辛基酚对MCF-7乳腺癌细胞周期及周期蛋白表达的影响

目的观察辛基酚对MCF-7乳腺癌细胞周期及周期蛋白表达的影响。方法以MTT试验、流式细胞分析、免疫细胞化学和RT-PCR等方法观察辛基酚对MCF-7乳腺癌细胞增殖、细胞周期相和细胞凋亡、CDK2和CDK4 mRNA表达、Cyclin D1 mRNA及蛋白表达的影响。结果4、8、16μmol/LOP作用MCF-7乳腺癌细胞72h时,细胞增殖率分别为107.31%、168.06%、62.00%,G0/G1期细胞分别为(52.46±6.67)%、(50.19±7.39)%、(67.31±5.47)%,对照组(55.27±7.53)%,细胞凋亡率分别为(4.84±1.12)%、(6.48±1.36)%、(19.26±3.57)%,对照组(5.56±1.125)%,16μmol/LOP减少了细胞中CDK2、CDK4、Cyclin D1 mRNA及Cyclin D1的表达。结论4、8μmol/LOP促进MCF-7乳腺癌细胞增殖,16μmol/LOP则抑制细胞增殖,促进细胞凋亡,这可能与OP影响细胞中CDK2、CDK4、Cyc-lin D1 mRNA及Cyclin D1表达有关。     

FoxO3a在三氧化二砷诱导的人乳腺癌MCF-7细胞凋亡中的表达和意义

目的 探讨FoxO3a在三氧化二砷(As2O3)诱导人乳腺癌细胞MCF-7凋亡过程中的表达变化及其可能的机制。方法 将不同浓度的As2O3(2.0、4.0、8.0μmol/L)与MCF-7细胞共同培养24h后,采用流式细胞术检测MCF-7细胞凋亡;采用免疫细胞化学及Western blot技术检测FoxO3a和Caspase 3蛋白的表达,各实验均设相应的阴性对照组。结果 经不同浓度As2O3作用后,MCF-7细胞凋亡率逐渐增加,分别为(6.26±0.94)%、(9.30±1.63)%和(13.02±3.82)%,且呈剂量依赖关系(rs=0.949, P<0.01);免疫细胞化学染色显示FoxO3a蛋白胞核表达增强,且Caspase-3蛋白表达增强（P<0.05);Western blot条带显示FoxO3a蛋白表达水平逐渐升高,且激活型Caspase-3蛋白表达水平逐渐升高（P<0.05)。结论 As2O3可通过增强FoxO3a蛋白的活性,激活Caspase-3蛋白的表达而诱导MCF-7细胞凋亡。     

Polyamidoamine dendrimer liposome-mediated survivin antisense oligonucleotide inhibits hepatic cancer cell proliferation by inducing apoptosis

Polyamidoamine dendrimer (PAMAM) is a new nanometer material, which can transfer the target genes to cells with high efficiency and lower toxicity. This study aims to evaluate antitumor effects of survivin antisense oligonucleotide (survivin-asODN) (carried by polyamidoamine dendrimer liposome) on hepatic cancer in nude mice. Hepatic cancer model was established by injecting SMMC-7721 cells subcutaneously into flanks of nude mice. Polyamidoamine dendrimer and liposome were mixed with survivin-asODN, respectively. The shape and size of complex were observed by transmission electron microscope, and zeta potential was measured by an analytical tool. Encapsulation efficiency and DNA loading level were determined by an ultraviolet spectrophotometer in centrifuging method. Expression of survivin in transplant tumor was measured by Western blotting. No significant difference appeared for diameter and envelopment ratio between PAMAM liposome-survivin-asODN and PAMAM-survivin-asODN (P?>?0.05). Both zeta potential and transfection efficiency in PAMAM liposome-survivin-asODN were higher than that in PAMAM-survivin-asODN complex (P?<?0.05). Expression of survivin protein and weight of tumors in transplanted tumors in PAMAM liposome-survivin-asODN group was less than that in PAMAM-survivin-asODN group (P?<?0.05). Cell apoptosis rate in PAMAM liposome-survivin-asODN group was higher than that of PAMAM-survivin-asODN group (P?<?0.05). In conclusion, polyamidoamine dendrimer liposome can deliver survivin-asODN into hepatic transplanted tumor cells effectively. Ployamidoamine dendrimer liposome-mediated survivin-asODN can inhibit hepatic cell proliferation by inducing apoptosis.     

