二氢杨梅素联合小剂量VP16抗肺癌研究

目的：研究二氢杨梅素(DMY)联合小剂量依托泊苷(VP16)治疗非小细胞肺癌的作用及机制,探索减毒高效联合化疗新方案。方法：以非小细胞肺癌A549细胞为研究对象,MTT实验摸索并确定单药作用浓度及时间(IC₂₀),之后分别给予不同药物(对照组、VP16组、DMY组、联合组)干预48 h;倒置显微镜下观察细胞增殖情况;Tunel实验检测各组细胞凋亡情况;Western blot实验检测凋亡相关蛋白PARP、Cleaved PARP、Caspase-3、Cleaved Caspase-3、Bax蛋白表达水平。结果：低剂量DMY(80μg·mL^-1)联合低剂量VP16(0.06μg·mL^-1)治疗后,对A549细胞的抑制作用显著增强,镜下可见细胞融合率显著降低,细胞固缩明显;Tunel实验显示联合组凋亡显著增强;Western blot结果显示联合组与单独用药组或与对照组比较Cleaved PARP、Cleaved Caspase-3、Bax蛋白表达显著增加,同时PARP、Caspase-3蛋白表达显著下降。结论：小剂量DMY...     

EP方案联合清肺胶囊治疗小细胞肺癌

目的　评价中成药清肺胶囊与EP方案 (VP16 +DDP)联合应用在小细胞肺癌治疗中的近期和远期疗效。方法　 12 6例小细胞肺癌的患者 ,随机分为单纯化疗组和化疗中药联合治疗组 ,对照组采用标准EP方案 ,联合治疗组用EP方案 +清肺胶囊 ,进行前瞻性对比观察和为期 5年的远期疗效评价。结果　联合治疗组获得 34 4 %的完全缓解率和 89 1%的总有效率 ,对照组分别为 2 4 2 % ,70 3% ,两组差异显著 (P <0 .0 5 ) ;远期疗效 1,2 ,3,5年的生存率联合组分别为 5 4 7% (35 /6 4 ) ,2 9 4 % (15 /5 1) ,19 4 % (7/36 ) ,15 1% (5 /33) ,对照组分别为 4 6 8% (2 9/6 2 ) ,11 4 % (5 /4 4 ) ,5 7% (92 /35 ) ,3 1% (1/32 )。两组比较 ,2 ,3,5年生存率均有统计学意义。 (P <0 .0 5 )。结论　用EP方案联合中药清肺胶囊治疗小细胞肺癌具有较好的耐受性和安全性 ,其近期疗效和远期疗效均优于单纯化疗组 ,说明化疗联合中药治疗小细胞肺癌是一条有效的治疗途径     

塞来昔布对非小细胞肺癌化疗的增敏作用及机制

目的 探讨塞来昔布对非小细胞肺癌(NSCLC)的化疗增敏作用及其机制.方法 应用噻唑蓝比色法(MTT)检测顺铂(DDP)、足叶乙甙(VP16)单独应用及与塞来昔布联合应用的增殖抑制率,并根据金氏公式计算Q值,评价两药的合用效应.应用流式细胞术检测各处理组的凋亡率.结果 DDP联合塞来昔布后对NSCLC抑制作用明显增强,二者合用有协同作用.VP16联合塞来昔布后对NSCLC抑制作用明显增强,二者合用有协同作用.塞来昔布与顺铂联合用药的凋亡率明显高于单用药组(P＜0.01).结论 塞来昔布可增强DDP、VP16对NSCLC的增殖抑制作用,具有化疗增敏作用,其机制可能与塞来昔布促进凋亡有关.     

