IP/MS结果显示,FADD与CUT样同源盒1(CUT-like homeobox 1,CUX1)蛋白存在相互作用,敲低FADD后CUX1表达水平升高。同时,在HNSCC细胞中敲除CUX1能够显著促进肿瘤细胞增殖能力。敲除CUX1可部分逆转FADD低表达引起的增殖抑制。结论·FADD在HNSCC中具有显著促癌作用,并与不良预后相关。FADD可通过与CUX1发生相互作用降低其表达水平进一步调控肿瘤细胞的增殖能力。展开更多
OBJECTIVE Platinum compounds such as cisplatin and carboplatin are frequently used as the first-line chemotherapy for the treatment of the head and neck squamous cell carcinoma(HNSCC).In the present study,we investiga...OBJECTIVE Platinum compounds such as cisplatin and carboplatin are frequently used as the first-line chemotherapy for the treatment of the head and neck squamous cell carcinoma(HNSCC).In the present study,we investigated whether garcinol,apolyisoprenylated benzophenone can chemosensitize HNSCC to cisplatin.METHODS The effect of garcinol and cisplatin on HNSCC was assessed by MTT,Western blotting,real time PCR,FACS,immunohistochemistry,DNA binding assay and xenograft mouse model.RESULTS We found that garcinol inhibited the viability of a panel of diverse HNSCC cell lines,enhanced the apoptotic effect of cisplatin,suppressed constitutive as well as cisplatin-induced NF-κB activation,and downregulated the expression of various oncogenic gene products(cyclin D1,Bcl-2,survivin and VEGF).In vivo study showed that administration of garcinol alone(0.5 mg·kg-1,ip five times/week)significantly suppressed the growth of the tumor,and this effect was further increased by cisplatin.Both the markers of proliferation index(Ki-67)and microvessel density(CD31)were downregulated in tumor tissues by the combination of cisplatin and garcinol.The pharmacokinetic results of garcinol indicated that good systemic exposure was achievable after ip administration of garcinol at 0.5and 2mg·kg-1 with mean peak concentration(cmax)of 1825.4 and 6635.7nmol·L-1 in the mouse serum,respectively.CONCLUSION Overall,our results suggest that garcinol can indeed potentiate the effects of cisplatin by negative regulation of various inflammatory and proliferative biomarkers.展开更多
文摘目的·检测Fas相关死亡结构域蛋白(Fas-associated protein with death domain,FADD)在头颈部鳞状细胞癌(head and neck squamous cell carcinoma,HNSCC)中的表达水平,并探究FADD促进HNSCC细胞增殖的分子机制。方法·利用GEPIA 2数据库分析肿瘤组织中FADD表达水平及其与预后的关系;通过对HNSCC组织进行免疫组织化学染色(immunohistochemistry staining,IHC),探究FADD在正常、不典型增生和肿瘤组织中的表达水平变化;构建稳定低表达FADD的人HNSCC Fadu、HSC3细胞株,并通过蛋白印迹实验和实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)方法进行验证;使用LiveCyte活细胞追踪系统、克隆形成、细胞活力检测等方法探究FADD对HNSCC细胞增殖水平的调控作用;使用免疫共沉淀串联质谱(co-immunoprecipitation mass spectrum,Co-IP/MS)鉴定与FADD发生相互作用的蛋白,并应用CRISPR/Cas9技术、LiveCyte活细胞追踪系统、蛋白印迹实验等方法对与FADD相互作用的蛋白进行进一步机制研究。结果·数据库分析显示FADD在头颈鳞癌中显著高表达,并与患者不良预后相关。免疫组化染色表明FADD在HNSCC患者正常组织、不典型增生及肿瘤组织中的表达水平呈现递增趋势。在HNSCC细胞中敲低FADD后,与对照组相比,细胞的增殖能力显著降低,形成克隆数减少。Co-IP/MS结果显示,FADD与CUT样同源盒1(CUT-like homeobox 1,CUX1)蛋白存在相互作用,敲低FADD后CUX1表达水平升高。同时,在HNSCC细胞中敲除CUX1能够显著促进肿瘤细胞增殖能力。敲除CUX1可部分逆转FADD低表达引起的增殖抑制。结论·FADD在HNSCC中具有显著促癌作用,并与不良预后相关。FADD可通过与CUX1发生相互作用降低其表达水平进一步调控肿瘤细胞的增殖能力。
基金The project supported by National Medical Research Council of Singapore and NUS Academic Research fund
文摘OBJECTIVE Platinum compounds such as cisplatin and carboplatin are frequently used as the first-line chemotherapy for the treatment of the head and neck squamous cell carcinoma(HNSCC).In the present study,we investigated whether garcinol,apolyisoprenylated benzophenone can chemosensitize HNSCC to cisplatin.METHODS The effect of garcinol and cisplatin on HNSCC was assessed by MTT,Western blotting,real time PCR,FACS,immunohistochemistry,DNA binding assay and xenograft mouse model.RESULTS We found that garcinol inhibited the viability of a panel of diverse HNSCC cell lines,enhanced the apoptotic effect of cisplatin,suppressed constitutive as well as cisplatin-induced NF-κB activation,and downregulated the expression of various oncogenic gene products(cyclin D1,Bcl-2,survivin and VEGF).In vivo study showed that administration of garcinol alone(0.5 mg·kg-1,ip five times/week)significantly suppressed the growth of the tumor,and this effect was further increased by cisplatin.Both the markers of proliferation index(Ki-67)and microvessel density(CD31)were downregulated in tumor tissues by the combination of cisplatin and garcinol.The pharmacokinetic results of garcinol indicated that good systemic exposure was achievable after ip administration of garcinol at 0.5and 2mg·kg-1 with mean peak concentration(cmax)of 1825.4 and 6635.7nmol·L-1 in the mouse serum,respectively.CONCLUSION Overall,our results suggest that garcinol can indeed potentiate the effects of cisplatin by negative regulation of various inflammatory and proliferative biomarkers.