呼肠孤病毒诱导乳腺癌微球体细胞凋亡

目的:探讨呼肠孤病毒对乳腺癌微球体细胞凋亡的影响。方法:以干细胞培养液培养乳腺癌细胞MCF-7和BT474,形成微球体,分别用呼肠孤病毒和表柔比星处理微球体,FCM检测MCF-7、BT474微球体细胞中CD44+CD24-/low细胞的比例,TUNEL法检测微球体细胞的凋亡,Western blotting检测MCF-7细胞及其微球体细胞中Ras的表达。结果:呼肠孤病毒对乳腺癌MCF-7和BT474微球体细胞的感染率显著高于亲本细胞(均P<0.05)。表柔比星作用下MCF-7和BT474微球体细胞中CD44+CD24-/low细胞比例明显升高[(8.1±0.4)%vs(18.0±4.5)%,P<0.01;(0.6±0.2)%vs(0.9±0.13)%,P<0.05]。呼肠孤病毒感染后MCF-7和BT474微球体细胞中CD44+CD24-/low细胞比例明显下降[(8.1±0.4)%vs(0.8±0.2)%,(0.6±0.2)%vs(0.2±0.1)%;均P<0.05]。MCF-7和BT474微球体细胞在呼肠孤病毒感染后凋亡率明显增加[(1.90±0.21)%vs(5.0±1.4)%,P<0.05;(0.20±0.1)%vs(1.0±0.16)%,P<0.05]。MCF-7细胞及其微球体细胞均高表达Ras,且两者水平相似。结论:与表柔比星相比,呼肠弧病毒对乳腺癌微球体细胞的抑制作用明显,还可明显诱导乳腺癌微球体细胞凋亡。     

Cisplatin’s tumoricidal effect on human breast carcinoma MCF-7 cells was not attenuated by American ginseng

Purpose We previously observed that American ginseng berry and ginsenoside Re attenuated cisplatin-induced emesis in a rat model, suggesting that the herb may have a value in treating chemotherapy-induced nausea/vomiting. However, it is not clear whether consuming ginseng concurrently with chemotherapy affects the efficacy of chemotherapeutic agents. In this study, we explored if the ginseng extract and its constituents, ginsenosides Rb₁, Rb₃, and Re, alter tumoricidal activity of cisplatin in human cancer cells. Methods Tumoricidal effects of cisplatin, and/or American ginseng berry extract (AGBE) and ginsenosides Rb₁, Rb₃, and Re, on human breast carcinoma MCF-7 cells were measured as cell proliferation in vitro. Cell counts were performed in MCF-7 cells pretreated with test agents for 72 h. Results Cisplatin decreased MCF-7 cell proliferation significantly in a concentration-dependent manner. Compared to control group, cisplatin reduced the cell proliferations to 56.5±3.3% at 1 μg/ml, to 36.6±2.4% at 5 μg/ml, and to 26.9±2.4% at 15 μg/ml (P<0.01). AGBE also inhibited the cell proliferation significantly, although in a less extended manner. When the berry extract at 0.5 mg/ml was used with cisplatin at 1 μg/ml, a significant enhancement of cisplatin’s activity was observed (35.8±2.5%; P<0.05). We also observed that Rb₁ did not change cisplatin’s activity; Rb₃, at a higher concentration, increased cisplatin’s anti-proliferation activity (48.0±1.2%; P<0.05); Re increased cisplatin’s activity (Re 0.1 mg/ml, 48.0±2.8%; Re 0.3 mg/ml, 31.9±2.2%, P<0.01). Conclusion Our data suggest that AGBE and the tested ginsenosides do not attenuate cisplatin’s tumoricidal activity in MCF-7 cells, but in fact may actually enhance it. Additionally, the ginseng extract and ginsenoside Re by themselves exerted anti-proliferative activity against MCF-7 cells. The herb might potentially serve a complementary role with the chemotherapeutic agents in treating cancer, in addition to decreasing chemotherapy-induced nausea/vomiting.     

