红景天对乳腺癌抑制作用的研究

目的:通过体外和体内实验研究红景天对乳腺癌的增殖抑制作用和浸润转移相关因子表达的影响。方法:采用不同浓度红景天提取物处理乳腺癌MDA-MB-435细胞,制作细胞增殖曲线,观察药物对肿瘤细胞增殖活性的影响;应用流式细胞仪检测红景天提取物对乳腺癌细胞的细胞周期阻滞作用;选择裸鼠乳腺癌MDA-MB-435细胞移植瘤模型,以免疫组织化学染色方法研究红景天提取物对裸鼠移植瘤内MMP-2和组织蛋白酶D(CathD)表达的影响。结果:红景天提取物可显著抑制MDA-MB-435细胞的增殖活性,该抑制作用随着作用时间的延长、药物浓度的增高更为明显。红景天提取物具有阻滞MDA-MB-435细胞增殖周期的作用,主要阻滞于S期,该阻滞作用呈浓度依赖性。在红景天提取物治疗组裸鼠乳腺癌移植瘤中,CathD蛋白表达明显低于生理盐水对照组(免疫组化H-评分均数治疗组为72±50.70,而对照组为168±64.19,P=0.03);治疗组移植瘤MMP-2蛋白表达明显低于生理盐水对照组(免疫组化H-评分均数治疗组为90±29.15,而对照组为176±65.80,P=0.04)。结论:红景天可能通过抑制细胞增殖、浸润能力等多途径对乳腺癌细胞产生一定的抑制作用,其在乳腺癌的防治中具有一定的临床意义。     

Quercetin inhibition of tumor invasion via suppressing PKC delta/ERK/AP-1-dependent matrix metalloproteinase-9 activation in breast carcinoma cells

Quercetin (QUE; 3,5,7,3',4'-tetrahydroxyflavone) has been shown to possess several beneficial biological activities including antitumor, anti-inflammation and antioxidant properties; however, the effects of QUE in preventing invasion by breast carcinoma cells are still undefined. Increases in the protein, messenger RNA and enzyme activity levels of matrix metalloproteinase (MMP)-9 were observed in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated MCF-7 cells, and these were blocked by QUE, but not by quercitrin or rutin. A translocation of protein kinase C (PKC)delta from the cytosol to the membrane followed by activation of extracellular signal-regulated kinase (ERK) and c-Jun/activator protein-1 (AP-1) by TPA was demonstrated, and TPA-induced MMP-9 activation and migration were inhibited by the pan PKC inhibitor, GF109203X, the specific PKCdelta inhibitor, rottlerin, an ERK inhibitor (PD98059) and an AP-1 inhibitor (curcumin). Application of QUE significantly suppressed TPA-induced activation of the PKCdelta/ERK/AP-1-signaling cascade. To elucidate the importance of hydroxyl (OH) substitutions to QUE's inhibition of tumor migration, several structurally related flavones of QUE including 3',4'-diOH, 3',4'-diOCH(3), 3,5,7-triOH, 3,4',4'-triOH, 3,3',4'-triOCH(3), luteolin and fisetin were used. Results suggested that OH groups at both C3' and C4' play central roles in QUE's inhibition of TPA-induced MMP-9 activation and migration, and an additional OH at C3, C5 or C7 may increase the inhibitory potency of the 3',4'-diOH flavone against TPA-induced MMP-9 activity and migration. The antitumor invasion and migration effects of breast carcinoma cells induced by QUE with the structure-activity relationship analysis were identified.     

大豆异黄酮摄入与乳腺癌发生风险的Meta分析

目的探讨大豆异黄酮摄入与乳腺癌发生风险的关系。方法应用Meta分析的方法,对近10年来国内外发表的关于大豆异黄酮摄入与乳腺癌发生风险的病例对照研究资料,以乳腺癌组与对照组大豆异黄酮摄入的优势比（OR）为效应指标,经一致性检验后进行OR合并,并进行偏倚评估。结果符合人选标准的病例对照研究一共有13项,病例数6328,对照数8987。以进食大豆异黄酮的量分为常食组与偶食组,经Meta分析后,合并后OR为0．79,经常食用大豆异黄酮人群乳腺癌发生风险低于不食或偶食者（Z=2．65,p=0．008）。结论大豆异黄酮摄入是乳腺癌发生的保护性因素,常进食者乳腺癌发生风险降低。     

