Effect of Artemisia species on cellular proliferation and apoptosis in human breast cancer cells via estrogen receptor-related pathway

OBJECTIVE： To investigate the mechanism underlying the anticancer effect of Artemisia species through the inhibition of cell growth and induction of apoptosis in breast carcinoma cells.METHODS： To evaluate the anticancer activity of methanol extracts of eight Artemisia species（Artemisia stolonifera, Artemisia selengensis, Artemisia japonica, Artemisia Montana, Artemisia capillaris,Artemisia sylvatica, Artemisia keiskeana, and Artemisia scoparia）, we first investigated the proliferation of estrogen receptor（ER）-positive MCF-7breast carcinoma cells exposed to 5 or 200 g/mL for72 h. Apoptosis induction was assessed by an Annexin V binding assay in cells exposed to extracts at a high concentration（200 g/mL）. To verify the mechanism of apoptosis, ER expression and its related signaling was investigated using an immunoblot assay under the same conditions.RESULTS： MCF-7 cells showed the strongest antiproliferative response to the tested extracts. Howev-er, a biphasic effect was observed： the extracts inhibited proliferation at high concentrations whereas they stimulated it at low ones. ER expression was similarly modulated by the extracts. However, all of the extracts induced apoptosis at a high concentration（200 g/mL）. Compared to the control level, exposure to the extracts resulted in a remarkable increase in the shift of cell populations.CONCLUSION： The present study suggests that the tested Artemisia species exerted their anticancer effects through the induction of apoptosis via an ER-related pathway.     

Effect of Artemisia species on cellular proliferation and apoptosis in human breast cancer cells via estrogen receptor-related pathway

Objective

To investigate the mechanism underlying the anticancer effect of Artemisia species through the inhibition of cell growth and induction of apoptosis in breast carcinoma cells.

Methods

To evaluate the anticancer activity of methanol extracts of eight Artemisia species (Artemisia stolonifera, Artemisia selengensis, Artemisia japonica, Artemisia Montana, Artemisia capillaris, Artemisia sylvatica, Artemisia keiskeana, and Artemisia scoparia), we first investigated the proliferation of estrogen receptor (ER)-positive MCF-7 breast carcinoma cells exposed to 5 or 200 g/mL for 72 h. Apoptosis induction was assessed by an Annexin V binding assay in cells exposed to extracts at a high concentration (200 g/mL). To verify the mechanism of apoptosis, ER expression and its related signaling was investigated using an immunoblot assay under the same conditions.

Results

MCF-7 cells showed the strongest antiproliferative response to the tested extracts. However, a biphasic effect was observed: the extracts inhibited proliferation at high concentrations whereas they stimulated it at low ones. ER expression was similarly modulated by the extracts. However, all of the extracts induced apoptosis at a high concentration (200 g/mL). Compared to the control level, exposure to the extracts resulted in a remarkable increase in the shift of cell populations.

Conclusion

The present study suggests that the tested Artemisia species exerted their anticancer effects through the induction of apoptosis via an ER-related pathway.     

Induction of G2/M arrest and apoptosis by water extract of Strychni Semen in human gastric carcinoma AGS cells

The seed of Strychnos nux-vomica (Loganiaceae) has been used in traditional Oriental medicine as a folk remedy for the treatment of cancer. However, the mechanism responsible for the anticancer effects of Strychni Semen is not clearly understood. The study tested whether and how the water extract of Strychni Semen (ESS) treatment would affect the growth of AGS human gastric carcinoma cells. ESS was found to inhibit the growth of AGS cells in a concentration-dependent manner. Cell cycle analysis showed G2/M phase arrest and apoptosis in AGS cells following ESS treatment. ESS-mediated G2/M arrest was found to be associated with up-regulation of cyclin A, Cdc2, tumor suppressor p53 and cyclin dependent kinase (Cdk) inhibitor p21(WAF1/CIP1), whereas the expressions of other G2/M regulatory proteins, including cyclin B1 and Cdk2, were down-regulated compared with the control. The induction of apoptotic cell death by ESS was associated with down-regulation of anti-apoptotic Bcl-2 and up-regulation of pro-apoptotic Bax expression. Further results indicate that caspase-3, caspase-8 and caspase-9 are all activated by ESS, together with cleavage of downstream caspase-3 target proteins. Taken together, the results of this study suggest the involvement of multiple signaling pathways targeted by ESS in mediating G2/M cell cycle arrest and apoptosis in AGS cells, and warrant further investigation.     

