Cyclic dinucleotides(CDNs) are known to activate stimulator of interferon genes(STING) and induce type I interferon responses, therefor possess great potentials to be of immunotherapeutic value for cancers and infecti...Cyclic dinucleotides(CDNs) are known to activate stimulator of interferon genes(STING) and induce type I interferon responses, therefor possess great potentials to be of immunotherapeutic value for cancers and infectious diseases. However, the existence of different single nucleotide polymorphism(SNP) of human STING(hSTING) gene poses an obstacle to achieve broad-spectrum activation by CDNs. We reported here the design and synthesis of a total of 36 CDNs, representing all structural variations, that contain four bases(A, G, C, U) and two linkage directions(2′-5′-linked and 3′-5′-linked phosphodiester).Through systematic evaluation of IFN-β induction with a dual-luciferase reporter assay, we discovered that wild type hSTING and two isoforms(HAQ and AQ) showed strong response while hSTING-R232 H and R293 Q exhibited the relatively weak response to CDNs stimulation. For the first time, we found that the c[G(2′,5′)U(2′,5′)] showed excellent activity against all five hSTING variants even equivalent to the endogenous ligand c[G(2′,5′)A(3′,5′)]. Furthermore, we have also demonstrated that 3′-3′CDNs with two 3′-5′ phosphodiesters showed higher serum and hydrolase stability than 2′-2′ CDNs with two 2′-5′ phosphodiesters and 2′-3′ CDNs with one 2′-5′ and one 3′-5′ phosphodiester. It is very interesting to note that 2′-2′ CDNs has been found for the first time to show strong activity. These findings will stimulate our exploration for the new functional role of CDNs, and provide guidelines to design CDNs based hSTING targeted drugs.展开更多
目的:探讨次乌头碱(HA)通过环磷酸鸟苷-腺苷酸合成酶(cGAS)/干扰素基因刺激因子(STING)信号通路对胃癌细胞增殖、迁移、侵袭和免疫逃逸的影响。方法:将BGC-823细胞分为对照组(NC组)、HA低剂量组(HA-L组,2μmol/L)、HA中剂量组(HA-M组,4...目的:探讨次乌头碱(HA)通过环磷酸鸟苷-腺苷酸合成酶(cGAS)/干扰素基因刺激因子(STING)信号通路对胃癌细胞增殖、迁移、侵袭和免疫逃逸的影响。方法:将BGC-823细胞分为对照组(NC组)、HA低剂量组(HA-L组,2μmol/L)、HA中剂量组(HA-M组,4μmol/L)、HA高剂量组(HA-H组,8μmol/L)、RU.521(cGAS抑制剂)组(1μmol/L)、HA-H+RU.521组(8μmol/L+1μmol/L),CCK-8、克隆形成实验检测细胞增殖;划痕愈合实验检测细胞迁移;Transwell实验检测细胞侵袭;ELISA检测细胞上清中趋化因子配体(CXCL)2、CXCL8水平;Western blot检测细胞中增殖细胞核抗原(PCNA)、基质金属蛋白酶(MMP)-9、cGAS、STING蛋白表达。将上述各组细胞分别与NK细胞共培养24 h,命名为NC共培养组、HA-L共培养组、HA-M共培养组、HA-H共培养组、RU.521共培养组、HA-H+RU.521共培养组,检测NK细胞杀伤力。结果:与NC组比较,HA-L组、HA-M组、HA-H组BGC-823细胞OD450(24 h)(0.87±0.08 vs 0.75±0.06、0.62±0.06、0.41±0.03)、克隆形成率[(47.75±2.13)%vs(41.12±1.81)%、(33.58±1.61)%、(19.95±0.84)%]、划痕愈合率[(43.37±2.08)%vs(36.65±1.54)%、(27.74±1.03)%、(13.36±0.62)%]、侵袭细胞数(78.85±3.67 vs 65.59±2.49、51.52±2.01、22.23±1.36)、CXCL2、CXCL8水平、PCNA、MMP-9蛋白表达降低,cGAS、STING蛋白表达升高(P<0.05);与NC组比较,RU.521组对应指标变化趋势与上述相反(P<0.05);与NC共培养组比较,HA-L共培养组、HA-M共培养组、HA-H共培养组NK细胞杀伤力增强,且呈剂量依赖性(P<0.05);与NC共培养组比较,RU.521共培养组对应指标变化趋势与上述相反(P<0.05);RU.521减弱了高剂量HA对BGC-823细胞增殖、迁移、侵袭、免疫逃逸的抑制作用。结论:HA可能通过激活cGAS-STING信号通路抑制胃癌细胞增殖、迁移、侵袭和免疫逃逸。展开更多
