BACKGROUND Trastuzumab constitutes the fundamental component of initial therapy for patients with advanced human epidermal growth factor receptor 2(HER-2)-positive gastric cancer(GC).However,the efficacy of this treat...BACKGROUND Trastuzumab constitutes the fundamental component of initial therapy for patients with advanced human epidermal growth factor receptor 2(HER-2)-positive gastric cancer(GC).However,the efficacy of this treatment is hindered by substantial challenges associated with both primary and acquired drug resistance.While S-phase kinase associated protein 2(Skp2)overexpression has been implicated in the malignant progression of GC,its role in regulating trastuzumab resistance in this context remains uncertain.Despite the numerous studies investigating Skp2 inhibitors among small molecule compounds and natural products,there has been a lack of successful commercialization of drugs specifically targeting Skp2.AIM To discover a Skp2 blocker among currently available medications and develop a therapeutic strategy for HER2-positive GC patients who have experienced progression following trastuzumab-based treatment.METHODS Skp2 exogenous overexpression plasmids and small interfering RNA vectors were utilized to investigate the correlation between Skp2 expression and trastuzumab resistance in GC cells.Q-PCR,western blot,and immunohistochemical analyses were conducted to evaluate the regulatory effect of thioridazine on Skp2 expression.A cell counting kit-8 assay,flow cytometry,a amplex red glucose/glucose oxidase assay kit,and a lactate assay kit were utilized to measure the proliferation,apoptosis,and glycolytic activity of GC cells in vitro.A xenograft model established with human GC in nude mice was used to assess thioridazine's effectiveness in vivo.RESULTS The expression of Skp2 exhibited a negative correlation with the sensitivity of HER2-positive GC cells to trastuzumab.Thioridazine demonstrated the ability to directly bind to Skp2,resulting in a reduction in Skp2 expression at both the transcriptional and translational levels.Moreover,thioridazine effectively inhibited cell proliferation,exhibited antiapoptotic properties,and decreased the glucose uptake rate and lactate production by suppressing Skp2/protein kinase B/mammalian target of rapamycin/glucose transporter type 1 signaling pathways.The combination of thioridazine with either trastuzumab or lapatinib exhibited a more pronounced anticancer effect in vivo,surpassing the efficacy of either monotherapy.CONCLUSION Thioridazine demonstrates promising outcomes in preclinical GC models and offers a novel therapeutic approach for addressing trastuzumab resistance,particularly when used in conjunction with lapatinib.This compound has potential benefits for patients with Skp2-proficient tumors.展开更多