曲妥珠单抗与依托泊苷序贯联合应用对乳腺癌细胞抑制作用的研究

目的：探讨体外试验中曲妥珠单抗（HER）与依托泊苷（VP16）联合的最佳序贯方式、HER对VP16作用的影响及机制。方法：利用MTT法及流式细胞技术分别测定HER、VP16及不同序贯方式联合应用对人乳腺癌细胞的抑制率及细胞周期的影响。结果：VP16化疗前应用HER组及两药同时应用组对乳腺癌细胞抑制率均明显高于单药VP16组（P〈0.01）,为相加作用,其中以HER作用6h后应用VP16组效果最好。细胞周期检测提示：VP16化疗前应用HER组及两药同时应用组对细胞S期的阻滞增强。结论：VP16化疗前6h应用HER对乳腺癌细胞的抑制率最高,为最佳序贯联合方式,其内在机制可能与细胞周期的变化有关。     

全反式维甲酸增强依维莫司对小细胞肺癌的体内外抗肿瘤作用

目的 探讨雷帕霉素靶蛋白(mTOR)抑制剂依维莫司与全反式维甲酸(ATRA)联用对小细胞肺癌细胞株NCI-H446增殖及迁移能力的影响.方法 将不同浓度ATRA分别和依维莫司联合作用于NCI-H446细胞,采用MTT试验检测药物作用24h、48h、72h后的细胞存活率;细胞划痕实验观测药物处理24h后的细胞迁移能力.不同药物浓度作用于荷瘤小鼠,通过裸鼠移植瘤模型观察全反式维甲酸与依维莫司的体内协同抑瘤作用.结果 MTT实验表明ATRA联合依维莫司抑制NCI-H446细胞增殖呈时间及浓度依赖性,高浓度联合用药组72h细胞存活率最低;划痕实验结果表明联合用药组抑制细胞迁移作用显著,ARTA药物浓度越高迁移抑制作用越强;裸鼠皮下抑制瘤模型中各组在给药3周后发现联合用药组抑瘤作用高于依维莫司组和对照组.结论 依维莫司与ATRA联合应用在抑制人小细胞肺癌细胞增殖、迁移方面具有协同作用,在小细胞肺癌治疗可能具有良好的临床应用前景.     

survivin基因在化疗药物诱导的肺癌细胞凋亡中的作用

目的　探讨survivin基因在化疗药物顺铂 (DDP)和足叶乙苷 (VP16 )诱导的肺癌细胞凋亡中的作用。方法　用肺腺癌A5 4 9细胞株进行培养传代 ,MTT试验检测DDP和VP16不同药物浓度对肺癌细胞生长的抑制作用 ,实验分为 :DDP、VP16和对照组。DDP和VP16组的肺癌细胞又分为高浓度和低浓度化疗药物处理组。用逆转率PCR检测sur vivin基因的表达。同时利用流式细胞术定量检测细胞凋亡。结果　不同浓度VP16和DDP对肺癌细胞生长均有明显的抑制作用 ,并有浓度和时间的依赖性。化疗药物组细胞凋亡率和survivin基因表达抑制率高于对照组 ,也有时间依赖性。结论　survivin基因的抑制在DDP和VP16诱导肺癌细胞凋亡中起重要作用     

ABT-737增强人卵巢癌细胞顺铂敏感性并诱导线粒体分裂

目的观察Bcl-2抑制剂ABT-737对人卵巢癌顺铂耐药(SKOV3/DDP)细胞株顺铂敏感性和细胞线粒体形态的影响,为降低人卵巢癌细胞顺铂耐药性的进一步研究提供依据。方法将处于对数生长期的SKOV3/DDP细胞随机分为对照组、顺铂组、ABT-737组和顺铂联合ABT-737组(联合组)。MTT法检测各组SKOV3/DDP细胞生存率,流式细胞术(flow cytometry,FCM)检测各组细胞凋亡率,MitoTracker Red荧光探针法共聚焦显微镜下观察线粒体形态变化,免疫荧光法、蛋白印记法(Western blot)检测细胞中线粒体动力相关蛋白Drp-1和线粒体外膜分子Fis1的表达。结果 MTT结果发现与对照组比较,顺铂组和联合组SKOV3/DDP细胞生存率明显降低(P0.05),且联合组细胞存活率明显低于顺铂组(P0.05)。流式细胞术检测结果显示顺铂组细胞凋亡率明显高于对照组,联合组细胞凋亡率高于顺铂组。MitoTracker Red荧光探针法检测发现顺铂组细胞胞浆线粒体较对照组细胞胞浆内阳性着色颗粒感增强,联合组趋势强于顺铂组。蛋白免疫印记法检测Drp-1和Fis1表达水平,结果显示与顺铂组比较,联合组细胞中Drp1和Fis1蛋白表达水平明显增强。免疫荧光法结果呈现相同趋势。结论 ABT-737能够增强SKOV3/DDP细胞的顺铂敏感性,且对线粒体分裂存在明显诱导作用。     