Cytotoxicity of antitumor platinum complexes withl-buthionine-(R,S)-sulfoximine and/or etanidazole in human carcinoma cell lines sensitive and resistant to cisplatin

Human 2008 ovarian carcinoma cells and the C13 CDDP-resistant subline and human MCF-7 breast carcinoma cells and the MCF-7/CDDP CDDP-resistant subline were exposed tol-buthionine-(S, R)-sulfoximine (50 M) for 48 h prior to and during exposure for 1 h to the antitumor platinum complexes,cis-diamminedichloroplatinum(II), carboplatin or D,L-tetraplatin and/or to etanidazole (1 mM) for 2 h prior to and during exposure for 1 to the antitumor platinum complexes. These modulators alone did not significantly alter the cytotoxicity of CDDP toward either parental line. A twofold enhancement in cytotoxicity was observed with carboplatin in the 2008 cells and with D,L-tetraplatin in both parental lines with the single modulators. The modulator combination (buthionine sulfoximine/etanidazole) was very effective along with D,L-tetraplatin in both the MCF-7 parent and MCF-7/CDDP cell lines where at the higher platinum complex concentrations there was 1.5 to 3 logs increased killing of cells by the drug plus the modulators compared with the drug alone. Similarly, when C13 cells were exposed to CDDP (100 M) or D,L-tetraplatin (100 M) along with buthionine sulfoximine and etanidazole there was a 2-log increase in cell killing compared with exposure to the platinum complex alone. Treatment of each of the four cell lines with buthionine sulfoximine decreased both the non-protein and total sulfhydryl content of the cells. Treatment with the combination of modulators did not produce a further decrease in cellular sulfhydryl content compared with buthionine sulfoximine alone. The total sulfhydryl content in MCF-7 cells and 2008 cells exposed to buthionine sulfoximine and etanidazole was 58% and 31% of normal and the total sulfhydryl content of MCF-7/CDDP cells and C13 cells treated the same way was 54% and 23% of normal, respectively. DNA alkaline elution was used to assess the impact of exposure to the modulators, buthionine sulfoximine and etanidazole, alone and in combination on the cross linking of DNA by the antitumor platinum complexes in the MCF-7 and MCF-7/CDDP cell lines. Overall, the increases in DNA cross linking factors were greater in the MCF-7 cells than in the MCF-7/CDDP cells. These results indicate a possible clinical potential for this modulator combination.This work supported by NIH grant P01-CA 38493.     

格尔德霉素在体内外增强人乳腺癌对阿霉素的敏感度

目的 探讨新型抗肿瘤药物格尔德霉素（GA）联合阿霉素（ADM）治疗乳腺癌的机制。方法 通过Western blot确定GA处理后AKT及HSP70表达变化,然后体外分别以GA/阿霉素（ADM）以及联合处理人乳腺癌MCF-7细胞,MTT法检测增殖变化,流式细胞仪检测细胞凋亡,在裸鼠模型体内检测GA联合ADM治疗后肿瘤大小变化并采用TUNEL法检测肿瘤细胞凋亡变化。结果 GA处理MCF-7细胞后AKT明显下降,HSP70无明显改变,GA联合ADM后,MCF-7细胞的增殖受到明显抑制,生存率为（51.90±6.80）% （P＜0.01),凋亡明显增加,凋亡率为（42.97±4.10）% （P＜0.05)。在裸鼠模型内肿瘤生长受到明显抑制,实验终点抑瘤率为（68.00±9.40）% （P＜0.05),联合治疗明显增加瘤内肿瘤细胞的凋亡,凋亡率为（14.70±3.30）% （P＜0.01)。结论 GA联合ADM能够显著增加ADM的治疗疗效,有望成为治疗乳腺癌的新型化疗联合方案。     

