泰索帝联合塞来昔布对肺腺癌细胞增殖的影响

目的探讨泰索帝、塞来昔布单药以及联合用药对非小细胞肺癌细胞株A549增殖及凋亡的影响。方法以人NSCLC细胞株A549作为研究对象,采用MTT,免疫组化以及FCM检测泰索帝和塞来昔布对非小细胞肺癌A549细胞增殖、细胞凋亡、细胞周期和COX-2蛋白表达的影响。结果泰索帝在体外对NSCLC细胞株A549的生长抑制作用呈剂量、时间依赖性,48h最高65%,最低10%,联合塞来昔布(12.5μmol/l,25μmol/l)可以提高泰索帝的抑制率;经高剂量塞来昔布组(>50μmol/l)处理的细胞COX-2蛋白表达呈阴性,而低剂量组(12.5μmol/l,25μmol/l)以及联合用药组细胞COX-2蛋白表达呈阳性;塞来昔布(12.5μmol/l,25μmol/l)作用后,G0/G1期细胞比例增加,S、G2/M期比例下降,联合泰索帝可以提高细胞的凋亡率。结论泰索帝在体外对非小细胞肺癌细胞株的生长有明显的抑制作用,联合塞来昔布可以提高抑制率,诱导细胞凋亡,影响细胞周期的分布,但不是通过影响细胞内环氧化酶-2蛋白表达这一途径实现。     

（125）I联合ADM对乳腺癌MCF-7细胞增殖、凋亡的影响

目的研究放射性粒子125I联合化疗药物多柔比星（阿霉素,ADM）对乳腺癌MCF-7细胞增殖、凋亡的影响。方法按2×2析因设计将乳腺癌敏感株MCF-7细胞随机分成A、B、C、D 4组,A组：空白对照组;B组：单纯125I粒子组;C组：单纯ADM组;D组：125I粒子＋ADM组。采用流式细胞术检测各组干预后的细胞周期分布及细胞凋亡率。结果 （1）单纯125I粒子近距离低剂量率持续照射后将MCF-7细胞阻滞于G2~M期1。25I联合ADM将MCF-7细胞周期主要集中在G0~G1期,同时伴有较大比例的细胞凋亡。（2）各组细胞的早期凋亡率分别为：A组（0.99±0.05）%、B组（19.22±4.92）%、C组（16.57±4.73）%、D组（3.16±1.08）%;各组晚期凋亡及坏死率为：A组（0.32±0.18）%、B组（3.16±1.39）%、C组（3.24±0.75）%、D组（28.99±7.96）%,D组晚期凋亡及死亡率明显提高。结论 125I放射性粒子能有效诱导乳腺癌细胞凋亡,与化疗药物ADM联合作用除诱导细胞凋亡,还可导致大量细胞死亡,具有协同、增效的作用。     

环氧化酶-2抑制剂对肺癌细胞的增殖抑制和放射增敏的实验研究

目的研究环氧化酶-2(COX-2)抑制剂塞来昔布对肺癌A549细胞增殖、凋亡的影响及其对A549细胞的放射增敏作用.方法噻唑蓝(MTT)比色法检测塞来昔布对肺癌A549细胞存活的影响;流式细胞分析法(FCM)检测对A549细胞周期时相影响及凋亡的作用;体外培养克隆形成率的方法检测塞来昔布对A549细胞的放射增敏作用.结果塞来昔布对肺癌A549细胞有明显的增殖抑制作用,这种抑制作用有时间和剂量依赖性,50μmol/L处理24和72 h的抑制率分别为(4.27±1.33)%、(10.63±2.23)%,100μmol/L处理分别为(33.47±8.02)%、(61.97±7.32)%,主要是抑制增殖,使细胞聚集在G0/G1期,100μmol/L处理72 h还有细胞死亡.塞来昔布无凋亡诱导作用,也不增加放射诱导的凋亡.体外培养克隆存活曲线分析显示,100μmol/L塞来昔布处理24和72 h降低了各个照射剂量点的存活分数(72 h比24 h作用强),但在照射高剂量区更明显.对照、100μmol/L处理24、72 h在2 Gy剂量点的存活分数(SF2)分别为(0.53±0.06)、(0.52±0.11)和(0.46±0.05),处理24和72 h的放射增敏比为1.27和1.7(D0值比).结论塞来昔布对肺癌A549细胞有明显的增殖抑制作用,呈时间和剂量依赖性,使细胞聚集在G0/G1期,但无诱导凋亡作用.塞来昔布对肺癌A549细胞有放射增敏作用,在高照射剂量区增敏作用更为明显.     

