熊果酸对乳腺癌细胞caspase-3和PARP表达的影响

目的:探讨三萜类中药成分熊果酸(UA)诱导乳腺癌细胞MCF-7的凋亡作用,并通过分析UA作用后MCF-7细胞的caspase-3和多聚ADP核糖多聚糖(PARP)蛋白表达的变化,探讨其诱导MCF-7细胞凋亡的分子机制。方法:采用细胞培养技术,用不同浓度的药物在一定的时间内处理细胞株,采用MTT法、活细胞原位光镜和荧光染色技术、流式细胞技术(FCM)、荧光免疫组织化学技术,图象分析技术,研究UA对caspase-3和PARP蛋白表达的影响,诱导细胞凋亡的作用。结果:UA剂量依赖性的抑制MCF-7细胞增殖,半数生长抑制剂量(IC₅₀)为(22.6±3.0)μmol.L^-1,诱导caspase-3和PARP表达增加,使细胞呈现典型的凋亡形态学特征,核质浓集,有凋亡小体。结论:UA诱导MCF-7细胞凋亡,其机制涉及到caspase-3和PARP依赖性凋亡调节信号通路。     

桔梗皂苷D联合伊马替尼对白血病耐药细胞K562/R的抑制增殖和作用机制研究

桔梗皂苷D(platycodin D,PD)对多种恶性肿瘤有显著抑制作用,对白血病细胞有明显的增殖抑制和诱导凋亡的作用,但其是否有效提高耐药细胞对伊马替尼的敏感性及其发挥功能的分子机制仍未阐明。为了研究PD与伊马替尼(imatinib,IM)联合用药对慢性粒细胞白血病耐药细胞株K562/R的作用及机制。细胞增殖实验检测桔梗皂苷D对伊马替尼增殖抑制功能的影响,采用CCK8测定PD和IM单药及联合用药对K562/R增殖的抑制作用;流式细胞术检测Annexin V-FITC/PI双标记细胞凋亡率的变化,Western blot法检测cleaved caspase-3,cleaved caspase-9,PARP,cleaved PARP,Bcr/abl,p-AKT,p-mTOR蛋白表达。结果显示桔梗皂苷D联合伊马替尼对K562/R细胞的增殖抑制作用和细胞凋亡率比单独用药组效果明显。Western blot法检测结果显示与单药组比较,联合用药组可以明显上调cleaved caspase-3,cleaved caspase-9,cleaved PARP蛋白表达,同时下调PARP,Bcr/abl,p-AKT,p-mTOR蛋白的表达。结果表明桔梗皂苷D可以提高耐药细胞对伊马替尼的敏感性,联合用药在抑制细胞增殖、诱导凋亡、抑制Bcr/abl蛋白和激活PI3K/AKT/mTOR信号通路方面明显优于单独用药。     

七叶皂苷钠抑制人白血病Jurkat细胞增殖的机制研究

目的研究七叶皂苷钠抑制人白血病Jurkat细胞增殖的作用及其机制。方法 MTT法分析七叶皂苷钠对Jurkat细胞增殖的抑制作用,Hoechst 33258染色、FITC-Annexin V/PI双染、DNA Ladder、流式细胞术检测细胞凋亡和细胞周期,Western blotting法分析凋亡相关蛋白变化。结果七叶皂苷钠呈质量浓度和时间相关方式抑制Jurkat细胞增殖;经七叶皂苷钠处理后的Jurkat细胞出现凋亡的形态学特征、DNA条带,Annexin V+/PI细胞(早期凋亡细胞)显著增加;七叶皂苷钠可活化Jurkat细胞中Caspase-8、Caspase-9、Caspase-3,引起PARP的切割,并减少Bcl-2蛋白的表达。结论七叶皂苷钠能有效地通过诱导细胞凋亡抑制Jurkat细胞增殖。     