Noxa基因转染乳腺癌MCF-7细胞的增殖抑制和促凋亡作用

目的 探讨Noxa基因转染乳腺癌MCF-7细胞的增殖抑制和促凋亡作用。方法 利用脂质体将真核表达载体pIRES2-EGFP-Noxa瞬时转染至乳腺癌MCF-7细胞,通过RT-PCR检测转染后Noxa基因mRNA的表达,Western blot检测转染后蛋白的表达,MTT 比色法测定细胞增殖的抑制,流式细胞仪检测细胞的凋亡和细胞周期的变化,Hoechst 33342染色检测细胞的凋亡情况。结果 Noxa基因转染后在乳腺癌MCF-7细胞中成功表达。转染后mRNA及蛋白表达持续上升,其转染后24h、48h、72h mRNA的相对灰度值分别为(0.347±0.031)、(0.703±0.041)、(1.044±0.033),差异具有统计学意义（P<0.05）。转染24h、48h、72h后蛋白表达的相对灰度值为(1.171±0.086)、(1.013±0.088)、(0.886±0.063),差异具有统计学意义（P<0.05）。Noxa基因的表达使得乳腺癌MCF-7细胞出现增殖抑制,其24h、48h、72h抑制率分别为（23.9±4.2）%、（36.6±3.0）%、（47.0±3.3）%,差异具有统计学意义（P<0.05）。流式细胞仪检测显示MCF 7细胞DNA合成受到抑制,细胞周期主要抑制在G0/G1 期。其转染24h、48h、72h的G0/G1期分别为（68.1±2.5）%、（72.6±1.5）%、（75.6±0.9）%,与阴性对照组相比,差异具有统计学意义（P<0.05）;其24h、48h、72h凋亡率分别为（11.5±0.9）%、（19.6±0.8）%、（25.4±0.7）%,组间差异有统计学意义（P<0.05）。Hoechst 33342染色显示Noxa基因转染后细胞出现凋亡,其24h、48h、72h凋亡率分别为（7.3±4.1）%、（16.8±3.3）%、（23.8±2.3）%,与阴性对照组相比,其差异具有统计学意义（P<0.05）。结论 Noxa基因转染乳腺癌MCF-7细胞后能够抑制细胞增殖并促进其凋亡。     

甲基莲心碱对耐阿霉素人乳腺癌细胞多药耐药性的逆转

Objective: To study the reversal effect of neferine on adriamycin (ADM) resistant human breast cancer cell line MCF-7/ADM. Methods: The cytotoxic effect of Nef or ADM was determined by 3-[4, 5-dimethylthiazol-2.-yl], 5-diphenyl tetraxolium bromid (MTT) assay. Apoptosis and the expression of P-glycoprotein (P-gp) were detected by flow cytometry (FCM). The intracellular ADM concentration was measured by HPLC. Results: Nef at 1, 5, 10 mol/L decreased the IC₅₀ of ADM to MCF-7/ADM from 11.63 g/mL to 4.59, 2.44, 0.27 g/mL respectively. MCF-7/ADM could resist the apoptosis induced by ADM while Nef (1-10 mol/L) could augment ADR-mediated apoptosis. Nef (10 mol/L) increased the accumulation of ADM up to 2.88 fold in MCF-7/ADM but not in sensitive cells MCF-7/S and reduced the expression of P-gp in MCF-7/ADM cells. Conclusion: Nef can circumvent multidrug resistance (MDR) of MCF-7/ADM cells and the mechanism was associated with the increase of intracellular accumulation of ADM and the reduced expression of P-gp in MCF-7/ADM cells.     

Tumor targeting and imaging of intraperitoneal tumors by use of antisense oligo-DNA complexed with dendrimers and/or avidin in mice.

