吉西他滨联合放射线照射对人胆管癌细胞放射增敏作用的实验研究

目的 研究和探讨吉西他滨联合放射线照射对胆管癌细胞的放射增敏作用.方法 取指数生长期的胆管癌细胞(QBC939),采用细胞克隆形成分析法检测吉西他滨单药毒性,确定IC10、IC50和IC90作为下一步实验的药物浓度.将QBC939细胞分为对照组、单纯药物组、单纯照射组及照射前、后吉西他滨IC10、IC50和IC90浓度下作用24 h组,X线照射剂量为0、1、2、4、6、8、10 Gy.采用多靶单击数学模型拟合细胞存活曲线,分别用D0、Dq比计算放射增敏比(SERD0、SERD).结果 吉西他滨对QBC939细胞的Ic10、IC50和IC90值分别为0.1、11.0、21.5 nmol/L.吉西他滨低浓度(IC10)时只在照后给药且较低照射剂量区域(≤2 Gy)表现出放射增敏作用(SERDq=1.52);中浓度(IC50)时照前给药增敏作用最广泛(SERD0=1.27,SERDq=116.93),照后给药只在较低剂量区域(≤2 Gy)有放射增敏作用(SERDq=81.85);高浓度(IC90)时照射前后给药均具有一定放射增敏作用,但照前给药的作用明显强于照后给药.结论 吉西他滨与X线联合应用具有一定的放射增敏作用,但应注意药物浓度及给药顺序.     

三氧化二砷对纤维肉瘤细胞放射增敏作用的研究

目的 研究和探讨三氧化二砷（As2O3）是否对纤维肉瘤细胞有放射增敏作用。方法以人纤维肉瘤细胞HTl080为实验对象，首先检测As2O3的单药毒性，确定IC10、IC50和IC90。放射增敏作用的实验分为空白对照组、单纯给药组、单纯照射组（包括1、2、4、6、8、10Gy剂量）、照射前加药组（于照射前24h加入设定浓度的As2O3，药物作用24h后进行照射）和照射后加药组（于照射后即刻加入设定浓度的As2O3，药物作用24h）。所有实验均重复3次。采用克隆形成分析法观察单纯照射和照射联合As2O3对细胞的杀伤作用。计算细胞的存活分数，用多靶单击模型进行拟合并做图。结果 HT1080细胞的IC10、IC50和IC90剂量分别为0．57、3．67和12．0μmol／L。无毒剂量的As2O3照射前给药增敏比（SER）为0．86（Do值比）、0．98（SF2值比），照射后给药SER为0．99（Do值比）、1．09（SF2值比）。IC50剂量的As2O3照射前给药SER为0．90（Do值比）、0．87（SF2值比），照射后给药SER为1．14（Do值比）、1．08（SF2值比）。IC90剂量的As2O3照射前给药和照射后给药的SER均为1．14（Do值比）、3．20（SF2值比），As2O3对低剂量照射的放射增敏作用好于高剂量照射（SERSF2〉SERDo）。结论 As2O3对HT1080纤维肉瘤细胞具有一定的放射增敏作用，为临床放疗和As2O3联合应用提供了实验依据。     

卡培他滨对人鼻咽癌细胞CNE-2放射增敏作用的实验研究

目的观察卡培他滨对人鼻咽癌细胞CNE-2的放射增敏作用,并探讨其可能的机制。方法用MTT法测定卡培他滨抑制细胞增殖20%的药物浓度(IC_(20)),以卡培他滨24 h IC_(20)值对CNE-2细胞分别处理3 h、6 h、12 h、24 h,依次设为4个卡培他滨+照射组,对其给予6MeV X线分别单次照射0、2Gy、4Gy、6Gy、8Gy,另设对照组为单纯照射组;用克隆形成试验计算各组的放射增敏比,得到细胞生存曲线;5组细胞均给予6MV X射线单次照射6Gy,流式细胞仪检测各组的细胞周期变化情况及凋亡率。结果卡培他滨的24 h IC_(20)值为0.26μg/mL,卡培他滨处理3 h、6 h、12 h、24 h后的放射增敏比分别为1.08、1.15、1.22和1.35;卡培他滨处理后的照射组可将细胞阻滞在S期,且作用24 h组的细胞凋亡率达45.9%;S期细胞比例及细胞凋亡率与卡培他滨作用时间成正相关。结论卡培他滨(24 h IC_(20))对CNE-2细胞有放射增敏作用,其增敏比随作用时间的延长而增大,24h后增敏作用达最强,主要通过诱导细胞S期阻滞及促进凋亡来影响增敏。     