白头翁皂苷D对乳腺癌MCF-7细胞的体内外抗肿瘤作用研究

目的探讨白头翁皂苷D对人乳腺癌MCF-7细胞的体内外抗肿瘤作用及其机制。方法采用MTT法和吉姆萨染色,考察白头翁皂苷D对人MCF-7细胞体外增殖抑制的影响;建立MCF-7细胞裸鼠移植瘤模型,考察白头翁皂苷D体内抗肿瘤作用;HE染色和透射电镜实验考察肿瘤组织形态学和超微结构的变化;采用Western Blot法检测MCF-7细胞中Bcl-2、Caspase-3及PI3K/AKT/mTOR途径相关蛋白PI3K-p85、p-AKT、p-mTOR、p-p70S6K的表达。结果白头翁皂苷D可抑制MCF-7细胞的增殖,且呈剂量依赖关系;裸鼠体内实验结果显示白头翁皂苷D可抑制肿瘤的生长,30.0 mg·kg~(-1)剂量组瘤体质量明显降低,与模型组比较,差异有统计学意义(P0.01);形态学观察结果也显示白头翁皂苷D对MCF-7细胞具有凋亡作用,且随着给药剂量的增加变化越明显;白头翁皂苷D(15.0,20.0,25.0μmol·L~(-1))不同剂量组均可抑制MCF-7细胞中Bcl-2、Caspase-3蛋白的表达,与对照组比较,差异均有统计学意义(P0.01);白头翁皂苷D还可下调PI3K/AKT/mTOR信号传导通路相关蛋白PI3K-p85、p-AKT、p-mTOR、p-p70S6K蛋白的表达,20.0,25.0μmol·L~(-1)剂量组与对照组比较,差异均有统计学意义(P0.05,P0.01)。结论白头翁皂苷D对MCF-7细胞有显著的体内外抗肿瘤作用,其机制可能是通过下调PI3K/AKT/mTOR信号传导通路诱导细胞凋亡。     

丁香活性组分抑制PI3K/Akt/mTOR通路诱导人结肠癌HCT116细胞凋亡的研究

筛选丁香活性组分(active fraction from clove, AFC)对人结肠癌细胞敏感的细胞株,研究AFC对其增殖、凋亡及PI3K/Akt/mTOR(phosphoinositide 3-kinase/Akt/mechanistic target of rapamycin pathway)信号通路的影响,揭示AFC诱导人结肠癌细胞凋亡的分子机制。实验采用CCK-8法检测不同浓度的AFC的细胞毒作用;采用Hoechst 33258荧光染色、Annexin V-FITC/PI双染法检测AFC诱导细胞凋亡的发生;Western blot法检测AFC单独或AFC联合IGF-Ⅰ(insulin-like growth factor-Ⅰ)作用于细胞后,凋亡相关蛋白caspase-3,caspase-9,PARP(poly ADP-ribose polymerase)以及PI3K/Akt/mTOR信号通路PI3K,p-PI3K,Akt, p-Akt, mTOR和p-mTOR蛋白表达情况。结果显示,AFC抑制作用最明显的是人结肠癌HCT116细胞,最佳作用时间为48 h; AFC处理HCT116细胞后,剂量依赖性地出现典型的细胞凋亡特征,如核染色质浓缩、核碎裂、凋亡小体的出现等;Annexin V-FITC/PI双染法结果显示,与对照组比较,50,100μg·mL~(-1) AFC组诱导HCT116细胞凋亡率显著上升(P0.001);凋亡相关蛋白caspase-9,cleaved caspase-3,cleaved PARP均被AFC以浓度依赖方式激活,cleaved caspase-3/procaspase-3,cleaved PARP/PARP,caspase-9/β-actin在100μg·mL~(-1) AFC组与对照组间比较,差异均有统计学意义(P0.001);p-PI3K,p-Akt, p-mTOR蛋白相对表达量呈浓度依赖性的递减,而Akt, mTOR在各组间无明显变化,p-PI3K/PI3K,p-Akt/Akt, p-mTOR/mTOR在50,100μg·mL~(-1) AFC组与对照组间比较,差异均有统计学意义(P0.01)。联合IGF-Ⅰ,削弱了AFC抑制PI3K/Akt/mTOR信号通路的作用,p-Akt/Akt, p-mTOR/mTOR在AFC组和AFC+IGF-Ⅰ组间比较,差异有统计学意义(P0.05);联合IGF-Ⅰ,AFC诱导的cleaved caspase-3,cleaved PARP蛋白表达减弱,cleaved caspase-3/procaspase-3,cleaved PARP/PARP在AFC组和AFC+IGF-Ⅰ组间比较,差异有统计学意义(P0.01)。综上,AFC浓度相关性地激活caspase级联反应诱导人结肠癌HCT116细胞发生凋亡,凋亡发生与PI3K/Akt/mTOR信号通路的抑制有关。     