To establish an effective nonviral gene delivery and a corresponding imaging method for i.p.-disseminated tumors, various oligonucleotide-carrier complexes were synthesized, and their in vitro and in vivo properties were examined. The 20-mer multiamino-linked oligonucleotide (oligo), synthesized as antisense against the c-erbB-2 sequence, and the 3'-biotinylated form of the same oligonucleotide (oligo-Bt) were (111)In labeled through a diethylenetriaminepentaacetic acid chelate. (111)In-oligo was mixed with generation 4 polyamidoamine dendrimer (G4) or with biotinylated G4 (G4-Bt), which are positively charged to form electrostatic complexes. (111)In-oligo/G4-Bt and (111)In-oligo-Bt were conjugated to avidin ((111)In-oligo/G4-Av and (111)In-oligo-Av, respectively). (111)In-oligo/G4, (111)In-oligo/G4-Av, (111)In-oligo-Av, and carrier-free (111)In-oligo (2.96 kBq/22.4-45.9 ng of oligo) were examined for internalization in vitro in human ovarian cancer cells (SHIN3). Biodistribution of (111)In-oligo-carrier complexes or (111)In-oligo was examined in normal (n = 4-7) or i.p. SHIN3 tumor-bearing (n = 6-10) mice 2-24 h after i.p. injection (74 kBq/125-300 ng). Scintigraphy of i.p. tumor-bearing and normal mice was performed at various times postinjection of (111)In-oligo-carrier complex or (111)In-oligo (1.85 MBq/2.2 ng). (111)In-oligo-carrier complexes bound to the tumor cells were internalized at a rate of 34-56% at 24 h. In vivo, G4, G4-Av, and Av significantly enhanced tumor delivery of (111)In-oligo [9.1, 14.5, and 24.4% of injected dose per g of tissue (ID/g) at 24 h; P < 0.05, < 0.01, and < 0.0001, respectively] compared with delivery without carrier (0.8% ID/g). Scintigrams of (111)In-oligo delivered to the i.p.-disseminated tumors by the carriers were successfully obtained. In conclusion, G4, G4-Av, and Av can effectively deliver (111)In-oligo to i.p.-disseminated tumors. (111)In-oligo-carrier complexes also have potential as tracers for imaging and monitoring of gene delivery.     

米非司酮逆转人乳腺癌细胞MCF-7/ADR耐多柔比星机制的探讨

目的 探讨米非司酮对乳腺癌耐药细胞株MCF-7/ADR的逆转耐药作用.方法 以亲本乳腺癌细胞MCF-7和耐多柔比星(阿霉素)乳腺癌细胞MCF-7/ADR为研究对象,分别应用MIF进行干预后,流式细胞仪检测MIF作用前后瘤细胞P-gp的表达、细胞内阿霉素蓄积量的变化以及细胞周期的分布.结果 (1)10 μmol/L MIF作用72小时后,MCF-7/ADR细胞P-gp表达率[(23.21±1.80)％]明显高于MCF-7细胞[(19.37±2.37)％,P＜0.05].(2)5 μmol/L ADR处理后,MCF-7/ADR细胞内ADR蓄积量为(47.13±4.11)％,低于MCF-7细胞[(60.24±2.61)％,P＜0.05].(3) 10 μmol/L MIF联合5 μmol/L ADR处理细胞,MCF-7/ADR和MCF-7细胞内ADR的蓄积量分别为(82.72±2.42)％及(88.63±2.75)％ (P ＞0.05);但均较单用ADR时升高(均P＜0.01).(4) MIF作用前,MCF-7/ADR细胞G0/G1期比例[(77.21±3.10)％]高于MCF-7细胞G0/G1期比例[(59.05±2.16)％,P＜0.05];MCF-7/ADR细胞S期比例明显低于MCF-7细胞(P＜0.05).经10 μmol/L MIF作用后,MCF-7细胞G0/G1期比例(75.28±2.53)％较MIF作用前明显升高(P＜0.05);S期比例则较MIF作用前显著降低(P＜0.05);MCF-7/ADR细胞G0/G1期比例和S期比例分别为(80.13±2.72)％及(13.52±1.03)％,与MIF作用前比较差异均无统计学意义(均P＞0.05);两种瘤细胞的G2/M期比例与MIF作用无关(P＞0.05).结论 (1)MIF可以逆转MCF-7/ADR的耐药性,其作用机制与降低细胞P-gp含量、增加细胞内ADR蓄积量有关.(2) 10 μmol/L浓度的MIF对MCF-7/ADR细胞周期分布影响不大.     

