Dauricine inhibits insulin-like growth factor-I-induced hypoxia inducible factor 1α protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

Aim:

To investigate the effects of dauricine (Dau) on insulin-like growth factor-I (IGF-I)-induced hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human breast cancer cells (MCF-7).

Methods:

Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-I for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell (HUVEC) tube formation assay. An in vitro invasion assay on HUVECs was performed.

Results:

Dau significantly inhibited IGF-I-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-I. Mechanistically, Dau suppressed IGF-I-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-I-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-I-induced invasion of HUVECs.

Conclusion:

Dau inhibits human breast cancer angiogenesis by suppressing HIF-1α protein accumulation and VEGF expression, which may provide a novel potential mechanism for the anticancer activities of Dau in human breast cancer.     

Elevation of cysteine consumption in tamoxifen-resistant MCF-7 cells

Tamoxifen (TAM) resistance is a main cause of therapeutic failure in breast cancers. Although methionine dependency is a phenotypic characteristic of tumor cells, the role of sulfur amino acid metabolism in chemotherapy resistance remains to be elucidated. This study compared metabolite profiles of sulfur amino acid metabolism from methionine to taurine or glutathione (GSH) between normal MCF-7 and TAM-resistant MCF-7 (TAMR-MCF-7) cells. TAMR-MCF-7 cells showed elevated levels and activities of enzymes involved in both transsulfuration from methionine to cysteine and metabolism of cysteine to GSH and taurine. Cysteine concentrations in TAMR-MCF-7 cells and medium conditioned by cell culture for 42 h were markedly decreased, while GSH, hypotaurine, and taurine concentrations in the medium were increased. These results show that TAMR-MCF-7 cells display enhanced cysteine utilization. The addition of propargylglycine, a specific cystathionine γ-lyase inhibitor, and buthionine sulfoximine, a specific γ-glutamylcysteine ligase inhibitor, to TAMR-MCF-7 cells, but not to MCF-7 cells, resulted in cytotoxicity after sulfur amino acid deprivation. These results suggest that cell viability of TAMR-MCF-7 cells is affected by inhibition of sulfur amino acid metabolism, particularly cysteine synthesis from homocysteine and GSH synthesis from cysteine. Additionally, the S-adenosylmethionine/S-adenosylhomocysteine ratio, an index of transmethylation potential, in TAMR-MCF-7 cells increased to ～3.6-fold relative to that in MCF-7 cells, a finding that may result from upregulation of methionine adenosyltransferase IIa and S-adenosylhomocysteine hydrolase. In conclusion, this study suggests that TAMR-MCF-7 cells display enhanced cysteine utilization for synthesis of GSH and taurine, and are sensitive to inhibition of cysteine metabolism.     

A positive circuit of VEGF increases Glut-1 expression by increasing HIF-1α gene expression in human retinal endothelial cells

Treatment of human retinal microvascular endothelial cells (HRMECs) with vascular endothelial growth factor 165 (VEGF₁₆₅) increased hypoxia-inducible factor 1α (HIF-1α), VEGF, and glucose transporter 1 (Glut-1) mRNA expression and Glut-1 protein localization to the membrane. In contrast, treatment of human retinal pigment epithelium cells with VEGF₁₆₅ did not induce HIF-1α, VEGF, and Glut-1 gene expression. Microvascular endothelial cells are surrounded by astrocytic end feet in the retina. Astrocyte-derived A-kinase anchor protein 12 overexpression during hypoxia downregulated VEGF secretion, and this conditioned medium reduced VEGF and Glut-1 expression in HRMECs, suggesting that communications between astrocytes and endothelial cells may be the determinants of the blood vessel network. In HRMECs, HIF-1α small interfering RNA transfection blocked the VEGF₁₆₅-mediated increase in VEGF and Glut-1 gene expression. Inhibition of protein kinase C (PKC) with inhibitor GF109203X or with a small interfering RNA targeting PKCζ attenuated the VEGF₁₆₅-induced Glut-1 protein expression and VEGF and Glut-1 mRNA expression. In addition, results of an immunoprecipitation assay imply an interaction between VEGF receptor 2 (VEGFR2) and PKCζ in HRMECs. Therefore, VEGF secretion by hypoxic astrocytes may upregulate HIF-1α gene expression, inducing VEGF and Glut-1 expression via the VEGFR2–PKCζ axis in HRMECs.     

