PFK15对人胃癌的抗肿瘤活性及潜在的机制研究
发布时间：2019-06-14 20:26
【摘要】：PFK15是糖酵解通路关键调节酶PFKFB3的特异性小分子抑制剂。PFKFB3通过调节F2,6P2的生成量而影响糖酵解过程中限速酶PFK-1的活性进而控制糖酵解通量。本文旨在考察PFK15在体内外模型中对胃癌的抗肿瘤作用和潜在的作用机制以及与sorafenib联合用药对肿瘤血管生成的影响。本文应用蛋白免疫印迹方法验证PFKFB3在胃癌细胞移植瘤、血管内皮细胞和正常组织中的蛋白表达水平以确定PFKFB3蛋白表达的组织特异性。考察PFK15对胃癌细胞和内皮细胞糖酵解通量中葡萄糖摄取和F2,6P2产物的影响以及F2,6P2对PFK15诱导的细胞增殖能力的影响以确定PFK15对PFKFB3的"on-target"效应。应用细胞活力分析、细胞凋亡分析、细胞周期分析、划痕实验、Transwell小孔穿梭实验、细胞骨架检测实验分别考察PFK15对胃癌细胞的增殖、凋亡、周期、迁移、侵袭和骨架的影响。应用内皮细胞管状结构形成实验、体外3D内皮细胞球状体出芽实验和小鼠Matrigel Plug实验考察PFK15对血管新生的影响;应用体内裸小鼠移植瘤模型分析PFK15对胃癌的体内抗肿瘤活性和抗血管生成作用;应用蛋白免疫印迹分析和免疫组化分析检测PFK15对肿瘤细胞增殖、凋亡、周期和血管新生相关蛋白和信号通路的影响。另外,本文还在体内和体外模型中考察了 PFK15与sorafenib联用对肿瘤血管新生的影响。结果表明,相比于其它正常组织,PFKFB3在胃癌移植瘤和内皮细胞中是过量表达的。PFK15在实验剂量范围内可剂量依赖性的降低胃癌细胞(MKN45和AGS)和内皮细胞(EA.hy926)中葡萄糖的摄取和F2,6P2的产生,从而降低糖酵解通量;而F2,6P2加入后可以消除PFK15诱导的细胞增殖抑制作用,说明PFKFB3为PFK15的特异性靶点。PFK15在实验剂量范围内剂量依赖性的抑制胃癌细胞MKN45和AGS的增殖,PFK15引起的G0/G1期细胞周期阻滞与阻断Cyclin-CDKs/Rb/E2F信号通路有关,引起的细胞凋亡与线粒体途径诱导的细胞凋亡有关,引起的对细胞的迁移和侵袭的影响与改变细胞内F-actin数量和排列、改变细胞相关黏附分子的蛋白表达有关。PFK15可显著抑制内皮细胞EA.hy926的增殖、管状结构的形成、球状体的出芽和小鼠植入的基质胶块中血管的生成。在MKN45和AGS裸小鼠移植瘤模型中,PFK15都能显著抑制移植瘤的肿瘤体积和瘤重的增长,PFK15还能显著降低移植瘤中CD31阳性的染色面积。另外,PFK15与sorafenib联用显著抑制内皮细胞EA.hy926的增殖、管状结构的形成、球状体的出芽、Matrigel Plug和移植瘤中血管的发生,并且联用组对血管新生的抑制作用大于任何单独用药组。综上所述,本研究初步阐明了 PFK15抗胃癌活性的作用机制以及PFK15与sorafenib联用的抗血管新生作用,为糖酵解抑制剂的进一步开发提供理论基础。
[Abstract]:PFK15 is a specific small molecule inhibitor of PFKFB3, which is the key regulator of glycolysis pathway. PFKFB3 affects the activity of rate-limiting enzyme PFK-1 during glycolysis by regulating the production of F2and 6P2, and then controls the glycolysis flux. The purpose of this study was to investigate the antitumor effect and potential mechanism of PFK15 on gastric cancer in vitro and in vivo, as well as the effect of sorafenib on tumor angiogenesis. In order to determine the tissue specificity of PFKFB3 protein expression in gastric cancer cell transplantation tumor, vascular endothelial cells and normal tissues, the expression level of PFKFB3 protein was confirmed by Western imprinting. The effects of PFK15 on glucose uptake and F26P2 products in glycolysis fluxes of gastric cancer cells and endothelial cells and the effects of F2and 6P2 on the proliferation induced by PFK15 were investigated to determine the "on-target" effect of PFK15 on PFKFB3. The effects of PFK15 on proliferation, apoptosis, cycle, migration, invasion and skeleton of gastric cancer cells were investigated by cell vitality analysis, apoptosis analysis, cell cycle analysis, scratch test, Transwell pore shuttle test and cytoskeleton detection. The effects of PFK15 on angiogenesis were investigated by endothelial cell tubular structure test, 3D endothelial cell bulbous sprouting test and mouse Matrigel Plug test in vitro, and the antitumor activity and anti-angiogenic effect of PFK15 on gastric cancer in vivo were analyzed by transplanting tumor model in nude mice. The effects of PFK15 on proliferation, apoptosis, cycle and neovascularization related proteins and signaling pathways of tumor cells were detected by Western imprinting and immunohistochemical analysis. In addition, the effects of PFK15 combined with sorafenib on tumor angiogenesis were also investigated in vivo and in vitro. The results showed that compared with other normal tissues, PFKFB3 was overexpressed in gastric cancer xenografts and endothelial cells. PFK15 could decrease glucose uptake and F26P2 production in gastric cancer cells (MKN45 and AGS) and endothelial cells (EA.hy926) in a dose-dependent manner, thus reducing glycolysis flux. The addition of F2and 6P2 could eliminate the inhibitory effect of PFK15 on cell proliferation, indicating that PFKFB3 was a specific target of PFK15. PFK15 inhibited the proliferation of MKN45 and AGS in a dose-dependent manner. The cell cycle arrest in G0/G1 phase induced by PFK15 was related to the blocking of Cyclin-CDKs/Rb/E2F signaling pathway, and the apoptosis was related to apoptosis induced by mtDNA pathway. The effect of PFK15 on cell migration and invasion was related to changing the number and arrangement of F-actin in cells and changing the protein expression of cell-associated adhesion molecules. PFK15 could significantly inhibit the proliferation of endothelial cells, the formation of tubular structure, the budding of bulbous bodies and the formation of blood vessels in matrix glue blocks implanted in mice. In both MKN45 and AGS nude mice, PFK15 could significantly inhibit the increase of tumor volume and tumor weight, and PFK15 could also significantly reduce the positive staining area of CD31 in nude mice. In addition, the combination of PFK15 and sorafenib significantly inhibited the proliferation of endothelial cell EA.hy926, the formation of tubular structure, the budding, Matrigel Plug of bulbous body and the occurrence of blood vessels in the transplantation tumor, and the inhibitory effect of the combined group on vascular regeneration was greater than that of any single treatment group. In summary, this study preliminarily clarified the mechanism of anti-gastric cancer activity of PFK15 and the antiangiogenic effect of PFK15 combined with sorafenib, which provided a theoretical basis for the further development of glycolysis inhibitors.