苦瓜皂苷对人乳腺癌MCF-7细胞增殖作用的影响

目的 研究苦瓜皂苷抑制乳腺癌MCF-7细胞的作用及其诱导凋亡的机制.方法 采用MTT法观察苦瓜皂苷对MCF-7细胞的生长抑制作用;HE染色观察皂苷作用后癌细胞的形态学变化;免疫荧光染色检测Bcl-2的阳性表达率变化;流式细胞术分析细胞周期及细胞凋亡率.结果 MTT法得到苦瓜皂苷与MCF-7细胞生长抑制率之间存在浓度依赖关系,半数致死量IC50为159.02 mg/L;HE染色观察到经苦瓜皂苷作用24h后的MCF-7细胞出现典型的凋亡形态学特征;免疫荧光检测到苦瓜皂苷作用细胞24h后,Bcl-2的阳性表达率为22.35％,远小于对照组75.61％;流式细胞术表明苦瓜皂苷处理细胞后,S期细胞数明显增加,细胞周期被阻滞在S期,随着皂苷作用时间的延长细胞凋亡率增加.结论 苦瓜皂苷可抑制体外培养MCF-7细胞乳腺癌细胞的增殖,其抑制作用与细胞凋亡机制有关.     

秦皮甲素通过抑制Ras/ERK通路诱导人肺癌细胞A549凋亡

目的探讨秦皮甲素通过抑制Ras/ERK通路诱导人肺癌细胞A549凋亡的作用。方法 MTT法检测肺癌细胞A549的增殖,流式细胞仪检测细胞凋亡,分光光度法检测半胱氨酸蛋白酶caspase3、8、9的活性,免疫印迹法检测K-Ras、细胞外信号调节激酶1/2(ERK1/2)、磷酸化细胞外信号调节激酶1/2(p-ERK1/2)蛋白表达的变化。结果秦皮甲素可抑制人肺癌细胞A549的增殖,IC50值为0.4 mmol/L,并诱导凋亡;caspase3和caspase9表达增强,caspase8表达无明显改变。p-ERK1/2和K-Ras蛋白表达减弱,而ERK1/2蛋白表达无明显变化。结论秦皮甲素通过显著抑制K-Ras的表达和ERK1/2的磷酸化水平诱导人肺癌细胞A549凋亡。     

桦褐孔菌醇诱导人乳腺癌MCF-7细胞凋亡的分子机制研究

目的研究真菌桦褐孔菌中的代表化合物桦褐孔菌醇对人乳腺癌MCF-7细胞的作用,为桦褐孔菌及桦褐孔菌醇在抗肿瘤方面的临床应用提供新的依据。方法桦褐孔菌醇由传统植物化学方法从桦褐孔菌中提取分离得到。乳腺癌细胞系MCF-7与不同浓度的桦褐孔菌醇共孵育,采用噻唑蓝(MTT)法检测细胞存活率;流式细胞术Annexin V/PI双染色检测细胞凋亡;Western blotting法检测半胱氨酸蛋白酶3(Caspase-3)和多聚ADP-核糖聚合酶(Poly ADP ribose polymerase,PARP)蛋白表达情况。结果桦褐孔菌醇能够抑制MCF-7细胞的生长,并可诱导MCF-7细胞产生凋亡。浓度为25、50、100μmol/L的桦褐孔菌醇作用MCF-7细胞48 h后,细胞凋亡率显著增加至28.62%、39.45%、53.18%,明显高于对照组凋亡率(3.21%);经过50μmol/L桦褐孔菌醇分别处理12、24、48 h后,MCF-7细胞Caspase-3的活性形式cleaved Caspase-3表达量显著增加;凋亡蛋白标志物PARP裂解量升高,且呈时间依赖性。结论桦褐孔菌醇能抑制MCF-7细胞的增殖并诱导其凋亡,其作用机制推测与激活细胞Casepase-3和PARP有关。     