蛋白激酶A活性升高对人肺腺癌A549细胞株增殖的影响

目的 探讨蛋白激酶A(PKA)活性升高对人肺腺癌A549细胞株增殖的影响.方法 用20、40、80μmol/L PKA活性激动剂Forskolin诱导A549细胞后,用噻唑蓝(MTT)比色法检测Forskolin作用下A549细胞增殖的变化;用流式细胞仪检测Forskolin作用下细胞周期分布的变化.结果 在Forskolin作用下,PKA活性升高使A549细胞的增殖显著下降(P<0.05);在40μmoL/L Forsko-lin作用A549细胞24 h后,流式细胞仪检测实验组G0/G1期细胞(82.14±1.18)%,S期细胞(5.45±0.22)%,G2/M期细胞(12.33±1.21)%;对照组G0/G1期细胞(64.50±2.18)%,S期细胞(8.54±0.45)%,G2/M期细胞(26.31±1.08)%,两组间比较差异有统计学意义(G1期P<0.05,S期P<0.05,G2期P<0.05).结论 PKA活性升高能够显著降低人肺腺癌A549细胞株的活力,使其处于G1期阻滞,细胞增殖受到明显抑制.     

重楼皂苷Ⅰ对肺腺癌A549细胞系放射增敏作用研究

[目的]探讨重楼皂苷Ⅰ对肺腺癌A549细胞的体外放射增敏作用及其可能机制.[方法]以肺腺癌A549细胞系为研究对象,MTT法检测重楼皂苷Ⅰ对A549细胞的抑制率,得到重楼皂苷Ⅰ对A549细胞的半数抑制浓度(IC50)及IC20、IC30,取IC20-30之间的重楼皂苷Ⅰ浓度用于放射增敏,随机分为对照组、单纯照射组、重楼皂苷Ⅰ、重楼皂苷Ⅰ+照射组,描绘细胞生长曲线、计算克隆形成率、流式细胞术检测细胞周期及凋亡率,Western Blot法检测survivin及p21waf/cip1蛋白表达.[结果]A549细胞在经重楼皂苷Ⅰ处理和照射后重楼皂苷Ⅰ+照射组A549细胞的增殖能力明显受抑(P＜0.01),克隆形成率明显下降(3.3％,P＜0.01),流式细胞术分析显示在重楼皂苷Ⅰ+照射组G2/M期阻滞(37.56％±1.99％,P＜0.01),凋亡率明显升高(9.34％±0.84％,P＜0.01).Western Blot检测survivin蛋白表达降低,而p21waf1/cip1蛋白表达增加.[结论]重楼皂苷Ⅰ具有较好的放射增敏作用,可能通过诱导细胞凋亡,细胞G2/M期阻滞,降低survivin蛋白表达,增加p21蛋白表达,从而产生放射增敏作用.     

