藻蓝蛋白亚基脂质体光动力学抗乳腺癌细胞的实验研究

目的:研究PEG修饰的藻蓝蛋白亚基光敏剂长循环脂质体介导的光动力疗法抗乳腺癌的效果.方法:采用薄膜水合-超声分散法制备PEG修饰的藻蓝蛋白亚基脂质体(PEG-PCS-lip),MTT法检测藻蓝蛋白亚基脂质体对MCF-7(人乳腺癌细胞)、MA-782(鼠乳腺癌细胞)的光动力杀伤效果,流式细胞术(FCM)分析其对肿瘤细胞周期的影响,并对乳腺癌模型鼠做PDT治疗.结果:PEG-PCS-lip介导的PDT作用对乳腺癌细胞有良好的光动力疗效,对MCF-7及MA-782细胞IC_(50)(半数抑制浓度)分别为68 μg/mL、70 μg/mL;FCM的结果显示,当PEG-PCS-lip 浓度为50 μg/mL时,MCF-7凋亡率约为30.5 %; 用10 mg/kg PEG-PCS-lip静脉注射荷瘤小鼠,光照剂量为208.2 J/cm~2时,其抑瘤率可达到72 %;组织切片观察PDT作用后的肿瘤组织,瘤体中间以细胞凋亡为主,瘤体外周以细胞坏死为主,由血管损伤而形成的空腔增多.结论:PEG-PCS-lip在体外对乳腺癌细胞有良好的光动力杀伤效果,肿瘤细胞凋亡及瘤血管破坏是导致肿瘤细胞死亡的主要原因.     

藻蓝蛋白亚基细胞渗透性及对肿瘤细胞光敏作用的研究

目的:研究藻蓝蛋白亚基对SP2/0、S180、COS7、C6的最佳渗透条件及由其介导的PDT对肿瘤细胞的抑制作用。方法:采用柱层析方法从藻蓝蛋白样品中分离得到藻蓝蛋白亚基,通过荧光显微镜观察其在细胞内的渗透特性,并以He-Ne激光器为激发光源,MTT法检测藻蓝蛋白亚基光敏作用对肿瘤细胞生长的抑制作用。结果:藻蓝蛋白α、α/β亚基在75μg/mL浓度下可以在4 h时充分地进入细胞,并可以在细胞内稳定2 h以上;藻蓝蛋白中的α,β亚基稳定性和作用不相同,β亚基荧光性强但易于降解,由α亚基介导的PDT作用比α/βPDT作用强;100μg/mLα亚基介导的PDT对SP2/0和S180两种悬浮细胞的抑制率可分别达到78.6%和39.8%,强于贴壁细胞C6和COS7的29.2%和17.8%。结论:藻蓝蛋白α亚基在合适的渗透条件下表现出较强的光动力学抗肿瘤效果,且PDT效果与光敏剂浓度、照射剂量及细胞类型相关。     

藻蓝蛋白色素肽光动力学抗肿瘤作用的实验研究

目的：探讨藻蓝蛋白色素肽的理化特性及光动力疗法（PDT）抗肿瘤效果。方法：用柱层析法从螺旋藻藻蛋白酶介产物中分离色素肽,并用MTT法及吖啶橙染色法检测色素肽对体外培养的小鼠肉瘤细胞S180及荷瘤小鼠PD。结果：分离出三种色素肽的分子量分别为17.4KD,7.72KD及6.6KD,它们的吸收光谱峰值分别位于618nm,580nm,605nm,荧光发射峰均在685nm处。MTT法检测证明,在浓度100μg/ml,用波长为580nm,600nm,照射剂量为28.8J/cm^2染料激光（氩离子为激光光源）照射,对肿瘤细胞的杀伤率达69．2％－80．2％;在色素肽CCP1,CCP3介导的PDT对小鼠移植瘤生长影响的实验发现,色素肽易被瘤细胞吸收,当激光照射剂量为120J/cm^2,肿瘤直径在0.5-0.7cm范围内,瘤体旁注射剂量为50μg的CPP1及CCP3光敏剂时,藻胆蛋白酶介产的的抑瘤率可达46％－81％,且被PDT作用后的细胞表现了典型的凋亡细胞特征。     