G-CSF对VP16诱导白血病细胞凋亡及Survivin mRNA表达的调控作用

目的：探讨粒细胞集落刺激因子（G—CSF）对足叶乙甙（VP16）诱导白血病细胞凋亡以及Survivin基因表达的影响。方法：用HL-60白血病细胞株进行传代培养，通过DNA凝胶电泳和流式细胞术（FCM）检测细胞凋亡，用RT—PCR方法检测Survivin基凶表达。结果：VP16可诱导HL-60细胞凋亡，下调Survivin表达：G—CSF可上调SurvivinmRNA表达，与VP16联合应用时，细胞凋亡率低于单独应用VP16组（P〈0．01），Survivin mRNA表达水平高于单用VP16组（P〈0．01）。结论：VP16诱导白血病细胞凋亡可能与其抑制Survivin表达有关；G—CSF能抑制VP16的促凋亡作用。     

沙利度胺不同浓度对人胆管癌耐药细胞株增殖及凋亡的影响

目的:考察沙利度胺(THD)不同浓度体外对人胆管癌耐药细胞株(RBE)增殖及凋亡的影响。方法:将RBE株分为6组(A组、B组、C组、D组、E组和F组)分别加入THD 0.0,12.5,25.0,50.0,100.0和200.0μg/m L,并设立阴性对照组,采用MTT法检测上述各组药物加入后24.0、48.0和72.0 h的细胞生存率,并采用流式细胞仪及Annexin V-FITC检测上述各组药物加入后48 h的细胞周期分布和细胞的凋亡率。结果:经MTT法检测结果显示,随着THD浓度的上升,各组细胞存活率下降,且C组、D组、E组和F组药物加入后48 h和72 h的细胞生存率低于药物加入后24 h的细胞生存率(P<0.05);经流式细胞术检测结果显示,随着THD浓度的上升,药物加入后48 h各组细胞G_0/G_1期细胞和细胞凋亡率上升而S期下降(P<0.05)。结论:沙利度胺可抑制RBE株的增殖,将RBE株阻滞在G_0/G_1期,并诱导RBE株细胞的凋亡。     

顺铂和依托泊苷下调肺癌细胞端粒酶量及端粒酶逆转录酶的表达

目的　观察肺癌化疗的一线药物顺铂 (DDP)和依托泊苷 (VP16 )对肺癌细胞端粒酶量和端粒酶逆转录酶基因表达和蛋白质表达的影响 ,从而进一步探讨这些药物在肺癌治疗中的作用机理。方法用不同浓度的化疗药物处理肺腺癌A5 49细胞 ,用噻唑蓝 (MTT)和流式细胞术观察化疗药物 (DDP和VP16 )对肺癌细胞的生长抑制和细胞凋亡情况 ,分别用端粒重复扩增 (TRAP)法、逆转录 聚合酶链反应 (RT PCR)法和Western印迹杂交检测处理后端粒酶量 ,以及其催化亚基端粒酶逆转录酶 (hTERT)基因和蛋白质的变化。结果　DDP和VP16明显抑制肺癌细胞生长 ,并诱导细胞凋亡 ,有剂量和时间依赖性 .DDP和VP16明显抑制端粒酶量和hTERT基因及蛋白表达 ,尤其以高剂量 (VP16 2 0 0mg·L- 1,DDP5 0mg·L- 1)和低剂量 (VP16 2 0mg·L- 1,DDP 5mg·L- 1)作用的d 5更为明显。结论　VP16和DDP可以抑制肺癌细胞端粒酶量 ,这种作用可能是通过抑制端粒酶的催化亚基hTERT的表达来实现的。端粒酶量测定可能有助于作为判断肺癌化疗时VP16和DDP疗效的观察指标     