白头翁皂苷D对乳腺癌MCF-7细胞的体内外抗肿瘤作用研究北大核心CSCD

目的探讨白头翁皂苷D对人乳腺癌MCF-7细胞的体内外抗肿瘤作用及其机制。方法采用MTT法和吉姆萨染色,考察白头翁皂苷D对人MCF-7细胞体外增殖抑制的影响;建立MCF-7细胞裸鼠移植瘤模型,考察白头翁皂苷D体内抗肿瘤作用;HE染色和透射电镜实验考察肿瘤组织形态学和超微结构的变化;采用Western Blot法检测MCF-7细胞中Bcl-2、Caspase-3及PI3K/AKT/mTOR途径相关蛋白PI3K-p85、p-AKT、p-mTOR、p-p70S6K的表达。结果白头翁皂苷D可抑制MCF-7细胞的增殖,且呈剂量依赖关系;裸鼠体内实验结果显示白头翁皂苷D可抑制肿瘤的生长,30.0 mg·kg^(-1)剂量组瘤体质量明显降低,与模型组比较,差异有统计学意义(P<0.01);形态学观察结果也显示白头翁皂苷D对MCF-7细胞具有凋亡作用,且随着给药剂量的增加变化越明显;白头翁皂苷D(15.0,20.0,25.0μmol·L^(-1))不同剂量组均可抑制MCF-7细胞中Bcl-2、Caspase-3蛋白的表达,与对照组比较,差异均有统计学意义(P<0.01);白头翁皂苷D还可下调PI3K/AKT/mTOR信号传导通路相关蛋白PI3K-p85、p-AKT、p-mTOR、p-p70S6K蛋白的表达,20.0,25.0μmol·L^(-1)剂量组与对照组比较,差异均有统计学意义(P<0.05,P<0.01)。结论白头翁皂苷D对MCF-7细胞有显著的体内外抗肿瘤作用,其机制可能是通过下调PI3K/AKT/mTOR信号传导通路诱导细胞凋亡。     

异乌药内酯对人乳腺癌MCF-7细胞的生长抑制作用及其机制研究

目的研究异乌药内酯对人乳腺癌MCF-7细胞生长的抑制作用及其作用机制。方法体外培养MCF-7细胞,分为对照组及不同浓度异乌药内酯处理组。采用MTT实验检测异乌药内酯对MCF-7细胞增殖的影响;采用流式细胞术和TUNEL染色法检测异乌药内酯对MCF-7细胞周期、线粒体膜电位及细胞凋亡的影响;采用Western blotting法检测与细胞凋亡密切相关的蛋白表达情况。结果异乌药内酯以时间和剂量依赖性抑制MCF-7细胞增殖、促进细胞凋亡;能够将MCF-7细胞周期阻滞在G2/M期,诱导线粒体膜电位去极化;上调cleaved Caspase-3和促凋亡蛋白Bax表达,抑制抗凋亡蛋白Bcl-2表达,诱导MCF-7细胞的凋亡。结论异乌药内酯对MCF-7细胞的增殖抑制作用呈剂量和时间依赖性,其作用机制可能是通过线粒体途径诱导细胞凋亡。     

Dehydrocorydaline inhibits breast cancer cells proliferation by inducing apoptosis in MCF-7 cells

Dehydrocorydaline is an alkaloid isolated from traditional Chinese herb Corydalis yanhusuo W.T. Wang. We discovered that it possessed anti-tumor potential during screening of anti-tumor natural products from Chinese medicine. In this study, its anti-tumor potential was investigated with breast cancer line cells MCF-7 in vitro. The anti-proliferative effect of dehydrocorydaline was determined by MTT assay and the mitochondrial membrane potential (Δ Ψ m) was monitored by JC-1 staining. DNA fragments were visualized by Hoechst 33342 staining and DNA ladder assay. Apoptotic related protein expressions were measured by Western blotting. Dehydrocorydaline significantly inhibited MCF-7 cell proliferation in a dose- dependent manner, which could be reversed by a caspase-8 inhibitor, Z-IETD-FMK. Dehydrocorydaline increased DNA fragments without affecting ΔΨm. Western blotting assay showed that dehydrocorydaline dose-dependently increased Bax protein expression and decreased Bcl-2 protein expression. Furthermore, dehydrocorydaline induced activation of caspase-7,-8 and the cleavage of PARP without affecting caspase-9. These results showed that dehydrocorydaline inhibits MCF-7 cell proliferation by inducing apoptosis mediated by regulating Bax/Bcl-2, activating caspases as well as cleaving PARP.     