Rhapontigenin inhibited hypoxia inducible factor 1 alpha accumulation and angiogenesis in hypoxic PC-3 prostate cancer cells

Hypoxia inducible factor 1 alpha (HIF-1α) is frequently over-expressed in the numerous types of cancer and plays an important role in angiogenesis. In the present study, the inhibitory mechanism of rhapontigenin isolated from Vitis coignetiae was investigated on HIF-1α stability and angiogenesis in human prostate cancer PC-3 cells. Rhapontigenin significantly suppressed HIF-1α accumulation at protein level but not at mRNA level in PC-3 cells under hypoxia. Also, rhapontigenin suppressed hypoxia-induced HIF-1α activation in various cancer cells, such as colorectal adenocarcinoma (SW620), breast adenocarcinoma (MCF-7), fibrosarcoma (HT-1080) and prostate carcinoma (LNCaP). Interestingly, rhapontigenin had more potency in inhibition of hypoxia-induced HIF-1α expression than that of resveratrol, a known HIF-1α inhibitor. In addition, rhapontigenin promoted hypoxia-induced HIF-1α degradation and cycloheximide (CHX) blocked protein synthesis. A prolyl hydroxylase (PHD) inhibitor dimethyloxalylglycine (DMOG) is usually utilized to examine whether prolyl hydroxylation is involved in inhibition of HIF-1α accumulation. Here, DMOG recovered HIF-1α accumulation inhibited by rhapontigenin. Immunoprecipitation assay also revealed that rhapotigenin enhanced the binding of hydroxylated HIF-1α to von Hippel-Lindau (VHL) tumor suppressor protein. Furthermore, rhapontigenin reduced vascular endothelial growth factor (VEGF) secretion in hypoxic PC-3 cells as well as suppressed tube formation in human umbilical vein endothelial cells (HUVECs) treated by the conditioned media of hypoxic PC-3 cells. However, anti-angiogenic effect of rhapontigenin in hypoxic PC-3 cells was reversed by DMOG. Taken together, these findings suggest that rhapontigenin inhibits HIF-1α accumulation and angiogenesis in PC-3 prostate cancer cells.     

乳腺癌组织中ING4、HIF-1α和HIF-2α表达及与肿瘤微血管新生的关系

目的 探讨乳腺癌组织中生长抑制因子4(ING4)、缺氧诱导因子-1α(HIF-1α)和缺氧诱导因子-2α(HIF-2α)表达及与肿瘤微血管新生的关系.方法 选取择期行手术治疗的乳腺癌病人107例,利用实时荧光定量PCR技术检测乳腺癌和癌旁组织中HIF-1α、HIF-2α、ING4和血管内皮生长因子(VEGF)基因表达,免疫组化染色检测乳腺癌和癌旁组织中微血管密度(MVD).结果 乳腺癌组织中HIF-1α mRNA、HIF-2α mRNA相对表达量均高于癌旁组织,而ING4 mRNA 相对表达量低于癌旁组织,差异有统计学意义(P<0.05);HIF-1α mRNA相对表达量与淋巴结转移有关(P<0.05),HIF-2α mRNA相对表达量与肿瘤大小、淋巴结转移有关(P<0.05),ING4 mRNA相对表达量与临床分期、淋巴结转移有关(P<0.05);乳腺癌组织中VEGF mRNA相对表达量和MVD计数均高于癌旁组织,均差异有统计学意义(P<0.05);Pearson相关分析显示,乳腺癌组织中HIF-1α mRNA、HIF-2α mRNA相对表达量均与VEGF mRNA相对表达量和MVD计数呈正相关(r=0.382、0.417和0.408、0.491,P<0.05),ING4 mRNA相对表达量均与VEGF mRNA相对表达量和MVD计数呈负相关(r=-0.395、-0.502,P<0.05).结论 乳腺癌组织中HIF-1α、HIF-2α基因表达升高,而ING4基因则被抑制,可能共同参与了乳腺癌组织中血管新生.     