蛇葡萄素钠协同卡铂抑制人肺腺癌SPC-A-1细胞增殖

目的:探讨蛇葡萄素钠(AMP-Na)单用及其与卡铂(CBP)合用对SPC-A-1人肺腺癌细胞增殖的抑制作用及可能的机制。方法:采用MTT比色法测定AMP-Na单用及其与CBP合用对SPC-A-1细胞的体外细胞毒活性;用流式细胞仪分析AMP-Na单用及与卡铂合用后对SPC-A-1细胞凋亡的影响。结果:体外细胞毒实验和流式细胞仪结果表明,AMP-Na对人肺腺癌SPC-A-1细胞增殖的抑制作用具有明显的量效关系,与CBP联用后凋亡率较卡铂单用明显上升,有协同抑制作用。结论:AMP-Na单用能够显著抑制人肺腺癌SPC-A-1细胞的增殖,与卡铂合用可以增强卡铂的活性,协同抑制SPC-A-1细胞的增殖;AMP-Na可能是通过诱导凋亡而发挥其抗肿瘤作用。     

地钱素C衍生物F41对子宫颈癌HeLa细胞凋亡的影响

目的对地钱素C(MC)羟基衍生物进行筛选,寻找具有高抗肿瘤活性的MC衍生物,并初步探讨其抗肿瘤的细胞与分子生物学机制。方法用MTT法观察56种MC羟基衍生物对子宫颈癌HeLa细胞的毒性作用,筛选其中细胞毒作用最强的MC衍生物,并比较其与MC对HeLa细胞存活的抑制作用。用倒置显微镜、DAPI染色和DNA梯带检测其对细胞凋亡的影响。用流式细胞术检测其对细胞周期的影响。用Western印迹法检测其对细胞周期和凋亡相关蛋白表达的影响。结果在56种MC衍生物中,F41对HeLa细胞毒性最强,抑制HeLa细胞存活的IC50为(11.31±2.13)μmol·L-1,明显低于MC〔IC50为(17.19±3.28)μmol·L-1〕(P<0.01)。F41和MC与HeLa细胞作用24,48和72h,F41对HeLa细胞存活的抑制作用均明显强于MC(P<0.05)。形态学和DNA梯带检测显示,F41处理后,HeLa细胞皱缩变小,有空泡和凋亡小体出现,胞核浓缩变小,DNA电泳呈梯状条带。流式细胞分析显示,F41 15μmol·L-1处理HeLa细胞24h,G2/M期细胞占总细胞的比例为(43.8±3.0)%,明显高于对照组的(13.1±1.6)%(P<0.01);G1期细胞比例为(34.8±3.8)%,明显低于对照组的(63.6±5.5)%(P<0.01)。Western免疫印迹结果表明,F41可使HeLa细胞内磷酸化细胞周期蛋白依赖性蛋白激酶1水平降低,细胞周期蛋白B1和P53蛋白表达增多。结论 F41可诱导HeLa细胞凋亡,抑制HeLa细胞分裂,其抗肿瘤活性可能强于MC。     

木果楝内酯化合物抑制人肺肿瘤细胞增殖活性及作用机制的研究

目的检测3种木果楝内酯化合物(Xylocarpin H,Xy-logranatin C和Xylomexicanin A)对人肺肿瘤细胞增殖的抑制作用,并进一步探讨其可能的作用机制。方法 MTT比色法检测细胞增殖抑制率;采用Western blot分析与凋亡相关的蛋白表达变化。结果 Xylogranatin C对人非小细胞肺癌细胞(A549、RERF-LC-jk和QG-56)以及人小细胞肺癌细胞(PC-6和QG-90)5种实验用细胞的增殖均显示较强的抑制活性;Western blot结果显示由Xylogranatin C处理的A549细胞中的p53、Bax和caspase-3的表达与对照组相比有明显的增加(P<0.05)。结论 Xylogranatin C具有较强的抑制人肺肿瘤细胞增殖作用,其作用机制可能与其诱导肿瘤细胞凋亡有关。     

Cytotoxic activity of a synthetic deoxypodophyllotoxin derivative with an opened D-ring