目的:通过免疫共沉淀与质谱分析技术从宫颈癌HeLa细胞中获取与S期激酶相关蛋白2(S-phase kinase-associated protein 2,SKP2)结合的蛋白质群体并预测其生物功能。方法:以免疫共沉淀与Western blotting技术建立SKP2免疫共沉淀体系,以SDS-...目的:通过免疫共沉淀与质谱分析技术从宫颈癌HeLa细胞中获取与S期激酶相关蛋白2(S-phase kinase-associated protein 2,SKP2)结合的蛋白质群体并预测其生物功能。方法:以免疫共沉淀与Western blotting技术建立SKP2免疫共沉淀体系,以SDS-PAGE和银染技术获得SKP2结合蛋白的特异条带,通过质谱分析技术获得可能与SKP2结合的蛋白群体,应用生物信息学技术对筛选得到的蛋白进行GO分析与KEGG分析。结果:HeLa细胞内存在一定水平的SKP2蛋白表达,可进行免疫共沉淀反应;成功建立了SKP2免疫共沉淀体系,并获得SKP2结合蛋白样品;针对差异的凝胶条带进行质谱分析,共鉴定出SKP2结合蛋白563个;设定筛选条件后,获得可信度较高的SKP2蛋白270个,进行GO分析与KEGG分析后初步预测了结合蛋白参与的细胞功能和信号通路。结论:从宫颈癌HeLa细胞中成功筛选获取SKP2结合蛋白,为后续筛选靶标结合蛋白和寻找细胞靶向药物奠定了基础。展开更多
背景与目的S期激酶相关蛋白2(Skp2)是细胞周期正性调节因子之一,它能促进周期蛋白依赖性激酶抑制剂p27的泛素化蛋白水解,在肿瘤中过表达,本研究旨在探讨非小细胞肺癌(non-small cell lung cancer NSCLC)中Skp2表达的临床意义及其与p27...背景与目的S期激酶相关蛋白2(Skp2)是细胞周期正性调节因子之一,它能促进周期蛋白依赖性激酶抑制剂p27的泛素化蛋白水解,在肿瘤中过表达,本研究旨在探讨非小细胞肺癌(non-small cell lung cancer NSCLC)中Skp2表达的临床意义及其与p27蛋白表达的关系。方法应用组织芯片和免疫组织化学方法检测Skp2和p27在68例NSCLC组织和17例正常支气管上皮细胞中的表达。结果Skp2仅在肺癌组织中表达,且与患者的组织学类型(P=0.039),肿瘤细胞的分化程度(P=0.016),性别(P=0.012)和吸烟与否(P=0.026)显著相关,而与患者的年龄和TNM分期无关。p27在正常支气管上皮细胞中均有表达,在肺癌组织中表达降低;Skp2阳性表达的患者中p27表达明显降低,两者呈负相关(P=0.021)。结论在NSCLC中,Skp2蛋白表达的增高与p27泛素化依赖的蛋白降解有关,提示Skp2蛋白过表达在NSCLC的发生和发展中可能起重要作用。展开更多
目的探讨人胃癌S期激酶相关蛋白2(S-phase k inase-assoc iated prote in 2,Skp2)表达的意义及与p27表达的关系。方法采用免疫组化方法检测138例原发性胃癌,配对癌旁胃黏膜,102例配对淋巴结转移胃癌组织,30例非典型增生,30例肠上皮化生...目的探讨人胃癌S期激酶相关蛋白2(S-phase k inase-assoc iated prote in 2,Skp2)表达的意义及与p27表达的关系。方法采用免疫组化方法检测138例原发性胃癌,配对癌旁胃黏膜,102例配对淋巴结转移胃癌组织,30例非典型增生,30例肠上皮化生(肠化),10例慢性浅表性胃炎和5例正常胃黏膜Skp2的表达及138例原发性胃癌p27的表达。结果Skp2表达的阳性率(%),肠化(12.68±0.86)及癌旁胃黏膜(19.32±1.22)均明显高于慢性浅表性胃炎(0.53±0.13)及正常胃黏膜(0.47±0.19)(P=0.000),后两者差异无显著性(P=0.716);非典型增生(16.74±0.82)明显高于肠化(P=0.000);原发性胃癌(31.34±2.17)明显高于非典型增生及癌旁胃黏膜(P值均=0.000);淋巴结转移胃癌组织(39.76±2.00)明显高于原发性胃癌(P=0.037)。胃癌Skp2的阳性率与分化程度(rho=0.315,P=0.000)、脉管内瘤栓(rho=0.303,P=0.000)及淋巴结转移(rho=0.254,P=0.000)呈正相关。胃癌Skp2表达与靶蛋白p27表达呈负相关(rho=-0.451,P=0.000)。结论Skp2蛋白过表达与胃癌的发生及转移有关;胃癌Skp2蛋白过表达与p27蛋白降解有关,提示Skp2蛋白过表达在胃癌发生、发展过程中可能起重要作用。展开更多
目的:研究非霍奇金淋巴瘤(non-hodgkin's lymphoma,NHL)中微小染色体维持蛋白2(Minichromosome maintenance protein 2,MCM2)与细胞间隙连接蛋白43(Connexin 43,Cx43)以及S期激酶相关蛋白2(S phase kinase-associated protein 2,Sk...目的:研究非霍奇金淋巴瘤(non-hodgkin's lymphoma,NHL)中微小染色体维持蛋白2(Minichromosome maintenance protein 2,MCM2)与细胞间隙连接蛋白43(Connexin 43,Cx43)以及S期激酶相关蛋白2(S phase kinase-associated protein 2,Skp2)在非霍奇金淋巴瘤中的表达及临床意义,以期为临床诊治非霍奇金淋巴瘤提供一定的参考。方法:选取2013年1月至2015年1月间入院诊治的非霍奇金淋巴瘤36例作为实验组,并选取同期入院诊治的淋巴结反应性增生18例作为对照组,应用免疫组化法检测MCM2、Cx43、Skp2的表达情况,同时分析MCM2、Cx43、Skp2的表达与非霍奇金淋巴瘤恶性程度、临床分期等的相关性。结果:实验组NHL患者MCM2阳性表达率为83.33%、Skp2阳性表达率为86.11%,显著高于对照组22.22%与27.78%的阳性表达率(P<0.01);NHL患者Cx43阳性表达率为22.22%,显著低于对照组61.11%阳性表达率(P<0.01)。NHL患者MCM2阳性表达与肿瘤恶性程度、临床分期及Ki67水平密切相关(P<0.05);NHL患者Cx43阳性表达与肿瘤恶性程度呈负相关(P<0.01);NHL患者Skp2阳性表达与肿瘤恶性程度及临床分期密切相关(P<0.05)。结论:MCM2、Cx43、Skp2的异常表达与非霍奇金淋巴瘤的恶性程度密切相关,联合检测MCM2、Cx43、Skp2可为非霍奇金淋巴瘤的诊治提供一定的参考。展开更多
基金Supported by Youth Fund of National Natural Science Foundation of China,No.81803575,No.31902287Kaifeng Science and Technology Development Plan Project,No.2203008+2 种基金Key Specialized Research and Promotion Project of Henan Province in 2023,No.232102311205Henan Medical Science and Technology Research Program Project,No.LHGJ20210801College Students Innovation and Entrepreneurship Training Program of Henan University,No.20231022007.