藻红蛋白亚基光敏剂对小鼠移植瘤作用的超微结构研究

目的：从形态学角度探讨藻红蛋白(R-PE)β亚基光动力学抗肿瘤效果及其作用机理。方法：用不同密度的波长为496nm的氩离子激光对S180小鼠移植瘤进行β亚基光动力学治疗,并对治疗后的瘤体进行透射电镜的形态学观察。结果：用100μg／m1的β亚基,在200J/cm2激光照射剂量条件下治愈了瘤体直径为0．5cm-0．7cm大小的小鼠移植瘤,发现瘤组织中引起细胞死亡的途经有差异,被PDT抑制的肿瘤内部细胞表现出典型的凋亡细胞特征。结论：R-PE β亚基具较强的光动力学抗肿瘤效果,光动力治疗机理可能涉及肿瘤内部细胞死亡主要是凋亡途径而瘤周为坏死,且与血管系统破坏及白细胞参与的抗炎症反应相关。     

柱孢鱼腥藻的藻蓝蛋白亚基和F_(678)色素蛋白

柱孢鱼腥藻的藻蓝蛋白包含有两个亚基。β亚基具有578nm吸收峰和600nm荧光发射峰,分子量19.80±0.40KD,可表明β亚基仅有一个载色团。α亚基具有578nm和630nm吸收峰及645nm的荧光发射峰,分子量17.35±0.38,α亚基可能有两个载色团。别藻蓝蛋白有635nm和650nm吸收蜂及664nm的荧光发射峰。具藻胆体的类囊体膜从高盐浓度缓冲液移至低盐浓度缓冲液时表现678nm荧光发射峰,可能柱孢鱼腥藻存在的F_(678)色素蛋白相当于GLazer(1975)的别藻蓝蛋白B。     

C-藻蓝蛋白对乳腺癌细胞的抗癌作用研究

C-藻蓝蛋白具有抗癌、抗氧化、抗炎活性等多种功能,然而其对乳腺癌细胞的抗癌作用及机制尚不明确.本研究应用不同浓度(0～500 μg/mL)的C-藻蓝蛋白处理人乳腺癌细胞系MDA-MB-468.研究显示,C-藻蓝蛋白以剂量依赖性方式抑制MDA-MB-468细胞的增殖并降低细胞的菌落形成能力.C-藻蓝蛋白通过上调了Fas和...     

藻蓝蛋白和藻红蓝蛋白β亚基半胱氨酸的定点突变及体外重组研究

为研究藻蓝蛋白 (PC)和藻红蓝蛋白 (PEC) β亚基 (分别简称为 β PC、β PEC)生物合成、结构与功能的关系 ,用MegaprimerPCR定点突变技术设计 β PC、β PEC中与藻蓝胆素连接的第二个半胱氨酸的定点突变蛋白质 β PC(C15 5I)和 β PEC(C15 5I)。将相应的基因片段亚克隆于表达载体pET 30a,并转化大肠杆菌BL2l(DE3)。经IPTG诱导后 ,β PC(C15 5I)和 β PEC(C15 5I)在大肠杆菌中均得到了高效表达。β PC(C15 5I)和 β PEC(C15 5I)与藻蓝胆素的重组结果表明两个突变体的结构基本没发生改变 ,有利于对 β PC和 β PEC的生物合成进行进一步研究。     

螺旋藻藻蓝蛋白光敏作用的研究进展

螺旋藻是一种广泛养殖的丝状体蓝藻。从螺旋藻中提取的藻蓝蛋白具有抗肿瘤和增强免疫等多种生物学功能,作为光敏剂,藻蓝蛋白可应用于肿瘤治疗。本文阐述了藻蓝蛋白的结构、光动力疗法的原理和藻蓝蛋白光敏作用的研究进展,并介绍了藻蓝蛋白在光动力疗法中的应用现状与前景。     