Death receptor-dependent and -independent pathways in anticancer drug-induced apoptosis of breast cancer cells

Apoptosis is a crucial event for anticancer drug efficacy. The signal pathways activated by anticancer drugs are classified as death receptor (DR) -dependent or -independent. The mechanisms by which anticancer drugs induce apoptosis via DR-dependent pathways are not fully understood. In the present study, we assessed differential activation of signal transduction pathways leading to apoptosis by anticancer drugs in breast cancer cell lines. Three breast cancer cell lines, MDA-MB-231, MCF-7, and MCF-7ADM, which is drug-resistant, were used. Drug sensitivity and apoptosis were assessed by MTT assay and TUNEL, respectively. Expression of apoptosis-related protein was assessed by Western blotting and RT-PCR. The sensitivities of MDA-MB-231 and MCF-7 cells to mitomycin C (MMC) and adriamycin (ADM) were similar. In contrast, sensitivity of MDA-MB-231 cells to paclitaxel (TXL) was 30-fold greater than that of MCF-7 cells, 0.03 micro M in MDA-MB-231 and 0.9 micro M in MCF-7 cells, respectively. Treatment with MMC increased expression of DR4 and Fas in a time-dependent manner in MDA-MB-231 cells. Treatment with ADM increased expression of DR4 and DR5 but not Fas, whereas treatment with TXL increased expression of Fas but not DR4 and DR5 in MDA-MB-231 cells. Although treatment of MCF-7 cells with ADM increased expression of DR4 and DR5 but not Fas, expression of DR4, DR5, and Fas by the drug-resistant cells did not change following treatment with ADM. Activation of Fas, DR4, and DR5 in drug-sensitive cells in response to anticancer drugs is dependent on the cytotoxic effect of each drug. In drug-resistant cells, apoptosis is induced via DR-independent pathways mediated by mitochondrial dysfunction.     

Triptolide inhibits matrix metalloproteinase-9 expression and invasion of breast cancer cells through the inhibition of NF-κB and AP-1 signaling pathways

Triptolide is a diterpenoid epoxide that is endogenously produced by the thunder god vine, Tripterygium wilfordii Hook F. Triptolide has demonstrated a variety of biological activities, including anticancer activities, in previous studies. Invasion and metastasis are the leading causes of mortality for patients with breast cancer, and the increased expression of matrix metalloproteinase-9 (MMP-9) has been shown to be associated with breast cancer invasion. Therefore, the aim of the present study was to investigate the effect of triptolide on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced cell invasion and MMP-9 expression in breast cancer cells. The expression of signal molecules was examined by western blotting, zymography and quantitative polymerase chain reaction; an electrophoretic mobility gel shift assay was also used, and cell invasiveness was measured by an in vitro Matrigel invasion assay. The MCF-7 human breast cancer cell line was treated with triptolide at the highest concentrations at which no marked cytotoxicity was evident. The results demonstrated that triptolide decreased the expression of MMP-9 through inhibition of the TPA-induced phosphorylation of extracellular signal-regulated kinase (ERK) and the downregulation of nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) activity. In addition, a Transwell assay revealed that triptolide reduced the ability of MCF-7 cells to invade Matrigel. These data demonstrate that the anti-invasive effect of triptolide is associated with the inhibition of ERK signaling and NF-κB and AP-1 activation, and suggest that triptolide may be a promising drug for breast cancer.     

HOXD3 enhances motility and invasiveness through the TGF-beta-dependent and -independent pathways in A549 cells

Homeobox genes regulate sets of genes that determine cellular fates in embryonic morphogenesis and maintenance of adult tissue architecture by regulating cellular motility and cell-cell interactions. Our previous studies showed that a specific member, HOXD3, when overexpressed, upregulates integrin beta3 expression in human erythroleukemia HEL cells and lung cancer A549 cells, and enhances their motility and invasiveness. We performed a microarray study of over 7075 genes to determine the mechanisms underlying the HOXD3-enhanced motility and invasiveness in A549 cells. RT-PCR-based tracking gene analyses highlighted a set of TGF-beta-upregulated genes, which included matrix metalloproteinase-2, syndecan-1, CD44, and TGF-beta-induced 68 kDa protein. Exogenous TGF-beta also caused this pattern of upregulation in A549 cells and enhanced their migratory and invasive activity, confirming the involvement of TGF-beta signaling. However, HOXD3 reduced the expression of TGF-beta-independent genes coding for desmosomal components such as desmoglein, desmoplakin and plakoglobin which are known to suppress tumor invasion and metastasis. These results suggest that HOXD3 enhances the invasive and metastatic potential of cancer cells through the TGF-beta-dependent and -independent pathways.     

