瑞香素调节Shh/Gli1信号通路对乳腺癌增殖、凋亡和化疗敏感性的影响

目的 探究瑞香素调节音猬因子(Shh)/胶质细胞瘤转录因子1(Gli1)信号通路对乳腺癌增殖、凋亡和化疗敏感性的影响。方法 将人乳腺癌细胞株MDA-MB-231细胞分为对照组、瑞香素组(40μmol/L瑞香素)、GANT61组(10μmol/L GANT61)、瑞香素+GANT61组(40μmol/L瑞香素和10μmol/L GANT61);CCK-8实验检测MDA-MB-231细胞增殖;划痕试验检测MDA-MB-231细胞迁移情况;在上述各分组细胞中加入1 mg/L阿霉素(Dox)处理48 h后CCK-8实验检测细胞化疗敏感性;流式细胞术检测MDA-MB-231细胞凋亡情况;Western blot检测MDA-MB-231细胞Shh/Gli1通路及凋亡相关蛋白表达水平。结果 与0μmol/L比较,随着瑞香素的剂量增加MDA-MB-231细胞存活率显著降低(P<0.05),而与40μmol/L比较,50μmol/L瑞香素处理MDA-MB-231细胞存活率无差异(P>0.05),因此选择40μmol/L瑞香素作为后续实验的干预条件;与对照组比较,瑞香素组和GANT61组OD4...     

褐藻素对乳腺癌MDA-MB-231细胞增殖和凋亡的影响

目的:探究褐藻素对人乳腺癌MDA-MB-231细胞的增殖和凋亡的影响。方法:不同浓度褐藻素处理MDA-MB-231细胞后,采用CCK-8法测定细胞抑制率、Live/Dead^TM细胞成像试剂盒进行活死细胞荧光分析,TUNEL法、Annexin V/PI染色法检测细胞凋亡,Western blotting方法检测Bcl-2、Bax、JAK2、p-JAK2、STAT3、p-STAT3蛋白的表达,采用试剂盒检测Caspase-9和Caspase-3/7活性。结果:褐藻素处理MDA-MB-231细胞24 h时,16μmol·L^-1褐藻素明显抑制细胞增殖,IC₅₀为68.79μmol·L^-1;处理48 h时,8μmol·L^-1褐藻素明显抑制细胞增殖,IC₅₀为38.08μmol·L^-1;褐藻素对乳腺癌细胞的细胞活力和增殖的抑制作用表现出一定的时间和剂量依赖性(P<0.05)。与阴性对照组相比,16,32,64μmol·L^-1     

冬凌草甲素通过PI3K/Akt通路诱导MDA-MB-231细胞的凋亡

目的：研究冬凌草甲素对人乳腺癌MDA-MB-231细胞增殖产生的影响,初步探讨其作用机理。方法：体外培养人乳腺癌MDA-MB-231细胞,采用6、12、24μmol/L冬凌草甲素对其进行处理,采用倒置显微镜进行细胞形态学观察,MTT比色法检测细胞存活率,流式细胞术检测细胞凋亡率,Western blotting检测凋亡相关蛋白procaspase-3、PARP及Akt、p-Akt、p-GSK3β表达的变化。结果：冬凌草甲素作用MDA-MB-231细胞24h后,可观察到细胞凋亡的形态学改变,以24μmol/L组最为明显。实验组与对照组相比,细胞存活率显著降低、凋亡率显著升高（P〈0.01）,具有时间和剂量依赖性,凋亡相关蛋白procaspase-3下调,caspase-3底物PARP被逐步剪切,并伴随p-Akt、p-GSK3β蛋白水平下调（P〈0.05）。结论：冬凌草甲素可有效抑制人乳腺癌MDA-MB-231细胞的增殖,诱导其凋亡,机制与PI3K/Akt通路的抑制有关。     