藻蓝蛋白与含叶绿素a脂质体之间的能量传递

制备了嗜热蓝藻优雅粘囊藻（Ｍｙｘｏｓａｒｃｉｎａ　ｃｏｎｃｉｎnａ　Ｐｒｉｎｔz）的藻蓝蛋白和含Ｃｈｌ　ａ脂质体,测定了它们的吸收光谱和低温荧光发射光谱,研究了藻蓝蛋白与含Ｃｈｌａ脂质体之间的能量传递。结果显示能量传递的效率随着脂质体膜表面电荷的改变而改变：当膜表面带负电荷时,能量传递效率降低;当膜表面带正电荷时,能量传递效率随着正电荷表面活性剂ｄｉｏｃｔａｄｅｃｙｌｄｉｍｅｔｈｙｌａｍｍｏｎｉｕｍ　ｃｈｌｏｒｉｄｅ（ＤＯＤＡＣ）的增加（从０到３０ｍｏｌ％）而升高。表明静电引力在它们的能量传递中起重要作用。     

ADIPOQ/adiponectin induces cytotoxic autophagy in breast cancer cells through STK11/LKB1-mediated activation of the AMPK-ULK1 axis

ADIPOQ/adiponectin, an adipocytokine secreted by adipocytes in the breast tumor microenvironment, negatively regulates cancer cell growth hence increased levels of ADIPOQ/adiponectin are associated with decreased breast cancer growth. However, its mechanisms of action remain largely elusive. We report that ADIPOQ/adiponectin induces a robust accumulation of autophagosomes, increases MAP1LC3B-II/LC3B-II and decreases SQSTM1/p62 in breast cancer cells. ADIPOQ/adiponectin-treated cells and xenografts exhibit increased expression of autophagy-related proteins. LysoTracker Red-staining and tandem-mCherry-GFP-LC3B assay show that fusion of autophagosomes and lysosomes is augmented upon ADIPOQ/adiponectin treatment. ADIPOQ/adiponectin significantly inhibits breast cancer growth and induces apoptosis both in vitro and in vivo, and these events are preceded by macroautophagy/autophagy, which is integral for ADIPOQ/adiponectin-mediated cell death. Accordingly, blunting autophagosome formation, blocking autophagosome-lysosome fusion or genetic-knockout of BECN1/Beclin1 and ATG7 effectively impedes ADIPOQ/adiponectin induced growth-inhibition and apoptosis-induction. Mechanistic studies show that ADIPOQ/adiponectin reduces intracellular ATP levels and increases PRKAA1 phosphorylation leading to ULK1 activation. AMPK-inhibition abrogates ADIPOQ/adiponectin-induced ULK1-activation, LC3B-turnover and SQSTM1/p62-degradation while AMPK-activation potentiates ADIPOQ/adiponectin's effects. Further, ADIPOQ/adiponectin-mediated AMPK-activation and autophagy-induction are regulated by upstream master-kinase STK11/LKB1, which is a key node in antitumor function of ADIPOQ/adiponectin as STK11/LKB1-knockout abrogates ADIPOQ/adiponectin-mediated inhibition of breast tumorigenesis and molecular analyses of tumors corroborate in vitro mechanistic findings. ADIPOQ/adiponectin increases the efficacy of chemotherapeutic agents. Notably, high expression of ADIPOQ receptor ADIPOR2, ADIPOQ/adiponectin and BECN1 significantly correlates with increased overall survival in chemotherapy-treated breast cancer patients. Collectively, these data uncover that ADIPOQ/adiponectin induces autophagic cell death in breast cancer and provide in vitro and in vivo evidence for the integral role of STK11/LKB1-AMPK-ULK1 axis in ADIPOQ/adiponectin-mediated cytotoxic autophagy.     

