Anticancer Activities of an Anthocyanin-Rich Extract From Black Rice Against Breast Cancer Cells In Vitro and In Vivo

Anthocyanins widely present in human diet and have a variety of health effects. This study investigates the anticancer effects of an anthocyanin-rich extract from black rice (AEBR) on breast cancer cells in vitro and in vivo. AEBR reduced the viability of breast cancer cell lines MCF-7 (ER⁺, HER2/neu^?), MDA-MB-231 (ER^?, HER2/neu^?), and MDA-MB-453 (ER^?, HER2/neu⁺) and induced apoptosis in MDA-MB-453 cells via the intrinsic pathway in vitro by activating caspase cascade, cleaving poly (ADP-ribose) polymerase (PARP), depolarizing mitochondrial membrane potential, and releasing cytochrome C. Oral administration of AEBR (100 mg/kg/day) to BALB/c nude mice bearing MDA-MB-453 cell xenografts significantly suppressed tumor growth and angiogenesis by suppressing the expression of angiogenesis factors MMP-9, MMP-2, and uPA in tumor tissue. Altogether, this study suggests the anticancer effects of AEBR against human breast cancer cells in vitro and in vivo by inducing apoptosis and suppressing angiogenesis.     

染料木黄酮影响MDA-MB-453细胞对紫杉醇化疗敏感性的机制

目的:探讨染料木黄酮(genistein,GEN)影响紫杉醇(paclitaxel,PTX)体外化疗HER2/neu高表达人乳腺癌细胞株MDA-MB-453敏感性的作用机制。方法:GEN和PTX单独或联合处理MDA-MB–453细胞,流式细胞仪检测细胞周期分布,免疫细胞化学法检测HER2/neu蛋白、Westernblot检测Akt、p-Akt、CyclinB1、CDK1蛋白表达的变化。结果:GEN和PTX单独作用时MDA-MB-453细胞分别阻滞于G1/S期和G2/M期,HER2/neu、总Akt和CDK1蛋白水平均没有明显变化,但GEN可显著降低p-Akt和CyclinB1蛋白水平,而PTX则明显增加CyclinB1蛋白水平,二者联合处理时GEN拮抗PTX增加CyclinB1蛋白水平和诱导G2/M期阻滞的作用。结论:GEN拮抗PTX增加cyclin B1蛋白水平和G2/M期阻滞的作用,可能是其降低PTX体外化疗MDA-MB-453细胞敏感性的机制之一。     

黑米花色苷抑制人乳腺癌细胞促血管生成因子表达的机制

目的探讨黑米花色苷抑制HER-2/neu高表达人乳腺癌细胞株MDA-MB-453促血管生成因子表达的分子机制。方法以黑米花色苷和表皮细胞生长因子(EGF)单独或联合处理MDA-MB-453细胞株,Western blot检测HER-2/neu及MAPK信号通路关键蛋白ERK-1/-2的磷酸化水平改变,免疫荧光检测NF-κB p65的细胞内定位,RT-PCR检测肿瘤细胞促血管生成因子(包括VEGF、MMP-2/-9和uPA)在mRNA水平的表达变化。结果黑米花色苷能够显著抑制HER-2/neu和ERK-1/-2蛋白的磷酸化,阻止NF-Κb p65向细胞核内转位,并在mRNA水平抑制各促血管生成因子的表达。结论黑米花色苷可能通过对HER-2/neu及其下游EGFR/Ras/MAPK信号通路的阻断最终实现对肿瘤细胞促血管生成因子表达的抑制作用。     

染料木黄酮对MDA-MB-453乳腺癌细胞uPA表达及PTK活性的影响研究

目的: 观察染料木黄酮(亦称三羟异黄酮,genistein,Gen)对MDA-MB-453乳腺癌细胞尿激酶型纤维蛋白溶酶原激活剂(urokinase-type plasminogen activator,uPA)表达及蛋白酪氨酸激酶(protein tyrosine kinase,PTK)活性的影响,探讨Gen抗HER-2/neu高表达乳腺癌血管生成的分子机制。方法: 5×10-5mol/L Gen处理MDA-MB-453细胞24、48、72 h后,应用Western blot、免疫沉淀、RT-PCR及激酶活性分析法检测Gen对MDA-MB-453乳腺癌细胞uPA表达、HER-2/neu受体蛋白磷酸化水平及PTK活性变化。结果: Gen处理MDA-MB-453细胞后,uPA的mRNA和蛋白表达量下调,HER-2/neu受体蛋白磷酸化水平降低,PTK活性下降,且这种作用具有时效性。结论: Gen能有效抑制乳腺癌细胞HER-2/neu受体的PTK活性和蛋白磷酸化水平,在转录和翻译水平下调uPA的表达,从而抑制HER-2/neu高表达乳腺癌血管生成。     