Caveolin-1-dependent and -independent uPAR signaling pathways contribute to ganglioside GT1b induced early apoptosis in A549 lung cancer cells

Urokinase receptor interacts with α5β1-integrin and enhances cancer cell proliferation and metastasis. Activation of α5β1-integrin requires caveolin-1 and is regulated by uPAR, which upregulates persistently the activated ERK necessary for tumor growth. In this study, we show that the ganglioside GT1b induces proapoptotic signaling through two uPAR-ERK signaling pathways in A549 lung cancer cells. GT1b downregulated the expression of α5β1 integrin, caveolin-1, fibronectin, FAK, and ERK, whereas GT1b upregulated the expression of p53 and uPAR, suggesting GT1b mediated depletion of caveolin-1 in uPAR-expressing A549 cells also disrupts uPAR/integrin complexes, resulting in downregulation of fibronectin-α5β1-integrin-ERK signaling. Following p53 siRNA treatment, FAK and ERK expression was recovered, meaning the presence of reentry uPAR-FAK-ERK signaling pathway. These findings reveal that GT1b is involved in both caveolin-1-dependent uPAR-α5β1-integrin-ERK signaling and caveolin-1-independent uPAR-FAK-ERK signaling. These results suggest a novel function of GT1b as a dual regulator of ERK by modulating caveolin-1 and p53.     

CSF-1 activates MAPK-dependent and p53-independent pathways to induce growth arrest of hormone-dependent human breast cancer cells

The CSF-1 receptor (CSF-1R) is expressed in >50% of human breast cancers. To investigate the consequence of CSF-1R expression, hormone-dependent human breast cancer cell lines, MCF-7 and T-47D, were transfected with CSF-1R. Unexpectedly, CSF-1 substantially inhibited estradiol (E2) and insulin-dependent proliferation of MCF-7 transfectants (MCF-7fms) and prevented cyclin E/cdk2 and cyclin A/cdk2 activation, consistent with a G1 arrest. In contrast, CSF-1 increased DNA synthesis in T-47D transfectants (T-47Dfms) alone and with E2 or insulin. In response to CSF-1, there was a marked and sustained upregulation of the cyclin-dependent kinase inhibitor, p21Waf1/Cip1, in MCF-7fms but not T-47Dfms. CSF-1 also markedly upregulated cyclin D1 in MCF-7fms. The coordinate increase in cyclin D1 and p21 had the effect of decreasing the specific but not absolute activity of cyclin D1/cdk4. p53 was not involved since CSF-1 induction of p21 was unaffected by dominant-negative p53 expression. ERK activation by CSF-1 was robust and sustained in MCF-7fms and to a much lesser extent in T-47Dfms. Using pharmacological and transient transfection approaches, we showed that ERK activation was necessary and sufficient for p21 induction in MCF-7fms. Moreover, activated MEK inhibited E2-stimulated cdk2 activity. Our findings indicate that the consequence of CSF-1R-mediated signals in human breast cancer cells is dependent on the genetic background of the particular tumor.     