一种AP-2α选择性剪切的发现及其在MCF-7细胞中的功能研究

目的研究AP-2α的一种选择性剪切及其对MCF-7细胞生物学功能的影响。方法构建AP-2α基因表达质粒,得到pAP-2α和一个C-端编码产物完全不同于AP-2α的选择性剪切pAP-2αas表达质粒。通过荧光素酶法检测pAP-2αas和pAP-2α对含存活素(survivin)启动子核心序列的荧光素酶报告质粒pLuc-441活性影响的差异;并通过药物敏感性实验观察pAP-2αas和pAP-2α对乳腺癌细胞MCF-7药物敏感性影响的差异。结果采用荧光素酶法检测共转染pAP-2αas和pLuc-441的MCF-7细胞中survivin启动子活性,与对照pAP-2α相比,pAP-2αas也能抑制pLuc-441活性,但抑制的强度低于前者;药物敏感性实验显示,在MCF-7细胞中,转染pAP-2αas显著提高了药物敏感性,与pAP-2α产生的生物学功能近似。结论 AP-2αas作为一种体内的特异性剪切形式,具有和通常AP-2α相似但不完全相同的功能,推测这是基因表达调控的特殊方式,AP-2α对基因表达的调节在某些情况下可以不通过直接结合启动子发挥作用。     

链霉素及H7对机械牵张离体心肌HIF-1α、VEGF的影响及机制探讨

目的研究链霉素及H7对机械牵张大鼠心肌组织低氧诱导分子-1α和血管内皮细胞生长因子表达的影响,并探讨二者在其中的作用机制。方法采用大鼠离体灌流心脏模型,膨胀左心室30min,RT-PCR法检测左室心肌细胞HIF-1α、VEGF mRNA的表达,免疫组化观察二者在心肌细胞中定位,Western blot检测HIF-1α蛋白的表达,利用链霉素作为牵张敏感离子通道(SACs)阻断剂研究SACs和PKC抑制剂H7在其中的可能作用。结果与不牵张组HIF-1α和VEGF mRNA无表达的比较,牵张可以明显增加HIF-1α和VEGF mRNA的表达(P<0·05或P<0·01);而链霉素、H7可以明显减少HIF-1α和VEGF mRNA的表达(P<0·05);但是二者不能完全抑制急性牵张刺激激活的HIF-1α和VEGF mRNA水平升高(P<0·05),HIF-1α和VEGF在胞质和胞核中均有表达,并检测到HIF-1α蛋白表达。结论链霉素、H7对膨胀左室致HIF-1α、VEGF表达有明显抑制作用,提示心室膨胀经SACs-PKC-激活胞内信号诱导HIF-1α、VEGF表达。同时链霉素并不能完全抑制HIF-1α、VEGF表达,提示膨胀心室致HIF-1α、VEGF表达进而引起心室肥厚尚有其他传导途径,仍需进一步研究。     

Andrographolide down-regulates hypoxia-inducible factor-1α in human non-small cell lung cancer A549 cells

Andrographolide (Andro), a diterpenoid lactone isolated from a traditional herbal medicine Andrographis paniculata, is known to possess multiple pharmacological activities. In our previous study, Andro had been shown to inhibit non-small cell lung cancer (NSCLC) A549 cell migration and invasion via down-regulation of phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway. Here we demonstrated that Andro inhibited the expression of hypoxia-inducible factor-1α (HIF-1α) in A549 cells. HIF-1α plays an important role in tumor growth, angiogenesis and lymph node metastasis of NSCLC. The Andro-induced decrease of cellular protein level of HIF-1α was correlated with a rapid ubiquitin-dependent degradation of HIF-1α, and was accompanied by increased expressions of hydroxyl-HIF-1α and prolyl hydroxylase (PHD2), and a later decrease of vascular endothelial growth factor (VEGF) upon the treatment of Andro. The Andro-inhibited VEGF expression appeared to be a consequence of HIF-1α inactivation, because its DNA binding activity was suppressed by Andro. Molecular data showed that all these effects of Andro might be mediated via TGFβ1/PHD2/HIF-1α pathway, as demonstrated by the transfection of TGFβ1 overexpression vector and PHD2 siRNA, and the usage of a pharmacological MG132 inhibitor. Furthermore, we elucidated the involvement of Andro in HIF-1α transduced VEGF expression in A549 cells and other NSCLC cell lines. In conclusion, these results highlighted the potential effects of Andro, which may be developed as a chemotherapeutic or an anti-angiogenesis agent for NSCLC in the future.     