Wasabi-Derived 6-(Methylsulfinyl)Hexyl Isothiocyanate Induces Apoptosis in Human Breast Cancer by Possible Involvement of the NF-κB Pathways

6-(methylsulfinyl)hexyl isothiocyanate (6-MSITC) is a bioactive ingredient of wasabi (Wasabia japonica), which is a popular spice in Japan. 6-MSITC has been reported to inhibit the proliferation of breast cancer and melanoma cell lines. We inoculated 30 female Balb-nu/nu mice with MDA-MB-231 or -453 cells, and orally administered varying concentrations of 6-MSITC for 12 days following tumor growth. The tumor volumes and tumor weights from mice inoculated with MDA-MB-231 cells, and the tumor volumes of MDA-MB-453 cells were significantly inhibited by 6-MSITC on Days 9 and 11 after drug administration. DNA fragmentation, DNA ladder, and caspase 3/7 activity performed in vitro revealed that 6-MSITC induced apoptosis of MDA-MB-231, MDA-MB-453, and MCF-7 cells. Furthermore, nuclear factor-κB (NF-κB) expression in the nuclei and phosphorylation of inhibitor κBα (IκBα) was downregulated by 6-MSITC in a concentration-dependent manner; however, this activity was not observed in MCF-7 cells. Moreover, this downregulation of phosphorylated IκBα by 6-MSITC in MDA-MB-231 and -453 cells supports its inhibitory effects on NF-κB activity. The expression of phosphorylated AKT (pAKT) reduced by 6-MSITC was confirmed in MDA-MB-231 cells. Thus, we conclude that 6-MITC promotes apoptosis of breast cancer cells by inhibiting NF-kB and therefore releasing its control of the PI3K/AKT pathway.     

赫赛汀及9-顺式视黄酸对乳腺癌联合抑制作用

目的 探讨赫赛汀联合9-顺式视黄酸对ErbB2阳性乳腺癌细胞周期进程及分布的影响及其相应的分子机制.方法 利用流式细胞仪检测经赫赛汀、9-顺式视黄酸单独和联合作用后,癌基因(ErbB2/neu)阳性的MDA-MB-453乳腺癌细胞周期进程和分布的变化.在此基础上,利用免疫印记和半定量PCR法对CDK2、Cyclin E、P27的表达进行检测,同时利用免疫沉淀法检测CyclinE/CDK2结合力的变化.结果 与对照组比较,一定剂量的赫赛汀和9-顺式视黄酸可将MDA-MB-453细胞的周期进程阻遏于G0/G1期从而抑制其增殖活性.经2者联合作用后,cyclin E、CDK2 的表达较对照组均有所下降,其中以CDK2的变化最为明显,而P27蛋白表达则明显升高.同时,2者联合作用还能有效降低CyclinE/CDK2的结合能力.结论 赫赛汀和9-顺式视黄酸可在体外分别通过改变Cyclin E、CDK2和P27的表达和活性起到协同抑制HrbB2/neu阳性乳腺癌的目的.     