Identification of signal transduction pathways involved in constitutive NF-kappaB activation in breast cancer cells

Nuclear factor-kappaB (NF-kappaB) is usually maintained in an inactive form in the cytoplasm through its association with inhibitor of kappaB (IkappaB) proteins, and is activated upon stimulation of cells with a variety of signals. However, constitutive activation of NF-kappaB is observed in a number of cancers including breast cancer. The signaling pathways that are involved in constitutive NF-kappaB activation remain largely unknown. Using breast cancer cell lines derived from transgenic mice that overexpress specific oncogene/growth factors in the mammary gland, we show that heregulin but not her2/neu, c-Myc or v-Ha-ras plays a major role in constitutive NF-kappaB activation. Her2/neu potentiated tumor necrosis factor alpha (TNFalpha)-inducible NF-kappaB activation whereas c-Myc potentiated 12-o-tetracecanyolphorbol-13-acetate (TPA)-induced NF-kappaB activation. Heregulin-mediated NF-kappaB activation correlated with phosphorylation of epidermal growth factor receptor (EGFR) and ErbB3 but not her2/neu. Tryphostin AG1517, which inhibits heregulin-mediated phosphorylation of EGFR, her2/neu and ErbB3 reduced NF-kappaB activation. In contrast, emodin, which blocks phosphorylation of her2/neu by heregulin, failed to reduce NF-kappaB activation. These results suggest that heregulin induces NF-kappaB independent of her2/neu. PI3 kinase/AKT, protein kinase A (PKA) and IkappaB kinase appear to be downstream signaling molecules involved in NF-kappaB activation as specific inhibitors of these kinases but not inhibitors of ERK/MAP kinase or protein kinase C reduced heregulin-mediated NF-kappaB activation. Based on these results, we propose that heregulin increases the expression of pro-invasive, pro-metastatic and anti-apoptotic genes in cancer cells through autocrine activation of NF-kappaB, which leads to invasive and drug-resistant growth of breast cancer.     

Soy isoflavones augment the effect of TRAIL-mediated apoptotic death in prostate cancer cells

Prostate cancer represents an ideal disease for chemopreventive intervention. Genistein, daidzein and equol, the predominant soy isoflavones, have been reported to lower the risk of prostate cancer. Isoflavones exert their chemopreventive properties by affecting apoptosis signalling pathways in cancer cells. Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is an endogenous anticancer agent that induces apoptosis selectively in tumour cells. Soluble or expressed in immune cells, TRAIL molecules play an important role in immune surveillance and defense mechanisms against tumour cells. However, various types of cancer cells are resistant to TRAIL-mediated apoptosis. We examined the cytotoxic and apoptotic effects of genistein, daidzein and equol in combination with TRAIL in LNCaP cells. Cytotoxicity was measured by MTT and LDH assays. Apoptosis was analyzed by flow cytometry and fluorescence microscopy using Annexin V-FITC. Mitochondrial membrane potential (ΔΨm) was evaluated by fluorescence microscopy using DePsipher staining. Flow cytometry detected the expression of death receptor TRAIL-R1 (DR4) and TRAIL-R2 (DR5) on cell surfaces. The soy isoflavones sensitized TRAIL-resistant prostate cancer cells to apoptotic death. The isoflavones did not alter death receptor expression, but significantly augmented TRAIL-induced disruption of ΔΨm in the LNCaP cells. We showed for the first time that the chemopreventive effects of soy foods on prostate cancer are associated with isoflavone-induced support of TRAIL-mediated apoptotic death.     

Pharmacologic activation of p53-dependent and p53-independent apoptotic pathways in Hodgkin/Reed-Sternberg cells.

The status of the p53 pathway in classical Hodgkin lymphoma (cHL) remains unclear, and a lack of proven TP53 mutations contrasts with often high expression levels of p53 protein. In this study, we demonstrate that pharmacologic activation of the p53 pathway with the murine double minute 2 (MDM2) antagonist nutlin-3 in Hodgkin lymphoma-derived cell lines leads to effective apoptosis induction and sensitizes the cells to other anticancer drugs. Cells with mutant p53 are resistant to nutlin-3, but sensitive to geldanamycin, a pharmacologic inhibitor of heat shock 90 kDa protein (HSP90), indicating that HSP90 inhibition can induce apoptosis in a p53-independent manner. Conversely, cells with defects in the HSP90/nuclear factor-kappa B pathway expressing wild-type p53 are more resistant to geldanamycin, but still sensitive to nutlin-3. Our results suggest that selective activation of p53 by MDM2 antagonists as a single agent or in combination with conventional chemotherapeutics and/or inhibitors of p53-independent survival pathways may offer effective treatment options for patients with cHL. Importantly, because nutlins and HSP90 inhibitors are non-genotoxic agents, their use might offer a means to reduce the genotoxic burden of current chemotherapeutic regimens.