基金the National Key Research and Development Program of China(2017YFD0200500)the National Natural Science Foundation of China(21740002,21837001)。
文摘Cyclic dinucleotides(CDNs) are known to activate stimulator of interferon genes(STING) and induce type I interferon responses, therefor possess great potentials to be of immunotherapeutic value for cancers and infectious diseases. However, the existence of different single nucleotide polymorphism(SNP) of human STING(hSTING) gene poses an obstacle to achieve broad-spectrum activation by CDNs. We reported here the design and synthesis of a total of 36 CDNs, representing all structural variations, that contain four bases(A, G, C, U) and two linkage directions(2′-5′-linked and 3′-5′-linked phosphodiester).Through systematic evaluation of IFN-β induction with a dual-luciferase reporter assay, we discovered that wild type hSTING and two isoforms(HAQ and AQ) showed strong response while hSTING-R232 H and R293 Q exhibited the relatively weak response to CDNs stimulation. For the first time, we found that the c[G(2′,5′)U(2′,5′)] showed excellent activity against all five hSTING variants even equivalent to the endogenous ligand c[G(2′,5′)A(3′,5′)]. Furthermore, we have also demonstrated that 3′-3′CDNs with two 3′-5′ phosphodiesters showed higher serum and hydrolase stability than 2′-2′ CDNs with two 2′-5′ phosphodiesters and 2′-3′ CDNs with one 2′-5′ and one 3′-5′ phosphodiester. It is very interesting to note that 2′-2′ CDNs has been found for the first time to show strong activity. These findings will stimulate our exploration for the new functional role of CDNs, and provide guidelines to design CDNs based hSTING targeted drugs.
文摘目的:探讨次乌头碱(HA)通过环磷酸鸟苷-腺苷酸合成酶(cGAS)/干扰素基因刺激因子(STING)信号通路对胃癌细胞增殖、迁移、侵袭和免疫逃逸的影响。方法:将BGC-823细胞分为对照组(NC组)、HA低剂量组(HA-L组,2μmol/L)、HA中剂量组(HA-M组,4μmol/L)、HA高剂量组(HA-H组,8μmol/L)、RU.521(cGAS抑制剂)组(1μmol/L)、HA-H+RU.521组(8μmol/L+1μmol/L),CCK-8、克隆形成实验检测细胞增殖;划痕愈合实验检测细胞迁移;Transwell实验检测细胞侵袭;ELISA检测细胞上清中趋化因子配体(CXCL)2、CXCL8水平;Western blot检测细胞中增殖细胞核抗原(PCNA)、基质金属蛋白酶(MMP)-9、cGAS、STING蛋白表达。将上述各组细胞分别与NK细胞共培养24 h,命名为NC共培养组、HA-L共培养组、HA-M共培养组、HA-H共培养组、RU.521共培养组、HA-H+RU.521共培养组,检测NK细胞杀伤力。结果:与NC组比较,HA-L组、HA-M组、HA-H组BGC-823细胞OD450(24 h)(0.87±0.08 vs 0.75±0.06、0.62±0.06、0.41±0.03)、克隆形成率[(47.75±2.13)%vs(41.12±1.81)%、(33.58±1.61)%、(19.95±0.84)%]、划痕愈合率[(43.37±2.08)%vs(36.65±1.54)%、(27.74±1.03)%、(13.36±0.62)%]、侵袭细胞数(78.85±3.67 vs 65.59±2.49、51.52±2.01、22.23±1.36)、CXCL2、CXCL8水平、PCNA、MMP-9蛋白表达降低,cGAS、STING蛋白表达升高(P<0.05);与NC组比较,RU.521组对应指标变化趋势与上述相反(P<0.05);与NC共培养组比较,HA-L共培养组、HA-M共培养组、HA-H共培养组NK细胞杀伤力增强,且呈剂量依赖性(P<0.05);与NC共培养组比较,RU.521共培养组对应指标变化趋势与上述相反(P<0.05);RU.521减弱了高剂量HA对BGC-823细胞增殖、迁移、侵袭、免疫逃逸的抑制作用。结论:HA可能通过激活cGAS-STING信号通路抑制胃癌细胞增殖、迁移、侵袭和免疫逃逸。