Antiproliferative effect of Erycibe elliptilimba on human breast cancer cell lines

Erycibe elliptilimba Merr. & Chun., family Convolvulaceae, is a Thai traditional medicine which has long been prescribed for various infectious and malignant diseases. Bio-assays of extracts from Erycibe elliptilimba Merr. & Chun. showed that a fraction (fraction 3) from an methanolic extract had an antiproliferative effect on SKBR3 and MDA-MB435 human breast cancer cells. The ED50 value of Erycibe elliptilimba Merr. & Chun. fraction 3 was 56.07 and 30.61 μg/ml for SKBR3 and MDA-MB435, respectively. After 48 h of exposure, this fraction at a concentration of 100 μg/ml significantly reduced cell proliferation in both cancer cells. In MDA-MB435 cells, cell cycle analysis showed that the herb extract fraction 3 induced the accumulation of cells in G2/M phase, whereas no significant change in cell cycle was detected in SKBR3 cells. The results indicated that the extract fraction 3 could induce cell cycle arrest in some way. However, further investigation is needed to assess the molecular mechanisms mediated anticancer activities of this plant.     

Methanol extract of the seaweed Gloiopeltis furcata induces G2/M arrest and inhibits cyclooxygenase-2 activity in human hepatocarcinoma HepG2 cells

It was previously reported that a methanol extract of Gloiopeltis furcata (MEGF), a kind of edible seaweed, inhibited the growth of several human cancer cell lines. In the present study, the effect of MEGF on the growth of human hepatocarcinoma HepG2 cells and its effect on the cyclooxygenases (COXs) expression were investigated. MEGF markedly reduced the viability of HepG2 cells and induced the G2/M arrest of the cell cycle in a concentration dependent manner. These effects were associated with the down-regulation of cyclin A, up-regulation of cyclin-dependent kinase (Cdk) inhibitor p21 (WAF1/CIP1) and dephosphorylation of Cdc25C. Furthermore, it was found that MEGF decreased the levels of COX-2 mRNA and protein expression without significant changes in the levels of COX-1, which was correlated with a decrease in prostaglandin E(2) (PGE(2)) synthesis. These findings indicate that MEGF may have a possible therapeutic potential in hepatoma cancer patients.     

Cell cycle arrest and cell apoptosis induced by Equisetum hyemale extract in murine leukemia L1210 cells

Ethnopharmacological relevance

Equisetum hyemale has been used as a traditional herbal medicine to treat various diseases such as hypertension, inflammatory diseases, acute stroke, bleeding and cancer. The present study aimed to investigate the anti-proliferative effect and the underlying mechanisms of E.hyemale extract on murine leukemia L1210 cells.

Materials and methods

The inhibitory effect of Ehyemale extract on L1210 cells was evaluated by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. Cell cycle distribution was evaluated with flow cytometry following PI (propidium iodide) staining. Apoptotic cell death was determined by Annexin V–FITC/PI and nuclear DAPI (4′-6-diamidino-2-phenylindole) staining. DNA damage and changes of mitochondrial membrane potential were also detected with flow cytometry analysis.

Results

E.hyemale extract exerted significant antiproliferative effects on L1210 cells in a dose- and time-dependent manner. Flow cytometry analysis showed that E.hyemale extract induced cell cycle arrest at G2/M phase in L1210 cells. Phosphatidylserine exposure, chromatin condensation, DNA damage and loss of mitochondrial membrane potential were observed clearly after treatment with Ehyemale extract.

Conclusion

The results in this study indicate that E.hyemale extract could inhibit L1210 cell proliferation through inducing G2/M arrest and cell apoptosis.     