文摘BACKGROUND Trastuzumab constitutes the fundamental component of initial therapy for patients with advanced human epidermal growth factor receptor 2(HER-2)-positive gastric cancer(GC).However,the efficacy of this treatment is hindered by substantial challenges associated with both primary and acquired drug resistance.While S-phase kinase associated protein 2(Skp2)overexpression has been implicated in the malignant progression of GC,its role in regulating trastuzumab resistance in this context remains uncertain.Despite the numerous studies investigating Skp2 inhibitors among small molecule compounds and natural products,there has been a lack of successful commercialization of drugs specifically targeting Skp2.AIM To discover a Skp2 blocker among currently available medications and develop a therapeutic strategy for HER2-positive GC patients who have experienced progression following trastuzumab-based treatment.METHODS Skp2 exogenous overexpression plasmids and small interfering RNA vectors were utilized to investigate the correlation between Skp2 expression and trastuzumab resistance in GC cells.Q-PCR,western blot,and immunohistochemical analyses were conducted to evaluate the regulatory effect of thioridazine on Skp2 expression.A cell counting kit-8 assay,flow cytometry,a amplex red glucose/glucose oxidase assay kit,and a lactate assay kit were utilized to measure the proliferation,apoptosis,and glycolytic activity of GC cells in vitro.A xenograft model established with human GC in nude mice was used to assess thioridazine's effectiveness in vivo.RESULTS The expression of Skp2 exhibited a negative correlation with the sensitivity of HER2-positive GC cells to trastuzumab.Thioridazine demonstrated the ability to directly bind to Skp2,resulting in a reduction in Skp2 expression at both the transcriptional and translational levels.Moreover,thioridazine effectively inhibited cell proliferation,exhibited antiapoptotic properties,and decreased the glucose uptake rate and lactate production by suppressing Skp2/protein kinase B/mammalian target of rapamycin/glucose transporter type 1 signaling pathways.The combination of thioridazine with either trastuzumab or lapatinib exhibited a more pronounced anticancer effect in vivo,surpassing the efficacy of either monotherapy.CONCLUSION Thioridazine demonstrates promising outcomes in preclinical GC models and offers a novel therapeutic approach for addressing trastuzumab resistance,particularly when used in conjunction with lapatinib.This compound has potential benefits for patients with Skp2-proficient tumors.
