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
目的检测IgA肾病(immunoglobulin A nephropathy,IgAN)患者血清Rho相关卷曲螺旋形成蛋白激酶2(rho associated coiled coil containing protein kinase 2,ROCK2)、视黄醇结合蛋白4(retinol-binding protein 4,RBP4)表达水平,分析二者在...目的检测IgA肾病(immunoglobulin A nephropathy,IgAN)患者血清Rho相关卷曲螺旋形成蛋白激酶2(rho associated coiled coil containing protein kinase 2,ROCK2)、视黄醇结合蛋白4(retinol-binding protein 4,RBP4)表达水平,分析二者在评估患者病情及预测预后中的价值。方法选取2016年6月至2019年6月98例中国贵航集团302医院已确诊收治的IgAN患者为IgAN组,其中轻度IgAN 36例、中度IgAN 32例、重度IgAN 30例;同期选择在中国贵航集团302医院体检的健康者100名为对照组。酶联免疫吸附法检测血清中ROCK2、RBP4水平;采用Kaplan-Meier生存曲线分析ROCK2、RBP4表达水平与IgAN患者预后的关系;Pearson相关性分析ROCK2与RBP4表达相关性;受试者工作特征曲线(receiver operating characteristic curve,ROC)分析ROCK2、RBP4对IgAN的诊断价值;比例风险回归模型法分析IgAN患者预后的影响因素。结果IgAN组血清中ROCK2、RBP4表达水平分别为(24.25±5.31)μg/L、(59.70±9.43)μg/L,显著高于对照组的(15.57±4.16)μg/L、(32.14±7.82)μg/L,差异具有统计学意义(t=12.818、22.404,均P<0.001);轻度、中度、重度IgAN患者血清中ROCK2水平分别为(18.69±4.36)μg/L、(23.75±5.31)μg/L、(31.46±5.62)μg/L,RBP4表达水平分别为(45.37±7.86)μg/L、(62.48±8.12)μg/L、(73.94±8.65)μg/L。随着病情的加重,IgAN患者血清ROCK2、RBP4表达水平逐渐升高,差异具有统计学意义(F=51.854、102.234,均P<0.05);IgAN患者血清中ROCK2、RBP4表达水平与Lee氏分级、IgA/C3比值、病情程度有关(P<0.05);ROCK2、RBP4高表达组患者预后生存率分别为60.00%、74.19%,均显著低于低表达组的89.71%、91.67%,差异具有统计学意义(log rankχ^(2)=4.674,P=0.031);ROC曲线结果显示,血清ROCK2诊断IgAN的曲线下面积(area under curve,AUC)为0.889,敏感度为80.61%,特异性为80.00%,截断值为18.79μg/L;RBP4诊断IgAN的AUC为0.986,敏感度为96.94%,特异性为95.00%,截断值为41.31μg/L;二者联合检测的AUC为0.997,敏感度为98.98%,特异性为94.86%。Pearson相关性分析显示,IgAN患者血清ROCK2与RBP4表达水平呈显著正相关(r=0.701,P<0.001);多因素比例风险回归模型法分析表明,IgA/C3(OR=2.683,95%CI:1.060~6.794)、ROCK2(OR=1.831,95%CI:1.027~3.264)、RBP4(OR=1.517,95%CI:1.104~2.084)均是IgAN患者预后生存的影响因素(均P<0.05)。结论ROCK2、RBP4在IgAN患者血清中高表达,二者与IgAN患者的临床病理特征、病情严重程度及预后关系密切,ROCK2、RBP4表达水平对IgAN的诊断具有一定的价值。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
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。