PUMA基因转染乳腺癌MCF-7细胞增强表柔比星致凋亡敏感性的研究

  目的  探讨PUMA基因转染是否增强乳腺癌MCF-7细胞对表柔比星致凋亡的敏感性。   方法  应用脂质体介导重组真核表达载体PUMA-pCDNA3和空载体pCDNA3质粒瞬时转染至乳腺癌MCF-7细胞中, G418筛选阳性细胞。将系列浓度(0.01~100μmol/L)的表柔比星分别作用于MCF-7、MCF-7/PUMA和MCF-7/pCDNA3细胞72 h, MTT法测定各组细胞的存活率并计算IC50, FCM、TUNEL法检测细胞凋亡情况, Western blotting检测各组细胞PUMA蛋白表达的变化。   结果  MCF-7、MCF-7/PUMA和MCF-7/pCDNA3细胞的表柔比星IC50分别为(13±1.4)、(1.8±0.2)和(10.7±1.3)μmol/L, MCF-7/PUMA细胞对表柔比星作用的敏感性增加了7.2倍。表柔比星以剂量依赖方式诱导MCF-7细胞凋亡, 但对MCF-7/PUMA细胞所诱导的凋亡比MCF-7和MCF-7pCDNA3更明显。低浓度表柔比星(0.1μmol/L)轻微引起MCF-7/pCDNA3[(1.15±0.26)%]和MCF-7细胞凋亡[(0.9±0.24)%], 但能诱导MCF-7/PUMA细胞明显凋亡[(6.44±1.46)%]; 高浓度表柔比星(1μmol/L)诱导各组细胞凋亡, 但表柔比星MCF-7/PU MA细胞凋亡率[(35.47±9.36)%]明显高于MCF-7[(12.6±3.73)%]和MCF-7/pCDNA3细胞[(15.2±5.17)%], 差异均有统计学意义(P < 0.01);FCM和TUNEL方法检测显示相同的结果。PUMA蛋白在MCF-7/PUMA细胞中的表达明显高于MCF-7和MCF-7/pCD NA3细胞。   结论  PUMA基因稳定转染明显地增强了乳腺癌MCF-7细胞增强表柔比星致凋亡作用的敏感性。      

5,4'-二-正辛烷氧基-7-二氟亚甲基异黄酮体外抗人类乳腺癌细胞作用

目的 探讨金雀异黄素(Gen)衍生物5,4'-二-正辛烷氧基-7-二氟亚甲基异黄酮(DOdFMG)体外抗人类乳腺癌细胞的作用.方法 平皿克隆形成法检测DOdFMG对人类乳腺癌MCF-7细胞抑制生长和增生作用,琼脂糖凝胶电泳法观测DOdFMG诱导MCF-7细胞凋亡作用,Western blotting 法测定DOdFMG对MCF-7细胞PTEN、pAkt、caspase-3蛋白的表达及活性.结果 DOdFMG抑制MCF-7细胞生长,呈剂量依赖性,比Gen具有更强的抗肿瘤活性,DOdFMG能诱导MCF-7细胞凋亡,DOdFMG处理的MCF-7细胞PTEN蛋白及caspase-3蛋白表达上调,pAkt蛋白表达下调,呈时间剂量依赖性.结论 DOdFMG具有抗人类乳腺癌MCF-7细胞的作用,其机制可能与抑制PKB/Akt通路的信号传导、激活PTEN及caspase-3有关,是一种新型治疗乳腺癌的候选药物.     

Dendrimer-encapsulated camptothecins: increased solubility, cellular uptake, and cellular retention affords enhanced anticancer activity in vitro

A biocompatible polyester dendrimer composed of the natural metabolites, glycerol and succinic acid, is described for the encapsulation of the antitumor camptothecins, 10-hydroxycamptothecin and 7-butyl-10-aminocamptothecin. The cytotoxicity of the dendrimer-drug complex toward four different human cancer cell lines [human breast adenocarcinoma (MCF-7), colorectal adenocarcinoma (HT-29), non-small cell lung carcinoma (NCI-H460), and glioblastoma (SF-268)] is also reported, and low nmol/L IC(50) values are measured. Cellular uptake and efflux measurements in MCF-7 cells show an increase of 16-fold for cellular uptake and an increase in drug retention within the cell when using the dendrimer vehicle.     