槐耳颗粒联合DC-CIK细胞对乳腺癌MDA-MB-231干细胞体内外杀伤作用研究

目的观察槐耳颗粒联合树突细胞(DC)-细胞因子诱导的杀伤细胞(CIK)对乳腺癌MDA-MB-231干细胞体内外的杀伤效果。方法用槐耳颗粒(500 mg/L)、槐耳颗粒(1 000 mg/L)与乳腺癌MDA-MB-231细胞作用后,荧光倒置显微镜观察MDA-MB-231细胞形态的变化,MTT比色法检测槐耳颗粒对MDA-MB-231细胞增殖的抑制情况,流式细胞仪检测乳腺癌MDA-MB-231细胞干细胞(CD44~+CD24~-细胞)所占的比例。采用乳酸脱氢酶(LDH)释放测定法检测DC-CIK细胞联合槐耳颗粒对乳腺癌MDA-MB-231细胞的杀伤作用。建立BalB/C荷MDA-MB-231乳腺癌干细胞裸鼠模型,随机分为模型组、槐耳颗粒组、DC-CIK组及槐耳颗粒与DC-CIK细胞连合组,槐耳颗粒组小鼠按0.33g/kgig给药,每天1次,连续3周;DC-CIK组小鼠尾iv DC-CIK细胞5×10~5个/g,2次/周,连续3周;联合治疗组同时按照单药给药方法联合治疗,观察各组荷瘤裸鼠生存状态,比较各组裸鼠体质量和瘤体积。结果槐耳颗粒(500mg/L)、槐耳颗粒(1000mg/L)作用于乳腺癌MDA-MB-231细胞36h后,可使乳腺癌MDA-MB-231细胞逐渐变小,核浓缩,抑制乳腺癌MDA-MB-231细胞的增殖,同时乳腺癌MDA-MB-231细胞中CD44~+CD24~-细胞比例明显降低。给予荷瘤裸鼠治疗结束后,联合治疗组的瘤质量明显低于2个单独治疗组。结论槐耳颗粒联合DC-CIK细胞对乳腺癌MDA-MB-231干细胞体内外杀伤效果均好于单独使组。     

阿帕替尼调控上皮间质转化抑制三阴性乳腺癌MDA-MB-231细胞侵袭

目的探讨阿帕替尼对人三阴性乳腺癌MDA-MB-231细胞侵袭作用的影响。方法 MTT法检测不同浓度的阿帕替尼(1、 2、 4、 8、 16μmol·L~(-1))作用于乳腺癌MDA-MB-231细胞48 h的细胞毒性,并采用Bliss法计算半数抑制浓度(IC50); Annexin V-FITC/PI流式细胞术检测阿帕替尼对MDA-MB-231细胞凋亡的影响;Transwell实验检测阿帕替尼对MDA-MB-231细胞侵袭能力的影响;Western blot检测阿帕替尼对上皮间质转化(EMT)标志性蛋白上皮钙黏素、神经钙黏素及波形蛋白表达的影响。结果 1、 2、4、 8、 16μmol·L~(-1)阿帕替尼可显著抑制MDA-MB-231细胞的增殖,增殖抑制率分别为(38.38±3.33)%、(53.17±4.31)%、(62.09±6.21)%、(80.97±7.50)%和(98.54±9.75)%。阿帕替尼作用48 h对MDA-MB-231细胞的IC50为1.96μmol·L~(-1)。与空白对照组相比,阿帕替尼可显著诱导MDA-MB-231细胞凋亡,显著抑制MDA-MB-231细胞侵袭基底膜的能力(P<0.05);阿帕替尼还可显著上调MDA-MB-231细胞上皮钙黏素的表达,下调神经钙黏素及波形蛋白的表达(P <0.05)。结论阿帕替尼可能通过调控EMT进而发挥抗三阴性乳腺癌MDA-MB-231细胞侵袭作用。     

三氧化二砷对MDA-MB-231细胞增殖、凋亡的影响

目的观察不同浓度的三氧化二砷对三阴性乳腺癌MDA-MB-231细胞增殖、凋亡的影响。方法噻唑蓝(MTT)比色法检测不同浓度(0.2、0.4、0.6、0.8、1.0μmol.L-1)的三氧化二砷作用于三阴性乳腺癌MDA-MB-231细胞24、48、72 h后的细胞增殖率,Western blot检测不同浓度的三氧化二砷作用于MDA-MB-231细胞24 h的PCNA水平。结果不同浓度的三氧化二砷作用于三阴性乳腺癌MDA-MB-231细胞24、48、72 h后,随着时间的推移和浓度的升高,细胞增殖抑制率增加,有浓度和时间依赖性,以1.0μmol.L-1三氧化二砷作用最明显,Western blot检测显示,与对照组比较,随着浓度的升高,PCNA蛋白降低,其中以1.0μmol.L-1三氧化二砷作用后最明显。结论三氧化二砷能促进MDA-MB-231细胞的凋亡,其机制可能与下调PCNA表达的促凋亡途径有关。     