Podophyllotoxin and its synthetic derivatives are valuable medicinal agents that have antitumor, insecticidal, and antifungal properties. We previously synthesized a deoxypodophyllotoxin derivative with an opened D-ring (DPD) exhibiting potent insecticidal activity. This article was firstly performed to identify the cytotoxicity of DPD toward human cancer cell lines (SGC7901, HeLa, and A549) and normal cell line (HEK293T) using MTT assay. DPD showed potent cytotoxicity against human cancer lines (HeLa and A549) and less cytotoxicity against normal cell lines HEK293T. DPD could also induce the cell cycle arrest at G2/M phase in HeLa cells and significantly increase the phosphorylation (Tyr 15) of CDC2 leading to inactivation of CDC2. The effects of DPD on cellular microtubule networks were detected using immunofluorescence technique in HeLa cells. The immunofluorescence results showed DPD influenced the arrangement and organization of cellular microtubule networks in HeLa cells. Microtubules are long, hollow cylinders made up of polymerized tubulin dimers. Total microtubules were separated after DPD treatment. Western blot results showed that the free polymerized tubulin dimers were obviously increased after DPD treatment. DPD may be a good drug candidate with the therapeutic potential to human cancer by affecting microtubule polymerization.     

In vitro antitumor effect of recombinant human tumor necrotizing factor on cultured human cancer cell lines and freshly isolated lung cancer cells by the human tumor clonogenic assay

Summary In vitro antitumor effects of human recombinant tumor necrotizing factor (rH-TNF) were examined against nine lung cancer cell lines including six non small and three small cell lung cancer, four stomach cancer cell lines and 30 freshly isolated lung cancer cell samples by the human tumor clonogenic assay. rH-TNF did not show any inhibitory effect on the colony formations of lung and stomach cancer cell lines, except for PC10 established from squamous cell carcinoma even at the high concentration. The overall response rate of fresh material was 11.5%. The colony formations of only two materials from 20 patients without prior chemotherapy were significantly suppressed by rH-TNF in vitro. Three specimens of adenocarcinoma exhibited more than 70% decrease in colony number by treating with 100 and 1000 u/ml of rH-TNF resulting in the response rate of 15.8% (3/19). From these results, it can be concluded that rH-TNF has modest direct cytotoxic effect on lung cancer, and additional study against adenocarcinoma of the lung might be warranted.     

MTT法测定紫杉醇与奥沙利铂在不同分化程度胃腺癌细胞株中的敏感性

目的:采用MTT法测定不同分化程度胃腺癌细胞株对紫杉醇、奥沙利铂的敏感性。方法:以不同浓度的紫杉醇、奥沙利铂分别处理MKN45(低分化)和SGC7901(中分化)2种胃腺癌细胞株,采用MTT法检测紫杉醇、奥沙利铂对胃腺癌细胞增殖的影响。结果:紫杉醇、奥沙利铂分别对2株胃腺癌细胞的增殖均具有显著的抑制作用,中分化的SGC7901细胞株对2种化疗药物的敏感性明显高于低分化的MKN45细胞株,而低分化的MKN45细胞株对紫杉醇更敏感。结论:不同分化程度的胃腺癌细胞株对紫杉醇、奥沙利铂敏感性不同。     

黄芩素联合紫杉醇对人肺癌细胞的生长抑制作用

目的 探讨黄芩素联合紫杉醇对人肺癌细胞株QG-56的影响。方法 以不同浓度的黄芩素、紫杉醇分别组成单药组和联合用药组,另设不加药的空白对照组,分别作用于QG-56细胞24,48,72和96 h,观察细胞数量和形态的变化,MTT法检测各组对QG-56细胞的增殖抑制作用,用流式细胞仪检测各组细胞凋亡率的改变。结果 不同浓度的各实验组作用后细胞数量明显减少、形态不规则,部分细胞核固缩和胞质减少,而对照组细胞生长状态良好。黄芩素、紫杉醇单用与联用均能抑制QG-56细胞增殖,呈剂量-时间依赖效应,联用组细胞抑制率较单药组明显增高,差异有统计学意义（P〈0.05）。黄芩素、紫杉醇单用与联用均能诱导QG-56细胞凋亡,联合组更为明显（P〈0.05）。结论 黄芩素可显著抑制人肺癌细胞株QG-56细胞生长,并能有效增强紫杉醇对人肺癌QG-56细胞的增殖抑制作用;两者具有协同作用。     

Astaxanthin enhances pemetrexed-induced cytotoxicity by downregulation of thymidylate synthase expression in human lung cancer cells