ROCK1基因敲除 对乳腺癌细胞系迁移及侵袭的影响

目的：通过成簇规律间隔的短回文重复序列（CRISPR/Cas9）系统在MCF-7乳腺癌细胞系中构建Rho相关蛋白激酶1（ROCK1）基因敲除模型,研究ROCK1敲除对乳腺癌细胞迁移及侵袭能力的影响。方法：设计并合成靶向ROCK1的向导RNA （sgRNA）寡核苷酸序列,长度为20 bp,构建到CRISPR单载体慢病毒上,感染并筛选稳定细胞株,采用划痕实验和Transwell实验分别检测细胞迁移和侵袭能力。结果：目的sgRNA寡核苷酸双链成功插入酶切后的GV392质粒载体中且测序正确;经Western blot鉴定ROCK1敲除的细胞株构建成功;ROCK1基因敲除后,与对照组细胞相比,其蛋白表达缺失。划痕实验结果显示ROCK1敲除细胞株24 h划痕愈合度为（60.600±0.047）%,对照组细胞划痕愈合度为（80.404±0.018）%,差异有统计学意义（P=0.003）。Transwell实验结果显示ROCK1敲除细胞株在24 h的迁移细胞数为（271.3±5.0）个,对照组的迁移细胞数为（448.3±5.5）个;48 h的迁移细胞数在实验组和对照组分别为（1.7±2.9）个和（298.3±5.7）个,差异有统计学意义（P=0.000）。结论：ROCK1基因敲除可明显抑制乳腺癌细胞的迁移及侵袭能力,提示ROCK1基因在乳腺癌侵袭和转移中可能发挥重要作用。     

Tautomycetin诱导乳腺癌耐药细胞MCF-7/ADR的凋亡及其机制

目的:研究tautomycetin对乳腺癌耐药细胞MCF-7/ADR增殖及凋亡的影响及其机制.方法:MTr法检测tautomycetin对MCF-7/ADR细胞增殖的影响,流式细胞术检测MCF-7/ADR细胞的凋亡,Western blotting法检测tautomycetin对MCF-7/ADR细胞caspase相关蛋白、Bcl-2、Cyto-C、P53蛋白表达和Akt磷酸化的影响.结果:Tautomycetin可剂量(0.01～100μmol/L)依赖性地抑制MCF-7/ADR细胞的增殖(P＜0.05),IC50值为(1.26±0.12)μmol/L;与对照组相比,tautomycetin (1 μmol/L)可诱导MCF-7/ADR细胞凋亡,早期凋亡比例由(0.67±0.18)％升高至(17.2±3.8)％,晚期凋亡比例由(0.96±0.23)％升高至(28.4±5.7)％(P＜0.05).Tautomycetin可活化MCF-7/ADR细胞中caspase-7和caspase-9,降低Bcl-2蛋白的表达,促进线粒体释放Cyto-C,降低p-Akt的水平,但对caspase-8和P53的表达没有影响.结论:Tautomycetin可阻断Akt活化,以P53非依赖的方式通过Cyto-C介导的通路诱导MCF-7/ADR细胞凋亡.     