【学位授予单位】：沈阳药科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2
本文编号：2499666
[Abstract]:PFK15 is a specific small molecule inhibitor of PFKFB3, which is the key regulator of glycolysis pathway. PFKFB3 affects the activity of rate-limiting enzyme PFK-1 during glycolysis by regulating the production of F2and 6P2, and then controls the glycolysis flux. The purpose of this study was to investigate the antitumor effect and potential mechanism of PFK15 on gastric cancer in vitro and in vivo, as well as the effect of sorafenib on tumor angiogenesis. In order to determine the tissue specificity of PFKFB3 protein expression in gastric cancer cell transplantation tumor, vascular endothelial cells and normal tissues, the expression level of PFKFB3 protein was confirmed by Western imprinting. The effects of PFK15 on glucose uptake and F26P2 products in glycolysis fluxes of gastric cancer cells and endothelial cells and the effects of F2and 6P2 on the proliferation induced by PFK15 were investigated to determine the "on-target" effect of PFK15 on PFKFB3. The effects of PFK15 on proliferation, apoptosis, cycle, migration, invasion and skeleton of gastric cancer cells were investigated by cell vitality analysis, apoptosis analysis, cell cycle analysis, scratch test, Transwell pore shuttle test and cytoskeleton detection. The effects of PFK15 on angiogenesis were investigated by endothelial cell tubular structure test, 3D endothelial cell bulbous sprouting test and mouse Matrigel Plug test in vitro, and the antitumor activity and anti-angiogenic effect of PFK15 on gastric cancer in vivo were analyzed by transplanting tumor model in nude mice. The effects of PFK15 on proliferation, apoptosis, cycle and neovascularization related proteins and signaling pathways of tumor cells were detected by Western imprinting and immunohistochemical analysis. In addition, the effects of PFK15 combined with sorafenib on tumor angiogenesis were also investigated in vivo and in vitro. The results showed that compared with other normal tissues, PFKFB3 was overexpressed in gastric cancer xenografts and endothelial cells. PFK15 could decrease glucose uptake and F26P2 production in gastric cancer cells (MKN45 and AGS) and endothelial cells (EA.hy926) in a dose-dependent manner, thus reducing glycolysis flux. The addition of F2and 6P2 could eliminate the inhibitory effect of PFK15 on cell proliferation, indicating that PFKFB3 was a specific target of PFK15. PFK15 inhibited the proliferation of MKN45 and AGS in a dose-dependent manner. The cell cycle arrest in G0/G1 phase induced by PFK15 was related to the blocking of Cyclin-CDKs/Rb/E2F signaling pathway, and the apoptosis was related to apoptosis induced by mtDNA pathway. The effect of PFK15 on cell migration and invasion was related to changing the number and arrangement of F-actin in cells and changing the protein expression of cell-associated adhesion molecules. PFK15 could significantly inhibit the proliferation of endothelial cells, the formation of tubular structure, the budding of bulbous bodies and the formation of blood vessels in matrix glue blocks implanted in mice. In both MKN45 and AGS nude mice, PFK15 could significantly inhibit the increase of tumor volume and tumor weight, and PFK15 could also significantly reduce the positive staining area of CD31 in nude mice. In addition, the combination of PFK15 and sorafenib significantly inhibited the proliferation of endothelial cell EA.hy926, the formation of tubular structure, the budding, Matrigel Plug of bulbous body and the occurrence of blood vessels in the transplantation tumor, and the inhibitory effect of the combined group on vascular regeneration was greater than that of any single treatment group. In summary, this study preliminarily clarified the mechanism of anti-gastric cancer activity of PFK15 and the antiangiogenic effect of PFK15 combined with sorafenib, which provided a theoretical basis for the further development of glycolysis inhibitors.
【学位授予单位】：沈阳药科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2
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1 朱伟;PFK15对人胃癌的抗肿瘤活性及潜在的机制研究[D];沈阳药科大学;2016年
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