紫杉醇联合放射线照射对肺腺癌细胞放射增敏作用的研究

目的 探讨化疗药紫杉醇是否对肺腺癌A973细胞有放射增敏作用.方法 取指数生长期的人肺腺癌细胞A973,采用成克隆分析法检测紫杉醇毒性,确定IC10、IC50和IC90剂量作为药物浓度.分析照射前后紫杉醇IC10、IC50和IC90浓度下作用24 h,照射剂最分别为0、1、2、4、6、8、10 Gy时的细胞存活分数,并用多靶单击模型拟合细胞存活曲线.采用流式细胞术分析不同浓度紫杉醇作用0、2、4、6、10、18、24 h,A973细胞周期分布变化.结果 紫杉醇对A973细胞的IC10、IC50和IC90剂量分别为0.5、2.6和8.7 nmol/L.IC10剂量紫杉醇照前给药增敏比为0.97(D0值比)、1.01(D0值比)、1.00(SF2值比),照后给药为0.97、1.02、1.02;IC50剂量照前给药为1.06、129.00、2.61,照后给药为0.94、129.00、2.14;IC90剂量照前给药为1.00、120.00、2.09,照后给药为0.98、120.00、2.09.IC10剂量紫杉醇对A973细胞无明显的G2+M期阻滞作用,而IC50和IC90剂量紫杉醇分别于2和18 h将A973细胞阻滞在G2+M期.结论 紫杉醇对肺腺癌细胞A973产生明显的放射增敏作用,且照射前后给药均有相似的增敏作用,中高浓度剂量联合小剂量X线照射增敏效果最好.     

三氧化二砷对人肺腺癌A549细胞的放射增敏作用

[目的]探讨三氧化二砷(As2O3)对人肺腺癌A549细胞的放射增敏作用.[方法]MTT法确定As2O3对A549细胞的半数致死浓度(LD50),采用集落形成法观察20％LD50的As2O3对A549细胞的放射增敏作用.[结果]细胞生长抑制随As2O3浓度的增加而增强.As2O3+照射组的细胞存活率低于单纯照射组,D0值小于单纯照射组(1.46Gy vs 2.03 Gy),Dq值也小于单纯照射组(0.49 Gy vs 1.35Gy),存活曲线肩区(Dq)变小,放射增敏比(SER)为1.39.[结论]As2O3对肺腺癌A549细胞有明显的放射增敏作用,抑制A549细胞的修复能力使照射后细胞存活分数降低是其放射增敏的可能机制.     

吉西他滨对人鼻咽癌细胞系CNE-1的放射增敏作用

目的:观察吉西他滨对鼻咽癌细胞CNE-1的放射增敏作用并探讨其作用机理.方法:用克隆形成法制作不同处理条件的细胞存活曲线,用流式细胞仪分析测定细胞周期分布.结果:单纯用药0.1μmol/L和2μmol/L吉西他滨短时间作用时(12小时)未见到肿瘤细胞毒性作用.两个浓度的吉西他滨处理CNE-1鼻咽癌细胞12小时和24小时后均见到放射增敏作用,放射增敏比(SERD0)及2Gy时的放射增敏比(SERSF2)分别为1.36,1.83;1.56,2.16及1.55,1.36,1.43,1.40.0.1μmol/L和2μmol/L的吉西他滨作用24小时后照射均见到G1期细胞阻滞.准阈剂量(Dq)值在2μmoL/L中比较低,特别是在作用24小时后照射组.结论:吉西他滨对CNE-1鼻咽癌细胞具有明显的放射增敏作用,并且在高剂量照射时(Gy)随着剂量的增加及作用时间的延长增敏作用愈加明显,其作用机制可能与该药能阻止细胞由G1期进入S期及在高浓度、长时间作用时亚致死性损伤修复比较少有关.     

羟基喜树碱放射增敏作用的离体研究

目的　应用克隆形成方法 ,研究羟基喜树碱 (HCPT)对人鼻咽癌细胞系 (CNE)和胃癌细胞系 (BGC 82 3)的放射增敏作用。方法　实验分为单纯照射组和照射加药组。照射加药组在照射后均立即给予HCPT 2 μg/ml(药物剂量为ID50 剂量 ) ,37℃孵箱内作用 4h。应用克隆形成方法 ,观察单纯照射和照射加HCPT对细胞的杀伤作用。计算细胞存活率 ,用单击多靶数学模型进行曲线拟合做图。结果　BGC 82 3细胞单纯照射组D0 值为 1 17Gy,Dq 值为 1 91Gy,N值为 5 14;照射加HCPT组D0 值为 0 95Gy ,Dq 值为 0 0 1Gy ,N值为 1 0 1;放射增敏比 (SER)为 1 2 3(1 17/ 0 95 )。CNE Ⅰ细胞单纯照射组D0 值为 1 6 0Gy ,Dq 值为 0 6 5Gy ,N值为 1 5 ;照射加HCPT组D0 值为 0 95Gy ,Dq 值为 0 0 1Gy ,N值为 1 0 1;SER为 1 6 8(1 6 / 0 95 )。结论　研究结果显示 ,HCPT具有一定的放射增敏作用 ,为临床的放疗和HCPT的联合应用提供了实验依据。     