四氢姜黄素增强姜黄素抗乳腺癌作用机制初探

目的探讨口服姜黄素体内抗乳腺癌的作用及机制。即其主要代谢产物——四氢姜黄素增强姜黄素抑制乳腺癌细胞生长的作用及机制。方法用MTT法及克隆形成法检测姜黄素与四氢姜黄素单独或联合作用对人乳腺癌MCF-7、MDAMB-231细胞增殖的影响;用PI染色结合流式细胞术检测姜黄素和四氢姜黄素单独或联合作用对人乳腺癌MCF-7、MDA-MB-231细胞周期分布的影响;用Annexin V/PI双染结合流式细胞术检测姜黄素和四氢姜黄素单独或联合作用对人乳腺癌MCF-7、MDA-MB-231细胞凋亡的影响;用Western印迹法检测姜黄素和四氢姜黄素单独或联合作用后人乳腺癌MCF-7、MDA-MB-231细胞中凋亡相关蛋白Fas、FADD、c FLIP、活化胱天蛋白酶3/8及PARP的表达水平。结果 10～40μmol·L~(-1)的四氢姜黄素对于两种人乳腺癌细胞生长抑制作用较弱,而相同剂量的姜黄素可明显抑制乳腺癌细胞生长,两药联用对于MCF-7和MDA-MB-231细胞的生长抑制作用随时间的延长和药物浓度的增加而增加,且对肿瘤细胞的抑制作用明显强于对应的单药处理组,且多个剂量组合的CI值小于1。与对照组相比,四氢姜黄素及姜黄素单加或联用均不影响MCF-7及MDA-MB-231细胞的周期分布,但却发现姜黄素或四氢姜黄素联合姜黄素处理48及72 h后,两种细胞中sub-G1期细胞凋亡峰显著上调,且两药联用组的细胞凋亡比率显著大于姜黄素单加组。进一步Annexin V/PI双染结合流式细胞术的结果显示,与对照组相比,四氢姜黄素组没有凋亡产生,姜黄素组可随时间依赖性的诱导两种乳腺癌细胞发生凋亡,而四氢姜黄素与姜黄素联用后,细胞凋亡比率较姜黄素组有了极显著的增加。与对照组相比,姜黄素可上调Fas表达,且明显减少原型胱天蛋白酶3、胱天蛋白酶8和PARP的蛋白表达,相反,剪切形式胱天蛋白酶3、胱天蛋白酶8和PARP明显增加,同时下调抗凋亡蛋白cFLIP的表达,但四氢姜黄素对于以上蛋白作用并不明显,而四氢姜黄素与姜黄素共同处理后可以明显增加姜黄素对于以上凋亡相关蛋白表达水平的改变。结论四氢姜黄素能够在体外增强姜黄素诱导的乳腺癌细胞凋亡,提示姜黄素及其体内代谢产物共同发挥抗乳腺癌作用,且其代谢产物可能具有增效作用。     

西黄丸抑制乳腺癌MDA-MB-231细胞迁移的分子机制研究

目的探讨西黄丸水提液抑制乳腺癌MDA-MB-231细胞迁移的分子机制.方法通过细胞划痕实验分析不同浓度(5、7.5、15 g/L)西黄丸水提液对乳腺癌MDA-MB-231细胞迁移能力的影响.利用表皮生长因子(EGF)单独或联合加入西黄丸水提液,作用于乳腺癌MDA-MB-231细胞,通过蛋白质印迹法检测纤维连接蛋白(FN...     