α-Linolenic Acid Reduces Growth of Both Triple Negative and Luminal Breast Cancer Cells in High and Low Estrogen Environments

Flaxseed, rich in α-linolenic acid (ALA), is a complementary breast cancer (BC) therapy; however ALA effectiveness and mechanism are unclear. Variation in cellular expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and estrogen (E2) levels may alter ALA effectiveness. This research determined the effect of ALA on growth, apoptosis, and phospholipid fatty acids of 4 BC cell lines with varying receptor expression ± E2. MCF-7 (ER+/PR+/HER2-), BT-474 (ER+/PR+/HER2+), MDA-MB-231 (ER-/PR-/HER2-) and MDA-MB-468 (ER-/PR-/HER2-) cells were incubated with ALA (50–200 μM) ± 1 nM E2 for 48–72 h. ALA dose-dependently reduced growth, measured by trypan blue exclusion, of all cells (55–80% with 75 μM), and this effect was not altered by E2. ALA (75 μM)+E2 induced apoptosis, measured by flow cytometry (up to 111.2%). Decreased growth and increased apoptosis is related to increased cell phospholipid % ALA (up to 25.1%), measured by gas chromatography. ALA is shown for the first time to reduce cell growth and induce apoptosis regardless of receptor expression and E2 environment, by incorporating into BC phospholipids, supporting the use of ALA and ALA-rich foods as a safe, inexpensive complementary therapy for a wide range of BC.     

他莫昔芬诱导体内外雌激素受体阳性乳腺癌细胞凋亡的周期特异性研究

目的研究他莫昔芬(TAM)诱导不同生长状态下雌激素受体阳性(ER( ))乳腺癌细胞凋亡的周期特异性并比较其差异。方法体外培养的ER( )乳腺癌细胞株MCF-7细胞株和原代培养的。ER( )乳腺癌细胞用TAM作用后，在流式细胞仪(FCM)上用亚G1峰(sub—G1)法检测细胞凋亡率，用细胞周期特异性细胞凋亡检测法(API)检测细胞凋亡的周期特异性。结果TAM诱导不同生长状态的ER( )乳腺癌细胞凋亡均为G0／G1期发生的周期特异性细胞凋亡，但TAM在体外诱导的乳腺癌细胞凋亡率高于在体乳腺癌细胞。结论在ER( )乳腺癌细胞中TAM在G0／G1期特异性诱导细胞凋亡，但在不同的生长状态下，TAM诱导细胞凋亡的效率不同。     

赫赛汀和9-顺式视黄酸联合用药对裸鼠转染HER2基因乳腺癌效应的研究

目的探讨赫赛汀(Herceptin,HER)和9-顺式视黄酸(9-cis-RA)单独和联合用药对HER2阳性乳腺癌细胞裸鼠移植瘤的协同抑制效应及其相应机制。方法将处于对数生长期的MCF-7HER2细胞皮下注射到Balb/c裸鼠体内,4w后,HER和9-cis-RA分别单独和联合处理裸鼠移植瘤模型3w,测量各组移植瘤的体积大小并计算出相应的q值。同时观察移植瘤的组织结构,并利用免疫组化法检测移植瘤细胞Ki-67蛋白的表达变化,以检测移植瘤细胞的增殖活性。结果HER和9-cis-RA联合作用组较对照和单独作用组能显著减小HER2阳性乳腺癌细胞裸鼠移植瘤的体积并有效改变其组织病理结构,同时还能在体内显著抑制HER2阳性乳腺癌细胞的增殖活性。结论联合应用HER和9-cis-RA可在体内有效协同发挥抑制HER2阳性乳腺癌细胞的作用。     

染料木黄酮对MDA-MB-453乳腺癌细胞VEGF表达的影响

目的:观察染料木黄酮(genistein,Gen)对MDA-MB-453乳腺癌细胞血管内皮生长因子(vascularendothelialgrowthfactor,VEGF)表达的影响,探讨Gen抗HER-2/neu高表达乳腺癌血管生成的分子机制。方法:5×10-5mol/LGen处理MDA-MB-453细胞24、48、72h后,应用免疫组织化学、Westernblot及RT-PCR法检测MDA-MB-453乳腺癌细胞VEGF的表达变化。结果:Gen处理MDA-MB-453细胞24、48、72h后,VEGF的mRNA和蛋白表达量随着处理时间的延长逐渐下降。结论:Gen能在转录和翻译水平下调HER-2/neu高表达乳腺癌细胞VEGF的表达,这可能是Gen抑制HER-2/neu高表达乳腺癌血管生成的机制之一。     

Citrus Limonin Lacks the Antichemotherapeutic Effect in Human Models of Breast Cancer