西妥昔单抗对乳腺癌MDA-MB-231细胞放射敏感性影响的研究

目的:探讨西妥昔单抗(C225)对乳腺癌MDA-MB-231细胞株的放射增敏作用及其机制。方法:体外培养人乳腺癌MDA-MB-231细胞。使用活细胞计数试剂盒(CCK-8)检测浓度分别为1、5、25、125和625nmol/L C225对乳腺癌MDA-MB-231细胞株的增殖抑制率,计算C225的半数抑制浓度(IC50),并将20%IC50作为后续实验的浓度;观察C225联合X射线照射对MDA-MB-231细胞株增殖抑制的影响,并评价两者的联合效应。采用克隆形成实验检测MDA-MB-231细胞存活率,观察C225对细胞放射增敏性的影响,按多靶单击模型拟合细胞存活曲线,计算反映放射敏感性指标的细胞存活分数(SF2)、平均致死剂量(D0)、准阈剂量(Dq)和放射增敏比(SER)。流式细胞仪检测细胞凋亡及细胞周期情况。结果:1)1、5、25、125、625nmol/L C225对细胞的增殖抑制率分别为(3.66±0.26)%、(16.48±1.78)%、(35.41±2.46)%、(47.84±1.67)%和(62.49±2.94)%,C225作用于细胞48h的IC50为(151±2.98)nmol/L。2、4、6和8Gy的照射对细胞的增殖抑制率分别为31.12%、47.95%、59.12%和68.54%;当30nmol/L的C225与上述剂量的照射联合使用,细胞增殖抑制率分别为66.27%、82.03%、89.86%和93.03%,两者表现出协同作用,P<0.05。2)克隆形成实验显示,C225+照射组与单纯照射组相比,克隆形成能力下降,SF2、D0及Dq均下降(P<0.05),放射增敏比(SERD0)为1.41。3)细胞凋亡实验结果显示,C225+0、2、4、6、8Gy照射组凋亡率分别为(11.76±0.81)%、(21.46±1.40)%、(38.23±1.76)%、(44.01±1.23)%和(50.41±1.87)%,均高于单独照射组相应剂量点的凋亡率,分别为(2.94±0.58)%、(10.72±0.34)%、(21.14±1.08)%、(24.12±0.98)%和(30.05±2.64)%,P<0.05。C225联合照射明显增强了照射诱导的细胞凋亡。4)细胞周期分布显示,单独使用C225使细胞阻滞于G0/G1期,并减少S期细胞比例,C225作用后G0/G1期及S期细胞比例分别为(58.35±1.66)%和(31.12±0.23)%,P<0.05;单独照射使细胞阻滞于G2/M期,G2/M期细胞比例为(28.25±1.55)%,P<0.05;C225与照射联合作用后,G0/G1期细胞比例进一步升高,为(59.90±2.21)%,S期细胞比例进一步降低,为(13.67±1.34)%,P<0.05。结论:C225对MDA-MB-231细胞具有放射增敏作用,其机制可能与C225增强放射线对细胞的生长抑制作用、抑制肿瘤细胞亚致死性损伤的修复、增加细胞凋亡和诱导细胞G0/G1期阻滞有关。     

番茄红素对肺癌A549细胞增殖和凋亡的影响及其机制研究

目的:研究番茄红素(lycopene,LP)对人非小细胞肺癌A549细胞增殖和凋亡的影响并探讨其机制.方法:体外培养并取对数生长期人非小细胞肺癌A549细胞,分别给予不同浓度的LP(2.5、5、10、20 μg/ml)和顺铂(40 μg/ml)进行干预,48 h后采用四甲基偶氮唑盐(MTT)比色法测定吸光度值并计算细胞增殖抑制率,流式细胞术(FCM)检测细胞周期和细胞凋亡状况,逆转录PCR法(RT-PCR)检测细胞中凋亡相关基因(BaxmRNA、bcl-2 mRNA)表达,免疫印迹法(Western blot)检测凋亡相关蛋白(caspase-3)表达.结果:与空白对照组比较,LP能够剂量依赖性地提高人非小细胞肺癌A549细胞增殖抑制率,延长细胞周期中G0/G1期并缩短G2/M期,提高A549细胞凋亡率(AI),上调Bax mRNA表达、下调bcl-2 mRNA表达,提高Bax/bcl-2比值,上调caspase-3蛋白表达.结论:LP具有抑制人非小细胞肺癌A549细胞增殖并促进其凋亡的作用,其机制可能与LP能够阻滞细胞周期并调节凋亡相关基因蛋白表达有关.     