白藜芦醇对CNE-1鼻咽癌细胞放射增敏效应的研究

目的:探讨白藜芦醇(RV)对鼻咽癌细胞CNE-1的放射增敏作用及其机制.方法:将实验分为空白对照组、单纯用药组(10、20和50 μmol/L RV)、单纯照射组(1、2、3、4、5和7 Gy)以及实验组(药物+照射).利用多靶单击模型拟合放射剂量-细胞生存曲线,检测RV对鼻咽癌细胞CNE-1的放射增敏效应.用流式细胞仪观察RV对细胞周期分布和细胞凋亡的影响,并观察细胞的形态学变化.结果:RV作用48 h后,10、20、50 μmol/L RV的放射增敏比分别为1.07、1.32和1.66,呈药物浓度依赖性.流式细胞仪检测显示,RV和照射均可使G2/M期细胞阻滞,凋亡值(AI)增加,单纯用药组(10、20和50 μmol/L RV)、单纯照射组(1、2、3、4、5和7 Gy)和实验组(药物+照射)的G2/M及AI值均随药物浓度的增加而增加,P值均＜0.01.形态学检测可见凋亡小体,且两者具有协同作用.结论:RV对人鼻咽癌细胞CNE-1具有放射增敏效应,其机制可能与RV抑制细胞修复、导致G2/M期细胞阻滞和诱导细胞凋亡有关.     

紫杉醇联合放射线照射对鼻咽癌细胞的放射增敏效应

目的探讨化疗药紫杉醇(PTX)联合放射线照射对鼻咽癌细胞的放射增敏作用。方法取指数生长期的CNE-I细胞,采用克隆形成分析法检测PTX的单药毒性,确定IC10、IC50和IC90剂量作为实验的药物浓度。分析照射前后PTX IC10、IC50和IC90浓度下作用24 h,照射剂量分别为0～10 Gy时对CNE-I细胞的放射增敏效应。采用流式细胞术分析不同浓度PTX作用0、2、6、12、18和24 h CNE-I细胞周期分布的变化。结果PTX对CNE-I细胞的IC10、IC50和IC90剂量分别为0.05、1.0和2.5 nmol/L。当小剂量照射前后,分别用0.05和1.0 nmoL/L PTX作用24 h,具有一定放射增敏作用。PTX浓度为2.5和10.0 nmol/L时,CNE-I细胞出现明显的G2/M期阻滞,高峰出现在18 h。结论在适当的结合序贯的基础上,PTX联合放射线照射对CNE-I细胞具有放射增敏效应,其增敏作用可能与PTX导致的细胞G2/M期阻滞相关。     

艾迪注射液对肺癌细胞A549放射增敏作用的实验研究

目的研究艾迪注射液对人肺腺癌细A549放射增敏作用及机制。方法（1）MTT法测定药物对细胞的抑制作用,计算半数抑制浓度IC50。（2）应用集落形成法观察药物对细胞的放射增敏作用。（3）流式细胞仪分析药物对细胞周期分布、凋亡的影响。结果药物作用48小时后,细胞生长受到抑制,且细胞抑制率随药物浓度的升高而增加。IC50为16.04 mg/mL。加药照射组与单纯照射组比较,细胞存活率下降,药物有放射增敏作用,随药物作用时间的延长放射增敏作用增强。单纯照射组、加药24小时、48小时后照射组三组D0值依次为2.13 Gy、2.04 Gy、1.64 Gy,Dq值依次为2.88 Gy、1.68 Gy、1.51 Gy,加药24小时及48小时后照射组放射增敏比SERD0分别为1.04、1.3,SERDq分别为1.71、1.91。流式细胞仪检测药物作用后G2/M期细胞增加,细胞凋亡率升高,药物作用时间延长以上变化更显著。结论艾迪注射液对肺癌细胞A549有抑制作用及放射增敏作用,其放射增敏作用机制可能为抑制细胞亚致死损伤修复,阻滞细胞于G2/M期,诱导细胞凋亡。     

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。     

Inhibition of S-adenosylhomocysteine hydrolase decreases cell mobility and cell proliferation through cell cycle arrest

S-adenosylhomocysteine hydrolase (AHCY) hydrolyzes S-adenosylhomocysteine to adenosine and l-homocysteine, and it is already known that inhibition of AHCY decreased cell proliferation by G2/M arrest in MCF7 cells. However, the previous study has not indicated what mechanism the cell cycle arrest is induced by. In this study, we aimed to investigate the different cell cycle mechanisms in both p53 wild-typed MCF7 and p53 mutant-typed MCF7-ADR by suppressing AHCY. We extensively proved that AHCY knockdown has an anti-proliferative effect by using the WST-1 assay, BrdU assay, and cell cytometry analysis and an anti-invasive, migration effect by wound-healing assay and trans-well analysis. Our study showed that down-regulation of AHCY effectively suppressed cell proliferation by regulating the MEK/ERK signaling pathway and through cell cycle arrests. The cell cycle arrest occurred at the G2/M checkpoint by inhibiting degradation of cyclinB1 and phosphorylation of CDC2 in MCF7 cells and at the G1 phase by inhibiting cyclinD1 and CDK6 in MCF7-ADR cells. Finally, we determined that AHCY regulates the expression of ATM kinase that phosphorylates p53 and affects to arrest of G2/M phase in MCF7 cells. The findings of this study significantly suggest that AHCY is an important regulator of cell proliferation through different mechanism in between MCF7 and MCF7-ADR cells as p53 status.