Background/Aims: Chemicals that interfere with reactive oxygen species metabolism can act as potential candidates for the treatment of cancer. Some of the glucosides of citrus limonin inhibit the endogenously generated reactive oxygen species. The aim is to study the interactions of limonin with chemotherapy. Methods: Breast cancer cell lines MCF-7 (p53 wild type) and MDA-MB-231 (p53 mutant) as well as the nontumorigenic epithelial cell line MCF-10 were used to screen the effect of limonin at 1-, 5- and 10-μM concentrations with camptothecin for apoptosis and NF?B, p38 and ERK-MAPK signaling kinase assays. The effect of cyclophosphamide and limonin on MDA MB 231 xenografts was also studied. Results: Our results indicate that limonin did not inhibit camptothecin-induced apoptosis in human breast cancer cells in vitro through noninterference of camptothecin-induced phosphorylation of p38 MAPK and ERK-MAPK. Using an in vivo model of human breast cancer, limonin in combination with cyclophosphamide was not found to inhibit the cyclophosphamide-induced tumor regression through a reduced mitotic index of tumor xenograft cells when compared to treatment with cyclophosphamide alone. Conclusion: Both in vitro and in vivo results suggest that limonin could be beneficial for breast cancer patients undergoing chemotherapy.     

Soy Isoflavone Genistein-Mediated Downregulation of miR-155 Contributes to the Anticancer Effects of Genistein

We previously reported that dietary genistein inhibits mammary tumor growth and metastasis of the highly metastatic MDA-MB-435 cancer cells in immunocompromised mice. The purpose herein was to characterize the role of the novel oncogenic microRNA (miRNA) miR-155 in the anticancer effects of genistein in metastatic breast cancer. The effect of genistein was determined on breast cancer cell viability, apoptosis, and expression of miR-155 and its targets. At low physiologically relevant concentrations, genistein inhibits cell viability and induces apoptosis in metastatic MDA-MB-435 and Hs578t breast cancer cells, without affecting the viability of nonmetastatic MCF-7 breast cancer cells. In parallel with reduced cell viability, miR-155 is downregulated, whereas proapoptotic and anticell proliferative miR-155 targets FOXO3, PTEN, casein kinase, and p27 are upregulated in MDA-MB-435 and Hs578t cells in response to genistein treatment. However, miR-155 levels remain unchanged in response to genistein in the MCF-7 cells. Ectopic expression of miR-155 in MDA-MB-435 and Hs578t cells decreases the effects of genistein on cell viability and abrogates the effects of genistein on apoptosis and expression of proapoptotic genes. Therefore, genistein-mediated downregulation of miR-155 contributes to the anticancer effects of genistein in metastatic breast cancer.     

活性维生素D_3协同他莫西芬对裸鼠乳腺癌细胞移植瘤的抑制效应

目的:探讨活性维生素D3联合他莫西芬对转染pVDRE-Tk-ERα质粒的MDA-MB-231乳腺癌细胞裸鼠移植瘤的协同抑制效应及其相应机制。方法:将重组pVDRE-Tk-ERα真核表达质粒转染到ER阴性的MDA-MB-231乳腺癌细胞内并将处于对数生长期的MDA-MB-231VDRE-ERα细胞皮下注射到Balb/c裸鼠体内,4w后,分别用0.5μg/kg的活性维生素D3和50mg/kg的他莫西芬单独及联合处理裸鼠移植瘤模型20d,测量各组裸鼠移植瘤的体积大小。利用HE染色观察移植瘤的组织结构,用免疫组化法检测移植瘤细胞Ki-67蛋白的表达变化以检测移植瘤细胞的增殖活性变化。结果:活性维生素D3和他莫西芬联合作用组较对照和单独作用组能够显著减小裸鼠移植瘤的体积并有效改变其组织病理结构,二者联合运用还能在体内显著抑制乳腺癌细胞的增殖活性。结论:活性维生素D3在体内可通过VDRE-Tk启动子诱导ERα的表达从而有效恢复雌激素阴性乳腺癌细胞对他莫西芬的敏感性。     

Protein vaccination with the HER2/neu extracellular domain plus anti-HER2/neu antibody-cytokine fusion proteins induces a protective anti-HER2/neu immune response in mice