肿瘤转移靶向肽修饰的阿霉素脂质体对高转移性乳腺癌细胞的靶向特异性研究

肿瘤转移日渐成为肿瘤治疗的重要靶标。本研究采用肿瘤转移靶向肽（TMT）与脂质材料（PEG-DSPE）偶联获得靶向化合物（TMT-PEG-DSPE）,用以构建靶向阿霉素脂质体（TMT-LS-DOX）。结果表明,TMT-LS-DOX呈现出良好的药剂学性质。选用高转移性乳腺癌细胞（MDA-MB-435S和MDA-MB-231）对该转移特异性递送系统进行评价,采用非转移性乳腺癌细胞（MCF-7）作为对照。游离TMT多肽浓度达100μg/mL时仍未显示出细胞毒性。与MCF-7相比,MDA-MB-435S及MDA-MB-231细胞对TMT-LS-DOX摄取增加,并经受体竞争性实验证明该促进作用由TMT介导。因此,TMT修饰的纳米载体可能成为增加化疗药物对高转移性乳腺癌特异性的一种新策略。     

HSPG和无HS-GAGs链HSPG体外抗乳腺癌的研究

目的：比较硫酸乙酰肝素蛋白聚糖（简称HSPG）和去硫酸乙酰肝素链（简称HS-GAGs链即HS链）的HSPG的体外抗人乳腺癌细胞MDA-MB-231的抗增殖和凋亡诱导作用。方法：对HSPG进行提取、纯化,硫酸乙酰肝素酶1水解HSPG的HS链;光学显微镜法观察两组分引起的细胞形态学的变化;MTT比色法检测两组分对肿瘤细胞生长曲线的影响;流式细胞术结合Annexin V-FITC及PI双染标记法观察两组分对肿瘤细胞凋亡的影响。结果：HSPG可致MDA-MB-231细胞脱落悬浮;MTT法检测结果显示HSPG对MDA-MB-231细胞增殖的抑制作用随浓度的增加而增强,而去HS链HSPG只有在高浓度时才呈现出抑制作用。HSPG能诱导MDA-MB-231的早期凋亡,而去HS链HSPG在高浓度（4 mg/mL）时对MDA-MB-231早期凋亡有诱导作用。结论：HSPG能诱导MDA-MB-231的早期凋亡和抑制细胞生长增殖。作用机制可能与HSPG的硫酸乙酰肝素链有关。     

hTERT反义寡核苷酸对肝癌SMMC-7721细胞株细胞增殖和端粒酶活性的影响

目的研究hTERT基因反义寡核苷酸（ASPSODN）对SMMC-7721细胞端粒酶活性及细胞增殖的影响。方法不同浓度的ASPSODN转染到SMMC-7721细胞株24h和48h后分别采用MTT法、TRAP-ELISA法检测SMMC-7721细胞的增殖活性及端粒酶活性。结果各浓度组的ASPSODN均能降低端粒酶活性,作用24h时,不同浓度均明显受抑,其中0.8μmol/L ASPSODN较0.2μmol/L和0.4μmol/L组抑制作用更明显（P〈0.05）,但再增大浓度抑制作用并不随着增加。作用48h后,抑制作用减弱,低浓度组（0.2、0.4μmol/L）端粒酶活性恢复到正常水平,而高浓度组（0.8、1.0、1.2μmol/L）抑制作用依然明显,端粒酶活性低于60%。MTT法检测显示,不同浓度ASPSODN组对SMMC-7721细胞的生长均有抑制作用,随着浓度的增加,抑制作用明显,但随着时间的延长,抑制作用增加不明显。结论 ASPSODN靶向hTERT能特异性抑制SMMC-7721细胞增殖,明显下调端粒酶活性,hTERT可能成为肝癌治疗的一个靶点。     

Inhibition of MMP-3 activity and invasion of the MDA-MB-231 human invasive breast carcinoma cell line by bioflavonoids

Aim:

Stromelysin 1 (matrix metalloproteinase 3; MMP-3) is an enzyme known to be involved in tumor invasion and metastasis. In this study, flavonoids from vegetables and fruits, such as quercetin, kaempferol, genistein, genistin, and daidzein, were tested for their ability to modulate the secretion and activity of MMP-3 in the MDA-MB-231 breast cancer cell line. In addition, we investigated the in vitro effects of flavonoids on MDA-MB-231 cell invasion.

Methods:

The toxic concentration range of flavonoids was evaluated using the MTT assay. The ability of MDA-MB-231 cells to invade was evaluated using a modified Boyden chamber system. The activity of MMP-3 was determined by casein zymography. The secretion of MMP-3 was evaluated using Western blotting, casein zymography and confirmed by ELISA.