Targeting LKB1 signaling in cancer

The serine/threonine kinase LKB1 is a master kinase involved in cellular responses such as energy metabolism, cell polarity and cell growth. LKB1 regulates these crucial cellular responses mainly via AMPK/mTOR signaling. Germ-line mutations in LKB1 are associated with the predisposition of the Peutz–Jeghers syndrome in which patients develop gastrointestinal hamartomas and have an enormously increased risk for developing gastrointestinal, breast and gynecological cancers. In addition, somatic inactivation of LKB1 has been associated with sporadic cancers such as lung cancer. The exact mechanisms of LKB1-mediated tumor suppression remain so far unidentified; however, the inability to activate AMPK and the resulting mTOR hyperactivation has been detected in PJS-associated lesions. Therefore, targeting LKB1 in cancer is now mainly focusing on the activation of AMPK and inactivation of mTOR. Preclinical in vitro and in vivo studies show encouraging results regarding these approaches, which have even progressed to the initiation of a few clinical trials. In this review, we describe the functions, regulation and downstream signaling of LKB1, and its role in hereditary and sporadic cancers. In addition, we provide an overview of several AMPK activators, mTOR inhibitors and additional mechanisms to target LKB1 signaling, and describe the effect of these compounds on cancer cells. Overall, we will explain the current strategies attempting to find a way of treating LKB1-associated cancer.     

TrkB Promotes Breast Cancer Metastasis via Suppression of Runx3 and Keap1 Expression

In metastatic breast cancer, the acquisition of malignant traits has been associated with the increased rate of cell growth and division, mobility, resistance to chemotherapy, and invasiveness. While screening for the key regulators of cancer metastasis, we observed that neurotrophin receptor TrkB is frequently overexpressed in breast cancer patients and breast cancer cell lines. Additionally, we demonstrate that TrkB expression and clinical breast tumor pathological phenotypes show significant correlation. Moreover, TrkB expression was significantly upregulated in basal-like, claudin-low, and metaplastic breast cancers from a published microarray database and in patients with triple-negative breast cancer, which is associated with a higher risk of invasive recurrence. Interestingly, we identified a new TrkB-regulated functional network that is important for the tumorigenicity and metastasis of breast cancer. We demonstrated that TrkB plays a key role in regulation of the tumor suppressors Runx3 and Keap1. A markedly increased expression of Runx3 and Keap1 was observed upon knockdown of TrkB, treatment with a TrkB inhibitor, and in TrkB kinase dead mutants. Additionally, the inhibition of PI3K/AKT activation significantly induced Runx3 and Keap1 expression. Furthermore, we showed that TrkB enhances metastatic potential and induces proliferation. These observations suggest that TrkB plays a key role in tumorigenicity and metastasis of breast cancer cells through suppression of Runx3 or Keap1 and that it is a promising target for future intervention strategies for preventing tumor metastasis and cancer chemoprevention.     

Molecular mechanisms of tumor suppression by LKB1

The LKB1 tumor suppressor gene is frequently mutated in sporadic lung adenocarcinomas and cervical cancers and germline mutations are causative for Peutz-Jeghers syndrome characterized by gastrointestinal polyposis. The intracellular LKB1 kinase is implicated in regulating polarity, metabolism, cell differentiation, and proliferation – all functions potentially contributing to tumor suppression. LKB1 acts as an activating kinase of at least 14 kinases mediating LKB1 functions in a complex signaling network with partial overlaps. Regulation of the LKB1 signaling network is highly context dependent, and spatially organized in various cellular compartments. Also the mechanisms by which LKB1 activity suppresses tumorigenesis is context dependent, where recent observations are providing hints on the molecular mechanisms involved.     