Previously protein vaccines consisting of the extracellular domain of HER2/neu (ECD(HER2)) were shown to elicit an immune response that does not provide protection against transplantable tumors expressing HER2/neu. Here, we showed that when mice were vaccinated with a mixture of human ECD(HER2) and anti-human HER2/neu IL-12, IL-2 or GM-CSF fusion proteins, significant retardation of the growth of a syngeneic carcinoma expressing rat HER2/neu, and long-term survivors were observed. Immune sera inhibited the in vitro growth of SK-BR-3, a human breast cancer overexpressing HER2/neu. Transfer of immune sera into mice challenged with TUBO also led to partial inhibition of tumor growth. Splenocytes from mice vaccinated with ECD(HER2) plus IgG3-(GM-CSF) incubated with ECD(HER2) demonstrated significant proliferation and IFN-gamma secretion. Taken together these results suggest that vaccines including ECD(HER2) and Ab-cytokine fusion proteins may be used to elicit both humoral and cell-mediated responses against HER2/neu.     

Kaempferol induced the apoptosis via cell cycle arrest in human breast cancer MDA-MB-453 cells

The aim of present study was to investigate the effects of kaempferol on cellular proliferation and cell cycle arrest and explore the mechanism for these effects in human breast carcinoma MDA-MB-453 cells. Cells were treated with kaempferol at various concentrations (ranging from 1 to 200 µM) for 24 and 48 hrs. Kaempferol significantly inhibited cancer cell growth in cells exposed to 50 and 10 µM of kaempferol and incubated for 24 and 48 hrs, respectively. Exposure to kaempferol resulted in cell cycle arrest at the G2/M phase. Of the G2/M-phase related proteins, kaempferol down-regulated CDK1 and cyclin A and B in cells exposed to kaempferol. In addition, small DNA fragments at the sub-G0 phase were increased by up to 23.12 and 31.90% at 10 and 50 µM incubated for 24 and 48 hrs, respectively. The kaempferol-induced apoptosis was associated with the up-regulation of p53. In addition, the phosphorylation of p53 at the Ser-15 residue was observed with kaempferol. Kaempferol inhibits cell proliferation by disrupting the cell cycle, which is strongly associated with the induction of arrest at G2/M phase and may induce apoptosis via p53 phosphorylation in human breast carcinoma MDA-MB-453 cells.     

Soy Consumption and Histopathologic Markers in Breast Tissue Using Tissue Microarrays

This study examined the relation of soy intake with hormonal and proliferation markers in benign and malignant breast tissue using tissue microarrays (TMAs). TMAs with up to 4 malignant and 4 benign tissue samples for 268 breast cancer cases were constructed. Soy intake in early life and in adulthood was assessed by questionnaire. The TMAs were stained for estrogen receptor (ER) α, ERβ, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2/neu), proliferating cell nuclear antigen (PCNA), and Ki-67 using standard immunohistochemical methods. Logistic regression was applied for statistical analysis. A higher percentage of women showed positive marker expression in malignant than in benign tissue. With one exception, HER2/neu, no significant associations between soy intake and pathologic markers were observed. Early life soy intake was associated with lower HER2/neu and PCNA staining of malignant tissue. In benign tissue, early life soy intake showed higher ER and PR expression, but no difference in proliferation markers. The results of this investigation provide some assurance that soy intake does not adversely affect markers of proliferation. TMAs were shown to be a useful tool for epidemiologic research.     

Combination of epitope-optimized DNA vaccination and passive infusion of monoclonal antibody against HER2/neu leads to breast tumor regression in mice

HER2/neu is an oncogene amplified and over-expressed in 20-30% of breast adenocarcinomas. Treatment with the humanized monoclonal antibody trastuzumab has shown efficacy in combination with cytotoxic agents, although resistance occurs over time. Novel approaches are needed to further increase antibody efficacy. In this study, we provide evidence in a mouse breast cancer therapeutic tumor model that the combination of active immunization with a modified HER2/neu DNA vaccine and passive infusion of an anti-HER2/neu monoclonal antibody leads to significant regression of established tumors. Our data indicate that combination therapy with a HER2/neu DNA vaccine and trastuzumab may have clinical activity in breast cancer patients.     