6-Methoxydihydrosanguinarine induces apoptosis and autophagy in breast cancer MCF-7 cells by accumulating ROS to suppress the PI3K/AKT/mTOR signaling pathway

6-Methoxydihydrosanguinarine (6-MDS) is a natural benzophenanthridine alkaloid extracted from Hylomecon japonica (Thunb.) Prantl. It is the first time to explore the effect and mechanism of 6-MDS in breast cancer. Network pharmacology, molecular docking, and molecular dynamics simulation technology were adopted to identify the potential targets and pathways of 6-MDS in breast cancer. Besides, cell proliferation, apoptosis, and western blotting assays were conducted to investigate the effect of 6-MDS on MCF-7 cells. Network pharmacology, molecular docking, and molecular dynamics simulation results confirmed the effect of 6-MDS on resisting breast cancer via the PI3K/AKT/mTOR signaling pathway. In addition, the functional experiments results demonstrated that 6-MDS inhibited proliferation and induced apoptosis and autophagy. The autophagy inhibitor chloroquine and the silence of Atg5 augmented the effect of 6-MDS on promoting apoptosis. Furthermore, 6-MDS suppressed the PI3K/AKT/mTOR signaling pathway, and the PI3K inhibitor LY294002 enhanced these changes and promoted the 6-MDS pro-apoptotic and autophagy effects. 6-MDS triggered the generation of reactive oxygen species. The pretreatment with antioxidant N-acetyl-L-cysteine reversed the changes induced by 6-MDS, including increases in apoptosis and autophagy and inhibition of the PI3K/AKT/mTOR pathway. In conclusion, 6-MDS induces the apoptosis and autophagy of MCF-7 cells by ROS accumulation to suppress the PI3K/AKT/mTOR signaling pathway.     

Induction of apoptotic cell death by Pharbitis nil extract in HER2-overexpressing MCF-7 cells

Aim of the study

We performed this study to investigate the anti-cancer activity of Pharbitis nil (PN) ethanol extract which has been used for herbal medicinal treatment against diseases in East Asia.

Materials and methods

We analyzed the effects of PN extract on proliferation of breast cancer cell lines, MCF-7 control vector (vec) and MCF-7 human epidermal growth factor receptor 2 (HER2) cells engineered to overexpress oncogenic HER2 via retroviral infection. We performed the proliferation assay to measure the growth rate of the cells. FACS analysis was used to analyze the cell cycle. Western blot analysis was used to investigate the effect of PN on the level and activation of intracellular molecules.

Results

We found that PN extract inhibited the proliferation of both MCF-7 vec and MCF-7 HER2 cells. This growth inhibition was accompanied with the increase of sub G0/G1 apoptotic fractions. When we check the efficiency of PN on the level of intracellular signaling molecules, we found that PN extract induced the inhibition of phosphorylation of HER2 and its downstream effectors, Akt and extracellular signal-regulated kinases (ERK). Active forms of both Akt and ERK were gradually decreased in PN-treated MCF-7 vec and MCF-7 HER2 cells suggesting that the growth suppressive activity of PN is related to signaling pathway. The level of cyclin D also diminished in PN-treated both cells suggesting that PN may inhibit the growth of MCF-7 vec and MCF-7 HER2 cells by perturbing cell cycle progression. It should be noted that PN decreased the growth rate of both MCF-7 vec and MCF-7 HER2 cells without changing the level and activation of p53.