曲古菌素A对人结肠癌HCT-116细胞的作用及分子机制

目的:观察曲古菌素A对人结肠癌HCT-116细胞的作用。方法:采用不同浓度曲古菌素A(75、150、300、600ng/ml)分别处理人结肠癌HCT-116细胞,CCK8法检测其对细胞增殖的影响;流式细胞仪检测细胞周期和凋亡的变化;蛋白免疫印迹法检测细胞周期相关基因CyclinD1和DMTF1的表达。结果:曲古菌素A(75、150、300、600ng/ml)组的细胞抑制率在24h、48h、72h分别为(11.88±1.7、24.17±1.40、37.32±1.85)%、(21.00±1.45、31.22±2.10、54.94±1.71)%、(37.69±2.67、43.86±3.20、71.93±5.79)%和(51.90±2.60、61.80±4.90、87.93±5.39)%,不同浓度的曲古菌素A组细胞抑制率均明显高于对照组(P<0.05),不同浓度的曲古菌素A组细胞抑制率比较也有统计学差异(P<0.05)。不同浓度的曲古菌素A作用48h后细胞以G0期居多,细胞阻滞在G0/G1→S期。不同浓度的曲古菌素A作用细胞48h后,凋亡率分别为(4.6±0.55)%、(37.06±7.89)%、(65.53±5.55)%、(75.93±3.28)%,各曲古菌素A组细胞凋亡率均明显高于对照组(P<0.05)。蛋白免疫印迹法显示曲古菌素A可下调CyclinD1表达、上调DMTF1表达。结论:曲古菌素A可明显抑制结肠癌HCT-116细胞增殖,其机制可能与调控细胞周期相关基因CyclinD1和DMTF1的表达、阻滞细胞周期、诱导细胞凋亡等有关。     

β-榄香烯乳联合照射对DNA—PKcs基因表达及凋亡的影响

目的探讨β-榄香烯乳影响肺腺癌A549细胞凋亡及DNA—PKcs基因表达的放射增敏机制。方法实验分为对照组、单纯照射组（4Gy照射）、单纯药物组（10μg/ml、20μg／mlβ-榄香烯乳）、药物＋照射组（10μg/ml、20μg/mlβ-榄香烯乳＋4Gy照射）。采用四氮唑蓝比色分析法（MTF法）检测β-榄香烯乳对肺腺癌A549细胞的半数抑制浓度（IC50）;克隆形成实验计算细胞存活率;采用流式细胞仪检测细胞周期与凋亡率;逆转录实时荧光定量PCR技术（RT—PCR）检测细胞DNA—PKcs基因mRNA表达量。结果MTF法测得β-榄香烯乳对A549细胞的IC50值为120μg/ml;克隆形成实验证明β-榄香烯乳对A549细胞有放射增敏作用;药物＋照射组细胞G2／M期比率及凋亡率明显高于单纯照射组及单纯用药组（P〈0．05）;药物＋照射组DNA—PKcs基因mRNA表达量与单纯照射及单纯用药组相比明显下降（P〈0．05）。结论β-榄香烯乳可通过促进A549细胞凋亡及抑制DNA—PKcs基因mRNA表达实现对A549细胞放射增敏作用。     

全反式维甲酸对人肺癌细胞诱导凋亡的作用

目的:研究全反式维甲酸(ATRA)对肺癌细胞生长的影响及其凋亡作用.方法:应用MTT法检测ATRA对体外培养人肺癌细胞株细胞A549的抑制率,光镜及透射电镜、流式细胞仪等检测ATRA对A549细胞诱导凋亡的作用.结果: 不同浓度的ATRA对A549细胞的增殖均有抑制作用,且量效关系明显.但大剂量主要导致细胞死亡,以10-5mol*L-1的ATRA作用效果最好,经10-5mol*L-1 ATRA作用7d后,A549细胞增殖抑制率达到60%.光镜及透射电镜下可见细胞恶性表型向正常表型发生逆转,并可观察到凋亡小体.流式细胞仪分析结果显示,ATRA处理组细胞周期延迟,G1期比例明显升高,S、G2期比例下降.结论:ATRA可抑制肺癌细胞的增殖,并诱导癌细胞凋亡.     