Flavanols from Japanese Quince (Chaenomeles Japonica) Fruit Inhibit Human Prostate and Breast Cancer Cell Line Invasiveness and Cause Favorable Changes in Bax/Bcl-2 mRNA Ratio

Polyphenols are natural compounds of high structural diversity which translates into a very wide spectrum of biological activities, including chemoprevention. Here we report that a Japanese quince fruit flavanol preparation (JQFFP) caused favorable changes in Bax/Bcl-2 mRNA ratio, which rendered normal and cancer cells more resistant and more sensitive, respectively, to apoptosis. DU145 human prostate cancer cells were characterized by the most advantageous Bax/Bcl-2 ratio. The growth and invasiveness of MDA-MB-231 human breast cancer cells were strongly suppressed by JQFFP, which was accompanied with a decrease in MMP-9 activity and stimulation of TIMP-1 expression. Importantly, JQFFP did not decrease normal human prostate PNT1A cell number, whereas Bax/Bcl-2 ratio decreased which implies increased resistance to apoptosis. In conclusion, JQFFP exhibited a potent antiproliferative effect against cancer cells, inhibited their invasiveness, and decreased expression level of several genes involved in apoptosis, angiogenesis, and metastasis.     

α-Lipoic acid reduces matrix metalloproteinase activity in MDA-MB-231 human breast cancer cells

α-Lipoic acid (LA), a naturally occurring molecule in animal and plant cells, is a potent antioxidant that reportedly exerts beneficial effects on cell proliferation and apoptosis in various cancer cell lines. However, the molecular mechanisms behind the antimetastatic property of LA are not well understood. The present study investigates the effect of LA on metastasis in a cell system. Our hypothesis is that LA inhibits metastasis via inhibition of matrix metalloproteinase (MMP) in vitro. MDA-MB-231 cells, a human breast cancer cell line, were treated with various concentrations of LA (0, 250, 500, or 1000 μmol/L) to measure metastasis, MMP activity, and mRNA expression. The viability of cells was examined by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay. The effect of LA on metastasis was evaluated using the motility, migration, and invasion assay in vitro. The activity and mRNA expression of MMP-2 and MMP-9 were measured. After LA treatment, cell motility and cell migration were significantly decreased (P < .05). α-Lipoic acid also reduced cell invasion through a Matrigel-coated chamber (P < .05). Activities of MMP-2 and MMP-9 were decreased by LA treatment in a dose-dependent manner. RT-PCR analysis confirmed the reduction in mRNA expression level of MMP-2 and MMP-9 by LA treatment. We conclude that in this cell culture model, LA treatment inhibits cancer metastasis, and this inhibition is likely due to the decrease in the activity and mRNA expression levels of MMP-2 and MMP-9 caused by LA.     

环氧合酶-2和HER-2/neu蛋白在卵巢上皮性癌中的表达及其临床意义

目的探讨环氧合酶-2(cyclooxygenase-2,COX-2)和表皮生长因子-2(v-erb-b2erythroblastic leukemia viral oncogene homolog2,HER-2/neu)蛋白在卵巢上皮性癌中的表达及其临床意义。方法利用免疫组化链酶素抗生物蛋白—过氧化物酶连接(streptavidin-perosidase,SP)法检测12例正常卵巢组织、18例卵巢良性上皮性肿瘤、10例卵巢交界性上皮性肿瘤和40例卵巢上皮性癌组织中COX-2和HER-2/neu蛋白的表达水平,并结合临床病理特征进行分析。结果①COX-2和HER-2/neu蛋白在卵巢正常组织、良性肿瘤、交界性肿瘤和上皮性癌中的阳性表达率分别为0%、5.6%、30.0%、85.O%和8.3%、11.1%、20.0%、52.5%,二者的表达率随肿瘤恶性程度增加而增加,差异有统计学意义(P<0.05)。②COX-2和HER-2/neu蛋白在卵巢上皮性癌中的表达均与临床分期、病理分级、组织学类型无关(P>0.05),与淋巴结转移有关(P<0.05),二者在卵巢上皮性癌组织中同时表达的阳性率为52.5%,呈正相关(r=0.44,P<0.05)。结论 COX-2和HER-2/neu蛋白与卵巢上皮性癌的发生发展密切相关,二者可能协同参与了卵巢癌的发生发展。