Conclusion

PN extract suppressed the proliferation rate of HER-2 overexpressing MCF-7 breast cancer cells inducing apoptotic cell death in vitro. Our data demonstrates that PN extracts contain useful anti-tumor activity especially against HER2 overexpressing breast cancer.     

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??OBJECTIVE To study the effects of suberoylanilide hydroxamic acid (SAHA) and TRAIL treatment on cell proliferation and apoptosis for ER positive breast cancer cell line MCF-7. METHODS Human breast cell lines (MCF-7) were evaluated for the expressions of cell viability,cell apoptosis and cell cycle by muse cell analyzer. The mRNA levels of related apoptotic factors in MCF-7 cells were detected by real time PCR and solid phase apoptosis antibody microarray. RESULTS After the combination with SAHA and TRAIL,the ability of cell proliferation and cell viability were depressed,and the cell apoptosis was induced. The cell cycle assay showed that the MCF-7 cells were arrested in G₀/G₁ phase with SAHA and TRAIL treatment. CONCLUSION The combinatorial treatment of SAHA and TRAIL has a significantly inhibitory effect on cell growths of ER positive breast cancer MCF-7 cell.     

Hederacolchiside A1通过调节PI3K / Akt / mTOR信号通路诱导肿瘤细胞凋亡

目的：Hederacolchiside A1在体外对各种肿瘤细胞表现出细胞抑制和细胞毒活性,但其机制尚不清楚。本研究主要探讨白头翁中提取分离的Hederacolchiside A1的抗肿瘤活性和机制。 方法：用MTT法检测Hederacolchiside A1对A549,SMMC-7721,BEL-7402和MCF-7细胞增殖的抑制作用。通过流式细胞术分析鉴定经Hederacolchiside A1处理的肿瘤细胞凋亡状况。 结果：根据Western blotting和JC-1染色结果：hederacolchiside A1降低肿瘤细胞线粒体膜电位和Bcl-2蛋白表达水平,并升高cleaved caspase-3蛋白表达水平。此外,hederacolchiside A1可有效地抑制磷脂酰肌醇（-3）激酶（PI3K）,蛋白激酶B（Akt）和雷帕霉素靶蛋白（mTOR）表达水平。体内研究表明,在肝癌H22移植瘤模型中,Hederacolchiside A1（3.0,4.5和6.0mg / kg,ip）可显著抑制肿瘤的重量;在人乳腺癌MCF-7移植瘤模型中,用Hederacolchiside A1（3.25,7.5和15.0mg / kg,ig）进行治疗,观察到类似的抑制活性。 结论：这些数据表明,Hederacolchiside A1可通过调节PI3K / Akt / mTOR信号传导途径诱导肿瘤细胞凋亡从而抑制肿瘤细胞的增殖。     

白藜芦醇联合姜黄素对SMMC-7721肝癌细胞作用

目的:观察白藜芦醇联合姜黄素对体外人肝癌细胞SMMC-7721增殖和凋亡的影响及相关信号通路.方法:不同浓度白藜芦醇、姜黄素及两药联合干预SMMC-7721细胞,MTT法检测细胞增殖,流式细胞术检测细胞凋亡、Hoechst 33258染色检测细胞凋亡形态变化,比色法检测半胱氨酸天冬氨酸蛋白酶(caspase)-3,caspase-8,caspase-9酶活性,Western blot法检测半胱氨酸天冬氨酸蛋白酶切割底物(PARP).结果:与对照组相比,白藜芦醇、姜黄素单独或联合作用SMMC-7721细胞均可抑制SMMC-7721细胞增殖,两药联合后抑制作用更显著.白藜芦醇、姜黄素联合较单独用药可增强SMMC-7721细胞凋亡,呈现凋亡形态改变,白藜芦醇、姜黄素及联合组细胞凋亡率分别为( 17.39±1.41)％,(14.96±2.23)％,(25.36±2.68)％;同时提高SMMC-7721细胞caspase-3,caspase-8及caspase-9活性,促使PARP蛋白剪辑.结论:白藜芦醇、姜黄素联合使用可增强对人肝癌细胞SMMC-7721的抗癌作用,并可能与caspase-8,caspase-9/caspase-3/PA RP信号通路介导细胞凋亡相关.