槲皮素、补骨脂素对乳腺癌细胞株MCF-7增殖的影响

目的探讨槲皮素(quercetin,Que)和补骨脂素(psor-alen,Pso)对人类乳腺癌细胞株增殖的影响。方法采用流式细胞术和蛋白印迹法检测槲皮素和补骨脂素对雌激素依赖性乳腺癌细胞MCF-7的影响,并以雌激素受体拮抗剂ICI182,780为工具药来评价槲皮素和补骨脂素发挥雌激素样作用与雌激素受体的关系。结果①槲皮素、补骨脂素在10μmol.L-1可使MCF-7细胞增殖指数明显升高,增加S期细胞的比例,与10-3μmon.L-1E2阳性对照组的变化趋势一致。当ICI182,780分别与E2、金雀异黄素、槲皮素、补骨脂素共孵育48 h后,E2、金雀异黄素、槲皮素、补骨脂素的增殖效应被抑制,细胞周期S期细胞数比例下降,G0/G1期细胞数比例上升。②10μmol.L-1槲皮素和10μmol.L-1补骨脂素均上调MCF-7细胞ERα蛋白水平,而对ERβ蛋白表达没有影响;当分别与ICI182,780共孵育MCF-7细胞ERα蛋白表达被拮抗。结论槲皮素和补骨脂素具有雌激素活性,此作用是通过雌激素受体(ER)介导的;产生的类似金雀异黄素促进MCF-7细胞增殖的作用是通过增加ERα表达实现的。     

Suppression of ERK1/2 and hypoxia pathways by four Phyllanthus species inhibits metastasis of human breast cancer cells

Chemotherapies remain far from ideal due to drug resistance; therefore, novel chemotherapeutic agents with higher effectiveness are crucial. The extracts of four Phyllanthus species, namely Phyllanthus niruri, Phyllanthus urinaria, Phyllanthus watsonii, and Phyllanthus amarus, were shown to induce apoptosis and inhibit metastasis of breast carcinoma cells (MCF-7). The main objective of this study was to determine the pathways utilized by these four Phyllanthus species to exert anti-metastatic activities. A cancer 10-pathway reporter was used to investigate the pathways affected by the four Phyllanthus species. Results indicated that these Phyllanthus species suppressed breast carcinoma metastasis and proliferation by suppressing matrix metalloprotein 2 and 9 expression via inhibition of the extracellular signal-related kinase (ERK) pathway. Additionally, inhibition of hypoxia-inducible factor 1-α in the hypoxia pathway caused reduced vascular endothelial growth factor and inducible nitric oxide synthase expression, resulting in anti-angiogenic effects and eventually anti-metastasis. Two-dimensional gel electrophoresis identified numerous proteins suppressed by these Phyllanthus species, including invasion proteins, anti-apoptotic protein, protein-synthesis proteins, angiogenic and mobility proteins, and various glycolytic enzymes. Our results indicated that ERK and hypoxia pathways are the most likely targets of the four Phyllanthus species for the inhibition of MCF-7 metastasis.     

心肌转染缺氧诱导因子-1α对大鼠心肌梗死的影响

目的评价心肌内注射缺氧诱导因子(HIF)-1α进行基因转染对大鼠心肌梗死的影响,包括HIF-1α、血管内皮生长因子(VEGF)的基因表达、血中VEGF水平、心肌毛细血管密度以及心肌梗死面积的变化。方法选择体质量在250~350 g的雄性大鼠72只进行实验,分为3组,第1组为转染腺病毒HIF-1α的Ad-HIF-1α组;第2组为转染空质粒的Ad-null组(转染空质粒组);第3组为假手术组。每组以术后的不同时间处死取材分3,7,14 d 3个亚组,每个亚组8只。建立心肌梗死模型。当左前降支被结扎后,在室游离壁的3个不同部位分别注射50μg Ad-HIF-1α和50μg的Ad-dull,然后关胸缝合。假手术组不结扎左前降支。分别在转染3、7及14d后观察HIF-1α和VEGF的基因表达、血中VEGF水平、心肌毛细血管密度以及心肌梗死面积的变化。结果转染腺病毒HIF-1α基因的心肌组织中第3天即可测出478 bp的HIF-1α及291 bp VEGF部分转录产物,第7、14天检测仍有表达,而Ad-dull组的心肌组织有表达,但相对于转染腺病毒HIF-1α基因的心肌组织表达明显较弱;血中VEGF水平在Ad-HIF-1α组比Ad-null组在37、d升高更显著(P<0.01);心肌转染组的毛细血管密度与未转染组相比明显增加(P<0.01);转染14 d后的心肌梗死面积在转染组为(23.8±2.2)%,明显小于对照组的(38.7±3.1)%(P<0.01)。结论心肌内注射HIF-1α进行基因转染可以增加HIF-1α和VEGF的基因表达,血中VEGF水平升高,心肌毛细血管密度明显增加,心肌梗死面积明显减少。