香加皮杠柳苷对MCF-7细胞周期及p21WAF1/CIP1表达的影响

 目的：研究香加皮杠柳苷（CPP）对人乳腺癌MCF-7细胞周期及p21^WAF1/CIP1表达的影响 ,探讨其抗肿瘤作用及作用机制。方法：采用MTT法检测不同浓度CPP（1.25、2.50、5.00、 10.00、20.00 ng/ml）作用不同时间（24、48、72 h）对MCF-7细胞的增殖抑制作用;应用流 式细胞术分析不同浓度CPP （2.50、5.00、10.00 ng/ml）分别作用于MCF-7细胞6、12、24、 48、72 h对肿瘤细胞周期的影响;并采用RT-PCR和免疫细胞化学技术检测细胞周期相关因子 p21^WAF1/CIP1的表达。结果：CPP能明显抑制MCF-7细胞的增殖,并呈浓度及时间依赖性,作用 于MCF-7细胞48 h的IC₅₀为（4.88±0.16）ng/ml。流式细胞术结果显示,CPP作用MCF-7细胞24 h时,G₀/G₁期细胞明显增多,而S期和G₂/M期细胞显著减少,与对照组相比差异有统计学意义 （P<0.05）,其中5.00 ng/ml组G₀/G₁期细胞由对照组的（49.33±3.25）%升高至（79.47± 2.40）%,S期和G₂/M期细胞由对照组的（28.47±1.59）%和（22.20±2.09）%分别下降至（10.13±3.26）%和（10.40±1.41）%。经CPP处理的MCF-7细胞中p21^WAF1/CIP1 mRNA的表达明显增强,p21^WAF1/CIP1/β-actin光吸度比值与对照组相比明显增高（P<0.05）。免疫细胞化学结果显示,CPP组MCF-7细胞中p21^WAF1/CIP1 蛋白的表达随作用浓度的增加而增强,其中10.00 ng/ml组肿瘤细胞p21^WAF1/CIP1的表达呈强阳性。结论：香加皮杠柳苷（CPP）具有显著的体外抗肿瘤作用,且有效剂量很小,其IC₅₀仅为（4.88±0.16）ng/ml。CPP可使MCF-7细胞发生G₀/G₁期阻滞,并可使细胞周期相关基因p21^WAF1/CIP1的mRNA及蛋白表达增强,提示阻滞细胞周期可能是CPP体外抗肿瘤的作用机制之一。     

Modulation of Adriamycin Resistance in Human Breast Carcinoma MCF-7 Cells in vitro and in vivo by Medroxyprogesterone Acetate

The combination effect of adriamycin (ADM) and medroxyprogesterone acetate (MPA) was examined in vitro against human breast carcinoma MCF-7 and its ADM-resistant line (MCF-7/ADM). MCF-7 cells, which are positive for estrogen receptors, progesterone receptors and high-affinity MPA-binding activity, were more susceptible to the growth-inhibitory activity of ADM or MPA than MCF-7/ADM cells. A combination effect of ADM and MPA was observed against MCF-7/ADM cells, which are negative for steroid receptors, and furthermore against human nasopharynx carcinoma KB and its ADM-resistant line KB-A1. This combination effect of ADM and MPA against MCF-7/ADM cells was demonstrated to be synergistic by using the median effect plot method. The activity of MPA was almost equivalent to that of chlormadinone acetate or tamoxifen, greater than that of progesterone, and less than that of verapamil. The accumulation of ADM in MCF-7/ADM cells was enhanced by treatment with 10 μ M MPA as well as 10 μ M verapamil. The efflux of accumulated ADM from MCF-7/ADM cells was also partially inhibited by treatment with MPA or verapamil. MPA augmented the growth-inhibitory activity of ADM against MCF-7/ADM tumors inoculated into nude mice, although statistical significance was not observed. It is suggested that the clinical advantage of the combination of MPA with ADM against advanced breast cancers may be partly explained by the modulation of ADM resistance by MPA.     

丁酸钠诱导MCF-7细胞凋亡的研究

目的:观察丁酸钠诱导培养的乳腺癌细胞MCF-7凋亡的影响。方法:用终浓度分别为0、1.25、2.5、5.0、10.0mmol/L的丁酸钠处理MCF-7细胞48h,用MTT比色法分析丁酸钠对细胞的抑制率,倒置显微镜观察细胞生长情况的改变,流式细胞术,末端脱氧核糖核酸转移酶介导的缺口末端标记法(TUNEL)及DNA琼脂糖凝胶电泳检测凋亡。结果:不同浓度的丁酸钠对MCF-7细胞有明显的抑制作用,光镜下观察到细胞贴壁性随作用时间延长而下降;流式细胞术检测不同浓度丁酸钠处理细胞的凋亡率分别为4.8%、9.4%、26.1%和51.9%;TUNEL检测可见阳性细胞胞体缩小,核固缩,呈棕黄色或黄褐色颗粒;DNA琼脂糖凝胶电泳检测到10mmol/L处理细胞出现“DNA ladders”。结论:丁酸钠可以诱导MCF-7细胞发生凋亡。