生血汤加减联合放疗对乳腺癌患者外周血癌胚抗原、基质金属蛋白酶-2、金属蛋白酶抑制剂-2的影响

目的 探讨生血汤加减联合放疗对乳腺癌患者外周血癌胚抗原(CEA)、基质金属蛋白酶-2(MMP-2)、金属蛋白酶抑制剂-2(TIMP-2)mRNA的影响.方法 将2005年2月至2008年1月来我院就诊的91例乳腺癌患者,按随机数字表法分为两组,对照组40例,采用放疗配合口服阿那曲唑片治疗;治疗组51例,在对照组治疗基础上加服中药生血汤治疗.结果 治疗组总有效率为82.3％、对照组为52.5％,两组总有效率比较,差异有统计学意义(x2=19.74,P＜0.05).治疗后对照组外周血CEA、MMP-2 mRNA表达量[分别为(7.50±4.32) ng/ml、(116.32±67.40) ng/ml]均高于治疗组[(7.20±3.22)ng/ml、(105.08±37.82) ng/ml,P＜0.05];TIMP-2/MMP-2比值(26.65±19.75)则低于治疗组[ (39.48±21.21),p＜0.05].结论 生血汤加减联合放疗可有效降低乳腺癌患者外周血CEA、MMP-2mRNA表达量,升高TIMP-2/MMP-2比.     

Gleditsia sinensis thorn extract inhibits human colon cancer cells: the role of ERK1/2, G2/M‐phase cell cycle arrest and p53 expression

The thorns of Gleditsia sinensis are used as a medicinal herb in China and Korea. However, the mechanisms responsible for the antitumor effects of the water extract of Gleditsia sinensis thorns (WEGS) remain unknown. HCT116 cells treated with the WEGS at a dose of 800 μg/mL (IC₅₀) showed a significant decrease in cell growth and an increase in cell cycle arrest during the G2/M‐phase. G2/M‐phase arrest was correlated with increased p53 levels and down‐regulation of the check‐point proteins, cyclinB1, Cdc2 and Cdc25c. In addition, treatment with WEGS induced phosphorylation of extracellular signal‐regulated kinase (ERK), p38 MAP kinase and JNK (c‐Jun N‐terminal kinases). Moreover, inhibition of ERK by treatment of cells with the ERK‐specific inhibitor PD98059 blocked WEGS‐mediated p53 expression. Similarly, blockage of ERK function in the WEGS‐treated cells reversed cell‐growth inhibition and decreased cell cycle proteins. Finally, in vivo WEGS treatment significantly inhibited the growth of HCT116 tumor cell xenografts in nude mice with no negative side effects, including loss of body weight. These results describe the molecular mechanisms whereby the WEGS might inhibit proliferation of colon cancer both in vitro and in vivo, suggesting that WEGS has potential as an anticancer agent for the treatment of malignancies. Copyright © 2010 John Wiley & Sons, Ltd.     

九香虫水提液化学成分及其对乳腺癌细胞增殖的抑制作用

目的研究九香虫水提液化学成分及其对乳腺癌细胞增殖的抑制作用。方法制备九香虫水提液,采用GC-MS法分析九香虫水提液化学成分,利用SDS-PAGE电泳分析其蛋白组成。采用MTT法与形态学观察法检测九香虫水提液对人乳腺癌细胞MDA-MB-453、HCC-1937及小鼠乳腺癌细胞4T1增殖的抑制作用。结果九香虫水提液主要化学成分为L-苹果酸、L-核糖、半乳糖酸等;蛋白含有量为0. 843 mg/m L;蛋白分子量主要分布在75、35、30、25、12 k Da;水提液对人乳腺癌MDA-MB-453、HCC-1937细胞及小鼠乳腺癌4T1细胞体外增殖具有显著抑制作用,且呈剂量相关性,IC50值分别为0. 142、0. 059、0. 190 g/m L。结论九香虫水提液主要为氨基酸、脂肪酸、糖类物质,蛋白含有量较高,对人乳腺癌MDA-MB-453、HCC-1937细胞及小鼠乳腺癌4T1细胞的体外增殖具有显著抑制作用。     