DLC-1基因在MCF-7人乳腺癌细胞系中低表达的机制探究

目的：探究DLC-1基因在MCF-7人乳腺癌细胞系中低表达的机制。方法：应用甲基化特异性PCR（MSP）检测人乳腺癌细胞MCF-7的DLC-1基因甲基化状态,不同浓度的5-氮杂-2＇-脱氧胞嘧啶（5-Aza-CdR）处理人乳腺癌细胞MCF-7,RT-PCR及Real-time PCR定量检测用药前后细胞中DLC-1基因mRNA表达水平变化。结果：DLC-1基因启动子区CpG岛呈甲基化状态,经过5-Aza-CdR处理后,DLC-1基因启动子区呈去甲基化状态,并且其mRNA恢复表达。结论：抑癌基因DLC-1 CpG岛甲基化是导致该基因低表达的原因之一,5-Aza-CdR能逆转DLC-1基因甲基化状态。     

乳腺癌SULT1A1、ICAM5基因单核苷酸多态性的研究

目的:探讨硫酸基转移酶(sulfotransferase,SULT)lA1、细胞间粘附分子(ICAM5)基因多态性与女性乳腺癌易感性的关系.方法:采外周血DNA后用等位基因特异性扩增法(allele specific amplification,ASA)检测青岛市200例正常对照者和160例乳腺癌患者的SULTIA1、ICAM5基因多态性分布,并进行统计学分析.结果:(1)SULTlA1 Arg/Arg、Arg/His、His/His三种基因型分布在对照组和病例组之间的差异无显著意义(P=0.103);病例组、对照组His等位基因频率分别为19.5%和9.2%(P=0.039),此差别有统计学意义;在淋巴结转移方面SULTIA1基因三种基因型在阴、阳性组间的差异有统计学意义(P=0.038).(2)ICAM5基因各基因型及等位基因分布频率在病例组和对照组间的差异无显著意义(P=0.245,P=0.294);从临床病例分型方面进一步分析,基因型GG与携带变异基因A的GA及AA基因型相比差异均无统计意义.结论:SULTlA1 His等位基因与汉族女性乳腺癌的发生可能相关.     

The PLAG1-GDH1 Axis Promotes Anoikis Resistance and Tumor Metastasis through CamKK2-AMPK Signaling in LKB1-Deficient Lung Cancer

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High-level expression of functional tumor suppressor LKB1 in Escherichia coli

The human LKB 1 tumor suppressor has been implicated as an important regulator of many cellular processes and signaling pathways, indicating that it could be a good candidate for anticancer drugs. The failure of its obtain high-level expression has been a major obstacle to study its protein structure and function in vitro. Here, we describe the high-level expression of human LKB 1 in Escherichia coli and show its kinase activity and anticancer effects on a tumor cell line. The gene encoding LKB 1 was optimized by replacing rare codons with codons frequently used in E. coli and synthesized with overlapping primers. The recombinant His-LKB 1 was expressed in hosts BL21（DE3） （BL） and Rosetta-gami（DE3）pLysS （RG）. His- LKB 1 from BL was present mainly as inclusion body. The soluble His-LKB 1 from RG accounted for 34. 1% of total proteins and the yield of purified His-LKB 1 was approximately 92 μg/ml. Purified His-LKB 1 protein from both hosts was functionally active, as shown by reversible autophosphorylation and kinase activity in the absence of any other associated kinase. The growth inhibitory ratio of the purified BL-derived and RG-derived His-LKB 1 on hepatic carcinoma SMMC-7721 cells was 24.97% and 45.68%, respectively, and both could produce significant cell-cycle arrest.     

Fine Needle Aspiration Cytodiagnosis As A Pre-Requisite For Primary Medical Treatment of Breast Cancer

The accurate non-surgical diagnosis of breast lumps allows assessment of breast cancer patients for conservation or neoadjuvant primary treatment before surgical intervention. We have analysed the accuracy of clinical assessment, fine needle aspiration cytology (FNAC), and mammography in over 868 women seen in a symptomatic breast clinic. Clinical examination by an experienced breast physician, together with FNAC, detected over 99% of the cancers with a 12% false positive rate. A cytological diagnosis of definite carcinoma was obtained in 69% of women with breast cancer with no false positive result from the women with benign conditions producing a 100% positive predictive value. These results indicate that it would be acceptable to give pre-surgical systemic endocrine or chemotherapy to women with positive cytology which is therefore a prerequisite for a neoadjuvant therapy programme.