Pemetrexed, a multitargeted antifolate agent, has demonstrated clinical activity in non-small cell lung cancer (NSCLC) cells. Increased expression of thymidylate synthase (TS) is thought to be associated with resistance to pemetrexed. Astaxanthin exhibits a wide range of beneficial effects including anti-cancer and anti-inflammatory properties. In this study, we showed that down-regulating of TS expression in two NSCLC cell lines, human lung adenocarcinoma H1650 and squamous cell carcinoma H1703 cells, with astaxanthin were associated with decreased MKK1/2-ERK1/2 activity. Enforced expression of constitutively active MKK1 (MKK1-CA) vector significantly rescued the decreased TS mRNA and protein levels in astaxanthin-treated NSCLC cells. Combined treatment with a MKK1/2 inhibitor (U0126 or PD98059) further decreased the TS expression in astaxanthin-exposed NSCLC cells. Knockdown of TS using small interfering RNA (siRNA) or inhibiting ERK1/2 activity enhanced the cytotoxicity and cell growth inhibition of astaxanthin. Combination of pemetrexed and astaxanthin resulted in synergistic enhancing cytotoxicity and cell growth inhibition in NSCLC cells, accompanied with reduced activation of phospho-MKK1/2, phopho-ERK1/2, and TS expression. Overexpression of MKK1/2-CA reversed the astaxanthin and pemetrexed-induced synergistic cytotoxicity. Our findings suggested that the down-regulation of MKK1/2-ERK1/2-mediated TS expression by astaxanthin is an important regulator of enhancing the pemetrexed-induced cytotoxicity in NSCLC cells.     

Formaldehyde cytotoxicity in three human cell types assessed in three different assays.

International standards for preclinical screening of the cytotoxicity of dental materials so far recommend the use of established cell lines. The aim of this study was to assess the relative susceptibility of human dental pulp fibroblasts (HPF), human buccal epithelial cells (HBE) and HeLa cervix cancer cells exposed to identical cytotoxic challenges. Formaldehyde, which may be released from dental materials such as dental composites, glassionomer cements, and endodontic sealers, was used as test chemical. Cytotoxicity data including dose-response relations and TC(50) values were assessed in three different assays: BrdU incorporation, neutral red uptake and MTT assays. HBE and HPF demonstrated statistically significant lower TC(50) values in both the neutral red and the BrdU assay in comparison to HeLa cells. In the MTT assay no statistically significant differences were observed between the cell types. In the two target-tissue cell types (HPF and HBE) the Neutral Red assay revealed lower TC(50) values in comparison to the BrdU assay. In HeLa cells no statistically significant differences were observed between the assays. In conclusion, the present study confirms that cytotoxicity data obtained by cell culture studies are influenced by both cell culture model and choice of assay. Under identical experimental conditions, human target tissue cells appeared to be more sensitive to formaldehyde toxicity than human HeLa cancer cells.     

新型6,7-二甲氧基-4-(2-氟苯氧基)喹啉类c-Met抑制剂的设计、合成及生物活性研究

基于卡博替尼(cabozantinib)和foretinib的化学结构,通过改变中间链,设计合成了一系列含有哌嗪酰胺的6,7-二甲氧基-4-(2-氟苯氧基)喹啉类c-Met抑制剂。这些化合物未见文献报道,其结构通过MS和NMR确证。采用酶联免疫吸附测定(ELISA)法和MTT法测定了目标化合物对c-Met的抑制活性和对人结肠癌细胞(HT-29)、人肺癌细胞(H460)、人非小细胞肺癌细胞(A549)和人胃癌细胞(MKN-45)的细胞毒性。结果表明,4-(4-氯苯基)-N-[4-[(6,7-二甲氧基-4-喹啉)氧基]-3-氟苯基]哌嗪-1-甲酰胺(1b)、4-(3-氯苯基)-N-[4-[(6,7-二甲氧基-4-喹啉)氧基]-3-氟苯基]哌嗪-1-甲酰胺(1h)和4-(2-氯苯基)-N-[4-[(6,7-二甲氧基-4-喹啉)氧基]-3-氟苯基]哌嗪-1-甲酰胺(1j)对HT-29、H460、A549和MKN-45细胞的抑制活性明显优于对照药卡博替尼。其中,化合物1h和1j对c-Met具有较强的抑制活性,其IC50值分别为0.007 2和0.009 8 μmol/L。