5F对肝癌细胞毒性、细胞凋亡及细胞周期的影响

目的调查半边旗(PsL)提取物Ent-11α-hydroxy-15-oxo-kaur-16-en-19-oic-acid(5F)对人类肝癌细胞Hep3B的抗癌效果。方法在不同的时间段,采用5F(0～80μg/ml)处理Hep3B细胞。通过MTT法检测细胞毒性,通过Annexin V-EGFP染色及caspase-3活性检测细胞凋亡,通过碘化丙锭染色检测细胞周期。结果 5F以剂量、时间依赖方式抑制Hep3B细胞增殖,Hep3B细胞Annexin V-EGFP阳性染色及caspase-3活化确定了5F诱导的细胞凋亡,5F阻滞细胞于G2期。结论 5F抑制Hep3B细胞增殖与细胞G2期阻滞及细胞凋亡有关。     

四君子汤提取物对人乳腺癌MDA-MB-468细胞生长的抑制作用

目的探究四君子汤提取物(Sijunzi Decoction extract,SDE)对人三阴性乳腺癌MDA-MB-468细胞生长的作用。方法采用不同质量浓度SDE作用于MDA-MB-468细胞,CCK-8实验和细胞划痕实验检测SDE对细胞增殖和迁移能力的影响;克隆形成实验检测SDE对细胞集落形成能力的影响;Hoechst33342染色技术和流式细胞术(FCM)检测SDE对细胞凋亡和周期的影响;Western blotting技术检测SDE对信号传导及转录激活蛋白3(STAT3)表达的影响。结果与对照组比较,SDE对MDA-MB-468细胞有一定的抑制作用(P0.05),且呈质量浓度和时间依赖性。克隆形成实验结果表明SDE能够抑制细胞克隆形成。细胞迁移实验结果显示,SDE中、高质量浓度能够明显减弱细胞划痕愈合能力(P0.001)。FCM检测凋亡结果显示,SDE各质量浓度组细胞早期凋亡率和总凋亡率逐渐上升,呈质量浓度依赖性,且中、高质量浓度的SDE能显著诱导细胞凋亡(P0.01、0.001)。SDE能够影响细胞周期,使G2期细胞显著减少(P0.01)。Westernblotting结果显示,SDE处理细胞后,STAT3蛋白表达水平显著降低。结论 SDE能够抑制MDA-MB-468细胞的增殖和克隆形成,促进其凋亡,使G2期细胞减少。其机制可能与调控STAT3通路有关。     

乳腺癌的ER、PR、C-erbB-2表达与钼靶X线表现相关性研究

目的探讨乳腺癌ER、PR、C-erbB-2因子表达与钼靶X线表现之间的相关性。方法分析87例乳腺患者钼靶X线中肿块、钙化、病变区密度和结构扭曲与免疫组织化学测定的ER、PR和C-erbB-2的表达的关系。结果87例乳腺癌患者中,肿块边缘毛刺征者ER、PR阳性表达率高;病变区有钙化和结构扭曲者C-erbB-2阳性表达率高;病变区高密度和结构扭曲ER、PR阳性表达率低;肿块大小与ER、PR和C-erbB-2表达均无关。结论乳腺癌钼靶X线征象和ER、PR及C-erbB-2的表达有密切关系,在一定程度上反映了ER、PR及C-erbB-2的表达状态,为乳腺癌的术前辅助内分泌治疗和预后评估提供了一定的信息。     

Effect of ingredients from Chinese herbs on enterovirus D68 production

Human enterovirus 68 (EVD68) is a primary causative agent for respiratory illness worldwide. Until now, there has been no available medication for treating EVD68‐related diseases. Rheum emodin, artemisinin, astragaloside, pseudolaric acid B, oridonin, and erianin are natural extracts from Chinese herbs that have traditionally been used for the treatment and prevention of epidemic diseases. Our results showed that pseudolaric acid B protected cells from EVD68‐induced cytopathic effects and decreased viral production. However, the same effects were not observed with rheum emodin, astragaloside, or artemisinin. Pseudolaric acid B inhibited EVD68 production by manipulating the host cell cycle in G2/M phase. Further, either oridonin or erianin related G2/M arrest also inhibited viral production. Due to inducing G2/M phase arrest, pseudolaric acid B, oridonin, and erianin might be good candidates for inhibiting EVD68 production, and Chinese herbs with natural compounds inducing G2/M arrest should be considered for the treatment of EVD68‐related diseases.     