Results:

Some putative flavonoids, ie, quercetin and kaempferol (flavonols), significantly inhibited the in vitro invasion of MDA-MB-231 cells in a concentration-dependent manner, with IC₅₀ values of 27 and 30 μmol/L, respectively. Quercetin and kaempferol also reduced MMP-3 activity in a dose-dependent manner, with IC₅₀ values in the range of 30 μmol/L and 45 μmol/L, respectively. None of the flavonoids had a significant effect on the secretion of MMP-3.

Conclusion:

These data show that the flavonols quercetin and kaempferol have higher anti-invasion potency and higher MMP-3 inhibitory activity than isoflavones genistein, genistin and daidzein. In contrast, neither flavonols nor isoflavones have any effect on MMP-3 secretion.     

HPMA copolymer-aminoglutethimide conjugates inhibit aromatase in MCF-7 cell lines

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) has already shown clinical activity in breast cancer patients. Moreover, we have recently found that an HPMA conjugate containing a combination of both Dox and the aromatase inhibitor aminoglutethimide (AGM) shows significantly increased anti-tumour activity in vitro. To better understand the mechanism of action of HPMA copolymer-AGM conjugates several models were used here to investigate their effect on cell growth and aromatase inhibition. Cytotoxicity of HPMA copolymer conjugates containing AGM, Dox and also the combination AGM-Dox was determined by MTT assay in MCF-7 and MCF-7ca cells. Androstenedione (5 x 10(- 8) M) stimulates the growth of MCF-7ca cells. Both free AGM and polymer-bound AGM (0.2-0.4 mg/ml) were shown to block this mitogenic activity. When MCF-7ca cells were incubated [(3)H]androstenedione both AGM and HPMA copolymer-GFLG-AGM (0.2 mg/ml AGM-equiv.) showed the ability to inhibit aromatase. Although, free AGM was able to inhibit isolated human placental microsomal aromatase in a concentration dependent manner, polymer-bound AGM was not, suggesting that drug release is essential for activity of the conjugate. HPMA copolymer conjugates containing aromatase inhibitors have potential for the treatment of hormone-dependant cancers, and it would be particularly interesting to explore further as potential therapies in post-menopausal women as components of combination therapy.     

Frondoside A inhibits human breast cancer cell survival, migration, invasion and the growth of breast tumor xenografts

Breast cancer is a major challenge for pharmacologists to develop new drugs to improve the survival of cancer patients. Frondoside A is a triterpenoid glycoside isolated from the sea cucumber, Cucumaria frondosa. It has been demonstrated that Frondoside A inhibited the growth of pancreatic cancer cells in vitro and in vivo. We investigated the impact of Frondoside A on human breast cancer cell survival, migration and invasion in vitro, and on tumor growth in nude mice, using the human estrogen receptor-negative breast cancer cell line MDA-MB-231. The non-tumorigenic MCF10-A cell line derived from normal human mammary epithelium was used as control. Frondoside A (0.01-5 μM) decreased the viability of breast cancer cells in a concentration- and time-dependent manner, with 50%-effective concentration (EC50) of 2.5 μM at 24h. MCF10-A cells were more resistant to the cytotoxic effect of Frondoside A (EC50 superior to 5 μM at 24 h). In the MDA-MB-231 cells, Frondoside A effectively increased the sub-G1 (apoptotic) cell fraction through the activation of p53, and subsequently the caspases 9 and 3/7 cell death pathways. In addition, Frondoside A induced a concentration-dependent inhibition of MDA-MB-231 cell migration and invasion. In vivo, Frondoside A (100 μg/kg/dayi.p. for 24 days) strongly decreased the growth of MDA-MB-231 tumor xenografts in athymic mice, without manifest toxic side-effects. Moreover, we found that Frondoside A could enhance the killing of breast cancer cells induced by the chemotherapeutic agent paclitaxel. These findings identify Frondoside A as a promising novel therapeutic agent for breast cancer.     