Biomarkers in clear cell renal cell carcinoma

The treatment of advanced renal cell carcinoma (RCC) has evolved significantly following the identification of the von Hippel–Lindau (VHL) gene and the function of its protein, and subsequent development of antiangiogenic therapies. A series of clinical trials resulted in the approval of three new agents with significant activity in this disease. Additional studies are now underway to identify subsets of patients most likely to benefit. This article reviews the current therapy for advanced RCC and the development of biomarkers in RCC. This requires the identification of disease characteristics at a clinical, genetic and molecular level associated with response and/or surrogate measures of clinical benefit. Currently, a variety of prognostic factors (lactate dehydrogenase, performance status, disease-free interval, hemoglobin and calcium levels) are utilized to predict the survival of RCC patients. The use of validated biomarkers in either serum/plasma, urine or tissue could enhance this process, as well as define at the molecular and genetic levels, factors associated with response to therapy and/or the development of resistance. Examples include plasma VEGF levels, VHL gene mutation status and carbonic anhydrase IX levels in tumor tissue, among others. Validation of such biomarkers is crucial in order for them to be clinically useful.     

Biomarkers in clear cell renal cell carcinoma

The treatment of advanced renal cell carcinoma (RCC) has evolved significantly following the identification of the von Hippel-Lindau (VHL) gene and the function of its protein, and subsequent development of antiangiogenic therapies. A series of clinical trials resulted in the approval of three new agents with significant activity in this disease. Additional studies are now underway to identify subsets of patients most likely to benefit. This article reviews the current therapy for advanced RCC and the development of biomarkers in RCC. This requires the identification of disease characteristics at a clinical, genetic and molecular level associated with response and/or surrogate measures of clinical benefit. Currently, a variety of prognostic factors (lactate dehydrogenase, performance status, disease-free interval, hemoglobin and calcium levels) are utilized to predict the survival of RCC patients. The use of validated biomarkers in either serum/plasma, urine or tissue could enhance this process, as well as define at the molecular and genetic levels, factors associated with response to therapy and/or the development of resistance. Examples include plasma VEGF levels, VHL gene mutation status and carbonic anhydrase IX levels in tumor tissue, among others. Validation of such biomarkers is crucial in order for them to be clinically useful.     

Non-small and small cell lung carcinoma cell lines exhibit cell type-specific sensitivity to edelfosine-induced cell death and different cell line-specific responses to edelfosine treatment

The unique signal transduction pathways that distinguish non-small cell lung carcinoma (NSCLC) from small cell lung carcinoma (SCLC) are poorly understood. We investigated the ability of edelfosine, an inhibitor of phosphatidylinositol-specific phospholipase C (PLC) to inhibit cell viability among four NSCLC cell lines and four SCLC cell lines. The differential sensitivity of cells to edelfosine's cytostatic and cytotoxic effects has been attributed to edelfosine-induced changes in the activities of many enzymes, including c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinases (ERK), p38 kinase, and poly(ADP-ribose) polymerase (PARP). To investigate the role of these enzymes in edelfosine-induced cytotoxicity, we correlated edelfosine-induced changes in enzyme activity and cell viability among the different NSCLC and SCLC cell lines. We found that NSCLC cells are much more susceptible to the cytotoxic effects of this drug than are SCLC cells. Three out of the four edelfosine-sensitive NSCLC cell lines (NCI-H157, NCI-H520, NCI-H522) exhibit G2/M arrest, significant apoptosis and some degree of JNK activation in response to drug treatment. In contrast, none of the SCLC cell lines exhibit edelfosine-induced G2/M arrest or significant apoptosis. A comparison of the edelfosine-induced effects among the sensitive and resistant lung cancer lines indicates that there is little correlation between edelfosine-induced cytotoxicity and altered activities of JNK, ERK, p38, or cleavage of PARP. These results demonstrate that edelfosine-induced changes in JNK, ERK, p38, or PARP are not good predictors of cell susceptibility to edelfosine-induced cytotoxicity. Thus, edelfosine-induced inactivation of PLC may disrupt signaling cascades downstream of PLC that are unique to individual cellular environments. These findings also identify edelfosine as one of the few potential chemotherapeutic agents that has a greater cytotoxic effect against NSCLC cells than SCLC cells.     