Anti-breast cancer effects and mechanisms of Xihuang pill on human breast cancer cell lines

Objective

To investigate the anti-breast cancer (BC) effects and mechanisms of action of Xihuang pill (XHP) by conducting in vitro experiments on human BC cell lines.

Methods

Two human BC cell lines (MCF-7 and MDA- MB231) were cultured and treated with XHP. Cell viability was detected using the 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay. Flow cytometry was used to measure the cell cycle and apoptosis. The cell cycle was analyzed with propidium iodide staining. Apoptosis was evaluated using the Annexin V-fluorescein isothiocyanate/propidium iodide method. Western blotting was used to analyze the expression of estrogen receptor (ER)-α and ER-β.

Results

XHP had growth-inhibitory effects on MCF-7 and MDA-MB231 cells with a half-maximal inhibitory concentration (IC50) of 10.14 mg/mL (MCF-7) and 8.98 mg/mL (MDA-MB231). Apoptosis was induced to some extent. Certain changes in the ER were caused. Upregulation of ER-α protein was found in MCF-7 cells. ER-β expression in MDA-MB231 cells was increased. Cell-cycle arrest was not observed in the two BC cell lines. ER-β expression in MCF-7 cells was unchanged. No ER-α expression was shown in MDA-MB231 cells.

Conclusion

These data suggest that XHP can affect cell viability and cause apoptosis, but that the cell cycle is not blocked. XHP has a certain impact on ER expression, but its mechanisms of action of anti-BC effects may not be due to regulation of ER expression.     

猪苓酮A通过调节Bcl-2/Bax信号通路诱导雌激素受体阴性的乳腺癌细胞凋亡研究

目的 研究猪苓酮A对雌激素受体（ER）阳性或阴性人乳腺癌细胞的凋亡作用及其可能的作用机制。方法 选择2株ER阳性人乳腺癌细胞（MCF-7细胞、BT474细胞）和1株ER阴性人乳腺癌细胞（MDA-MB-453细胞）,用不同浓度的猪苓酮A分别处理24 h,或以50 μmol/L的猪苓酮A分别处理6、12、24、48 h后,分别采用噻唑蓝（MTT）法测定3株乳腺癌细胞的增殖情况;采用流式细胞仪检测细胞凋亡和细胞周期;采用Western blottig法检测与细胞凋亡密切相关的Bcl-2家族蛋白表达情况。结果 猪苓酮A可以时间和剂量依赖性地抑制ER阴性MDA-MB-453细胞的增殖;而对于ER阳性MCF-7、BT474细胞增殖均无明显影响;细胞周期检测显示猪苓酮A可以使MDA-MB-453细胞的生长阻滞在G₁或G₂/M期;50 μmol/L猪苓酮A作用于MDA-MB-453细胞24 h后,细胞出现明显凋亡,而MCF-7、BT474细胞则没有出现明显凋亡;Western blotting检测发现50 μmol/L猪苓酮A处理组ER阴性MDA-MB-453细胞中促凋亡蛋白Bax、Bad表达量升高,而抗凋亡蛋白Bcl-2、Bcl-w表达量降低。结论 猪苓酮A能抑制ER阴性乳腺癌细胞的增殖并诱导其凋亡,其作用机制与调控Bcl-2家族蛋白的表达量有关。     

针药结合对乳腺增生大鼠乳腺组织及雌激素受体亚型表达的影响

目的:研究针药结合对大鼠乳腺增生病模型的抑制作用及作用机制。方法:复制乳腺增生病模型,造模后随机分为针刺组、中药组、针药组、对照组、模型组,除正常组、模型组外,其他治疗组分别行针刺、中药、针药结合及西药三苯氧胺治疗。每日治疗1次,9次一疗程,治疗3个疗程(30天)。观察乳腺组织的形态变化,测定腺泡腔直径及面积,免疫组化检测雌激素受体亚型(ERα、ERβ)的表达。结果:模型组腺泡腔直径及面积均较正常组增大(均P<0.01);治疗组腺泡腔直径及面积均较模型组减小(P<0.05,P<0.01);针刺和中药可上调ERβ、下调ERα表达。结论:针刺和中药均对大鼠乳腺增生有抑制作用,针药结合的抑制作用最强,基本接近三苯氧胺水平,其治疗机制可能与上调ERβ、下调ERα表达有关。     