文摘目的:通过免疫共沉淀与质谱分析技术从宫颈癌HeLa细胞中获取与S期激酶相关蛋白2(S-phase kinase-associated protein 2,SKP2)结合的蛋白质群体并预测其生物功能。方法:以免疫共沉淀与Western blotting技术建立SKP2免疫共沉淀体系,以SDS-PAGE和银染技术获得SKP2结合蛋白的特异条带,通过质谱分析技术获得可能与SKP2结合的蛋白群体,应用生物信息学技术对筛选得到的蛋白进行GO分析与KEGG分析。结果:HeLa细胞内存在一定水平的SKP2蛋白表达,可进行免疫共沉淀反应;成功建立了SKP2免疫共沉淀体系,并获得SKP2结合蛋白样品;针对差异的凝胶条带进行质谱分析,共鉴定出SKP2结合蛋白563个;设定筛选条件后,获得可信度较高的SKP2蛋白270个,进行GO分析与KEGG分析后初步预测了结合蛋白参与的细胞功能和信号通路。结论:从宫颈癌HeLa细胞中成功筛选获取SKP2结合蛋白,为后续筛选靶标结合蛋白和寻找细胞靶向药物奠定了基础。
文摘背景与目的S期激酶相关蛋白2(Skp2)是细胞周期正性调节因子之一,它能促进周期蛋白依赖性激酶抑制剂p27的泛素化蛋白水解,在肿瘤中过表达,本研究旨在探讨非小细胞肺癌(non-small cell lung cancer NSCLC)中Skp2表达的临床意义及其与p27蛋白表达的关系。方法应用组织芯片和免疫组织化学方法检测Skp2和p27在68例NSCLC组织和17例正常支气管上皮细胞中的表达。结果Skp2仅在肺癌组织中表达,且与患者的组织学类型(P=0.039),肿瘤细胞的分化程度(P=0.016),性别(P=0.012)和吸烟与否(P=0.026)显著相关,而与患者的年龄和TNM分期无关。p27在正常支气管上皮细胞中均有表达,在肺癌组织中表达降低;Skp2阳性表达的患者中p27表达明显降低,两者呈负相关(P=0.021)。结论在NSCLC中,Skp2蛋白表达的增高与p27泛素化依赖的蛋白降解有关,提示Skp2蛋白过表达在NSCLC的发生和发展中可能起重要作用。
文摘目的探讨人胃癌S期激酶相关蛋白2(S-phase k inase-assoc iated prote in 2,Skp2)表达的意义及与p27表达的关系。方法采用免疫组化方法检测138例原发性胃癌,配对癌旁胃黏膜,102例配对淋巴结转移胃癌组织,30例非典型增生,30例肠上皮化生(肠化),10例慢性浅表性胃炎和5例正常胃黏膜Skp2的表达及138例原发性胃癌p27的表达。结果Skp2表达的阳性率(%),肠化(12.68±0.86)及癌旁胃黏膜(19.32±1.22)均明显高于慢性浅表性胃炎(0.53±0.13)及正常胃黏膜(0.47±0.19)(P=0.000),后两者差异无显著性(P=0.716);非典型增生(16.74±0.82)明显高于肠化(P=0.000);原发性胃癌(31.34±2.17)明显高于非典型增生及癌旁胃黏膜(P值均=0.000);淋巴结转移胃癌组织(39.76±2.00)明显高于原发性胃癌(P=0.037)。胃癌Skp2的阳性率与分化程度(rho=0.315,P=0.000)、脉管内瘤栓(rho=0.303,P=0.000)及淋巴结转移(rho=0.254,P=0.000)呈正相关。胃癌Skp2表达与靶蛋白p27表达呈负相关(rho=-0.451,P=0.000)。结论Skp2蛋白过表达与胃癌的发生及转移有关;胃癌Skp2蛋白过表达与p27蛋白降解有关,提示Skp2蛋白过表达在胃癌发生、发展过程中可能起重要作用。
文摘目的:研究非霍奇金淋巴瘤(non-hodgkin's lymphoma,NHL)中微小染色体维持蛋白2(Minichromosome maintenance protein 2,MCM2)与细胞间隙连接蛋白43(Connexin 43,Cx43)以及S期激酶相关蛋白2(S phase kinase-associated protein 2,Skp2)在非霍奇金淋巴瘤中的表达及临床意义,以期为临床诊治非霍奇金淋巴瘤提供一定的参考。方法:选取2013年1月至2015年1月间入院诊治的非霍奇金淋巴瘤36例作为实验组,并选取同期入院诊治的淋巴结反应性增生18例作为对照组,应用免疫组化法检测MCM2、Cx43、Skp2的表达情况,同时分析MCM2、Cx43、Skp2的表达与非霍奇金淋巴瘤恶性程度、临床分期等的相关性。结果:实验组NHL患者MCM2阳性表达率为83.33%、Skp2阳性表达率为86.11%,显著高于对照组22.22%与27.78%的阳性表达率(P<0.01);NHL患者Cx43阳性表达率为22.22%,显著低于对照组61.11%阳性表达率(P<0.01)。NHL患者MCM2阳性表达与肿瘤恶性程度、临床分期及Ki67水平密切相关(P<0.05);NHL患者Cx43阳性表达与肿瘤恶性程度呈负相关(P<0.01);NHL患者Skp2阳性表达与肿瘤恶性程度及临床分期密切相关(P<0.05)。结论:MCM2、Cx43、Skp2的异常表达与非霍奇金淋巴瘤的恶性程度密切相关,联合检测MCM2、Cx43、Skp2可为非霍奇金淋巴瘤的诊治提供一定的参考。