Berberine inhibits growth of the breast cancer cell lines MCF-7 and MDA-MB-231

The effects of berberine on the behavior of breast tumors have not yet been established. To determine whether this compound is useful in the treatment of breast cancer, we analyzed the impact of berberine on the human breast cancer cell lines MCF-7 and MDA-MB-231 cells. Berberine was added to proliferating MCF-7 and MDA-MB-231 cells in culture. Following treatment, changes in cell growth characteristics such as proliferation, cell cycle duration, and the degree of apoptosis were assayed. Following berberine treatment, a time-dependent reduction in proliferation was observed in both cell lines at differing concentrations: 20 microM for MCF-7 and 10 microM for MDA-MB-231 cells. Annexin V staining showed an increase in apoptosis in both cell lines of 31 % in MCF-7 and 12 % in MDA-MB-231 cells compared to their respective controls. In addition, 12 % of the MCF-7 cells were arrested at G0/G1, compared to 62 % of control cells. These results demonstrate that treatment with berberine inhibits growth in both MDA-MB-231 and MCF-7 cells. In addition, they show that this partly occurs through the induction of apoptosis in MDA-MB-231 cells, and through both cell cycle arrest and induction of apoptosis in MCF-7 cells. Thus, berberine may be a novel therapeutic drug for breast cancer.     

Plumbagin attenuates cancer cell growth and osteoclast formation in the bone microenvironment of mice

Aim： To investigate the effects of plumbagin, a naphthoquinone derived from the medicinal plant Plumbago zeylanica, on human breast cancer cell growth and the cancer cell-induced osteolysis in the bone microenvironment of mice.
Methods： Human breast cancer cell subline MDA-MB-231SA with the ability to spread and grow in the bone was tested. The cell proliferation was determined using the CCK-8 assay. Apoptosis was detected with Annexin V/PI double-labeled flow cytometry. Red fluorescent protein-labeled MDA-MB-231SArfp cells were injected into the right tibia of female BALB/c-nu/nu mice. Three days after the inoculation, the mice were injected with plumbagin （2, 4, or 6 mg/kg, ip） 5 times per week for 7 weeks. The growth of the tumor cells was monitored using an in vivo imaging system. After the mice were sacrificed, the hind limbs were removed for radiographic and histological analyses.

Results： Plumbagin （2.5–20 μmol/L） concentration-dependently inhibited the cell viability and induced apoptosis of MDA-MB-231SA cells in vitro （the IC50 value of inhibition of cell viability was 14.7 μmol/L）. Administration of plumbagin to breast cancer bearing mice delayed the tumor growth by 2–3 weeks and reduced the tumor volume by 44%–74%. The in vivo imaging study showed that plumbagin dose-dependently inhibited MDA-MB-231SArfp cell growth in bone microenvironment. Furthermore, X-ray images and micro-CT study demonstrated that plumbagin reduced bone erosion area and prevented a decrease in bone tissue volume. Histological studies showed that plumbagin dose-dependently inhibited the breast cancer cell growth, enhanced the cell apoptosis and reduced the number of TRAcP-positive osteoclasts.

Conclusion： Plumbagin inhibits the cell growth and induces apoptosis in human breast cancer cells in mice bone microenvironment, leading to significant reduction in osteolytic lesions caused by the tumor cells.     

T-2 toxin immunotoxicity on human B and T lymphoid cell lines

T-2 toxin belongs to a group of mycotoxins synthesized by Fusarium fungi that are widely encountered as natural contaminants in cereals. Human lymphoid cell lines of T (MOLT-4) or B (IM-9) lineage were used to characterize the cytotoxic effects mediated by T-2 at different concentrations (0.1 pg/ml to 1 microg/ml). After 24 h, membrane damage was observed by Trypan blue dye exclusion in IM-9 cells with a 50% cytotoxic concentration (CC50) of 0.2 ng/ml, whereas CC50 for MOLT-4 cells was 0.6 microg/ml (gmicro). At a T-2 concentration of 0.01 microg/ml, apoptosis was seen in MOLT-4 cells by Annexin V binding as early as after 4 h. T-2 toxin determined sustained (48 h) immunosuppression on both cell lines, as evaluated by BrdU and MTT assays. Cytotoxicity appeared to be due to early apoptosis in MOLT-4 cells, as indicated by increased Annexin V binding and activation of caspase-3, and to direct cell membrane damage in IM-9 cells.