桥接整合因子1通过AKT-mTOR通路诱导非小细胞肺癌H1975细胞周期阻滞

目的：研究桥接整合因子1（bridging intergrator 1,Bin1）基因过表达后对非小细胞肺癌细胞株H1975细胞周期的影响及其作用机制。方法：构建携带Bin1基因的CMV-MCS-GFP-SV40-Neomycin-Bin1质粒,并转染H1975细胞（Bin1+组）,另设置空白质粒转染组（Bin1-组）及空白对照组（Ctrl组）,利用RT-PCR和Western blotting分别检测3组细胞中Bin1在mRNA和蛋白质水平的表达情况。流式细胞术检测不同处理组H1975细胞周期的变化,Western boltting分别检测各组中AKT、mTOR磷酸化水平及细胞周期相关蛋白（周期蛋白D1、CDK4、Rb）的表达情况。结果：与Bin1-组、Ctrl组比较,Bin1+组H1975细胞中Bin1在mRNA、蛋白水平表达明显上调（均P<0.05）; H1975细胞阻滞在G1期\[（60.53±1.89）% vs（46.14±1.56）%、（47.33±2.07）%,均P<0.05\]; Bin1+组H1975细胞内p-AKT、p-mTOR表达下调（均P<0.05）,AKT、mTOR表达变化无统计学差异（P>0.05）;周期蛋白D1、CDK4的表达量均明显下调(P<0.05),Rb表达量明显增加（P<0.05）。结论：Bin1基因在H1975细胞株过表达后明显诱导细胞周期阻滞,其机制可能是通过抑制AKT-mTOR通路及其细胞周期相关蛋白实现的。     

Landscape of immune cell infiltration in clear cell renal cell carcinoma to aid immunotherapy

The tumor microenvironment, comprised of tumor cells and tumor-infiltrating immune cells, is closely associated with the clinical outcome of clear cell renal cell carcinoma (ccRCC) patients. However, the landscape of immune infiltration in ccRCC has not been fully elucidated. Herein, we applied multiple computational methods and various datasets to reveal the immune infiltrative landscape of ccRCC patients. The tumor immune infiltration (TII) levels of 525 ccRCC patients using a single-sample gene were examined and further categorized into immune infiltration subgroups. The TII score was characterized by distinct clinical traits and showed a significant divergence based on gender, grade, and stage. A high TII score was associated with the ERBB signaling pathway, the TGF-β signaling pathway, and the MTOR signaling pathway, as well as a better prognosis. Furthermore, patients with high TII scores exhibited greater sensitivity to pazopanib. The low TII score was characterized by a high immune infiltration level of CD8⁺ T cells, T follicular helper cells, and regulatory T cells (Tregs). Moreover, the immune check point genes, including CTLA-4, LAG3, PD-1, and IDO1, presented a high expression level in the low TII score group. Patients in the high TII score group demonstrated significant therapeutic advantages and clinical benefits. The findings in this study have the potential to assist in the strategic design of immunotherapeutic treatments for ccRCC.