Danshen (Salvia miltiorrhiza) extract inhibits proliferation of breast cancer cells via modulation of Akt activity and p27 level

Danshen is widely used in traditional Chinese medicine, often in combination with other herbs. To check the effect of Danshen on the proliferation of breast cancer cells, Danshen extract was used to treat MCF‐7 and MCF‐7 HER2 cells, the latter of which overexpresses HER2. HER2 is a receptor tyrosine kinase, and is involved in signal transduction pathways leading to tumor cell proliferation. MTT and cell proliferation assays revealed that Danshen strongly inhibited the proliferation of both MCF‐7 vec cells and MCF‐7 HER2 cells. Flow cytometry analyses indicated that Danshen induced cell cycle delay in the G1 phase. HER2 expression was shown to confer resistance to Danshen‐induced inhibition of proliferation and cell cycle delay, suggesting that HER2 is responsible for the resistance to Danshen. Danshen treatment induced the down‐regulation of Akt phosphorylation and an increase in p27 in MCF‐7 vec and MCF‐7 HER2 cells. Nevertheless, MCF‐7 HER2 cells were more resistant to the Danshen‐induced inhibition of Akt phosphorylation and p27 up‐regulation. Copyright © 2009 John Wiley & Sons, Ltd.     

红芪对血液流变性的影响

目的：研究红芪对血液流变性的影响。方法：采用正常、寒凝血瘀(肾上腺素 冰水应激)、气虚血瘀(灌服番泻叶)模型大鼠,观察红芪醇提物和水提物对血液粘度、血沉、红细胞压积、电泳时间等血液流变学指标影响,并考察其对ADP诱导家兔体外血小板聚集的影响。结果：红芪醇提物可明显降低正常大鼠高切(80s^-1)和低切(20s^-1)下全血比粘度,红芪水提物可明显减轻正常大鼠体外血栓的干重,显降低肾上腺素加冰水浴所致血瘀模型大鼠体外血栓湿重及干重,对血栓长度、全血比粘度及血沉等指标有一定抑制趋势;二均可明显降低番泻叶所致气虚血瘀模型大鼠红细胞压积。缩短其红细胞电泳速率;体外125、250、500μm／m1可剂量依赖性地抑制ADP引起的家兔血小板聚集。结论：红芪具有一定活血化瘀作用。     

补骨脂酚诱导人乳腺癌MCF-7细胞S期周期阻滞及机制研究

目的研究补骨脂酚对人乳腺癌MCF-7细胞生长抑制作用及其机制。方法采用噻唑蓝法(MTT法)考察补骨脂酚对MCF-7细胞生长的影响;采用流式细胞术检测补骨脂酚处理后细胞周期分布情况及活性氧(ROS)的产生;利用荧光显微镜观察补骨脂酚对MCF-7细胞核的影响;采用蛋白免疫印迹法检测补骨脂酚作用下MCF-7细胞凋亡、周期相关蛋白和丝裂原活化蛋白激酶(MAPK)家族蛋白的表达;引入ROS清除剂及MAPK家族蛋白抑制剂考察其在补骨脂酚作用下对MCF-7细胞生长抑制率和细胞周期相关蛋白表达的影响。结果补骨脂酚可以呈时间、剂量依赖性抑制MCF-7细胞生长,其抑制作用明显强于阳性药5-氟尿嘧啶。补骨脂酚可诱导MCF-7细胞产生ROS,同时上调p-p53及p21蛋白表达水平,下调细胞周期相关蛋白CDK2和CyclinA2表达水平,进而引起细胞发生S期阻滞。而上述影响可被ROS清除剂Tiron逆转,表明ROS参与补骨脂酚诱导的S期阻滞。加入p38MAPK抑制剂SB203580后,细胞产生的ROS水平降低,表明ROS的产生依赖于p38MAPK途径。结论补骨脂酚抑制MCF-7细胞增殖的作用与其引起细胞发生S期阻滞有关,ROS参与S期阻滞过程。