条件培养液中的活性组分

条件培养液中含有细胞分泌的活性物质,因此,条件培养液可再现体外培养细胞或组织的微环境,促进或抑制来源不同的细胞或组织的生长、增殖和分化,也被用于研究各种生理或病理现象的机制。当前,国内外研究较多的有乳腺癌细胞系MDA-MB-231细胞、骨髓内皮细胞、坐骨神经和肝细胞等的条件培养液。     

The anti-inflammatory activities of Tanshinone IIA, an active component of TCM, are mediated by estrogen receptor activation and inhibition of iNOS

Tanshinone IIA (Tan IIA) is a major compound extracted from a traditional herbal medicine Salvia miltiorrhiza BUNGE, which is used to treat cardiovascular diseases, cerebrovascular diseases and postmenopausal syndrome. It has also been shown to possess anti-inflammatory activity. Since Tan IIA has a similar structure to that of 17β-estradiol (E₂), the present study was undertaken to characterize the estrogenic activity of Tan IIA and to demonstrate a functional role of this activity in RAW 264.7 cells. In transient transfection assay, Tan IIA (10 μM) increases ERE-luciferase activity in an estrogen receptor (ER) subtype-dependent manner when either ERα or ERβ were co-expressed in Hela cells. In LPS-induced RAW 264.7 cells, Tan IIA exerts anti-inflammatory effects by inhibition of iNOS gene expression and NO production, as well as inhibition of inflammatory cytokine (IL-1β, IL-6, and TNF-α) expression via ER-dependent pathway. Therefore, it could serve as a potential selective estrogen receptor modulator (SERM) to treat inflammation-associated neurodegenerative and cardiovascular diseases without increasing the risk of breast cancer.     

uPAR expression under hypoxic conditions depends on iNOS modulated ERK phosphorylation in the MDA-MB-231 breast carcinoma cell line

Urokinase plasminogen activator receptor （uPAR） plays a major role in cancer-invasion and metastasis and uPAR expression is correlated with a poor prognosis in various cancer types. Moreover, the expression of uPAR is increased under hypoxic conditions. Nitric oxide （NO） and its metabolites produced by inducible nitric oxide synthase （iNOS） are important products ofhypoxic stress, and NO may activate or modulate extracellular signal regulated kinase （ERK）. Here, we evaluated uPA, uPAR, and activated ERK levels under hypoxic conditions, and the modulatory effects of iNOS and NO in the MDA-MB-231 human breast cancer cell line. Cells were incubated in a hypoxic or normoxic incubator and treated with PD98059 （a MEK 1/2 inhibitor, which abrogates ERK phosphorylation） and aminoguanidine （a selective iNOS inhibitor）, uPAR expression, ERK phosphorylation, and uPA activity were found to be increased under hypoxic conditions. Moreover, when cells were treated with PD98059 under hypoxic conditions, uPAR was downregulated, whereas aminoguanidine markedly increased ERK phosphorylation in a dose dependent manner. Furthermore, aminoguanidine increased uPAR expression and prevented the inhibition of uPAR expression by PD98059. These results demonstrated that uPAR is induced by hypoxia and that increased uPAR expression is mediated by ERK phosphorylation, which in turn is modulated by iNOS/NO in MDA-MB-231 cells. We conclude that iNOS/NO downregulates the expression of uPAR under hypoxic conditions via ERK pathway modulation.     

Actin and Arp2/3 localize at the centrosome of interphase cells

Although many actin binding proteins such as cortactin and the Arp2/3 activator WASH localize at the centrosome, the presence and conformation of actin at the centrosome has remained elusive. Here, we report the localization of actin at the centrosome in interphase but not in mitotic MDA-MB-231 cells. Centrosomal actin was detected with the anti-actin antibody 1C7 that recognizes antiparallel (“lower dimer”) actin dimers. In addition, we report the transient presence of the Arp2/3 complex at the pericentriolar matrix but not at the centrioles of interphase HEK 293T cells. Overexpression of an Arp2/3 component resulted in expansion of the pericentriolar matrix and selective accumulation of the Arp2/3 component in the pericentriolar matrix. Altogether, we hypothesize that the centrosome transiently recruits Arp2/3 to perform processes such as centrosome separation prior to mitotic entry, whereas the observed constitutive centrosomal actin staining in interphase cells reinforces the current model of actin-based centrosome reorientation toward the leading edge in migrating cells.     

融合蛋白1hFVII-LDM的制备及其对乳腺癌细胞的抑制作用

该文主要研究了强化融合蛋白lhFⅦ-LDM对乳腺癌MDA-MB-231细胞的抑制作用。通过PCR和重叠PCR的方法构建了pET19b-lhFⅦ-LDP表达载体,重组质粒转化BL21,经IPTG诱导后表达并用Co~(2+)亲和层析纯化lhFⅦ-LDP融合蛋白,Western blot检测融合蛋白的正确性,再通过分子重组的方法将lhFⅦ-LDP与力达霉素(LDM)的活性发色团(AE)组装成为强化融合蛋白lhFⅦ-LDM。通过免疫共沉淀实验鉴定lhFⅦ-LDP与组织因子(TF)的特异性结合作用,利用平板克隆形成实验观察lhFⅦ-LDM对细胞增殖的影响,采用Hoechst33342染色检测lhFⅦ-LDM诱导MDA-MB-231细胞凋亡情况,建立了人乳腺癌裸鼠肿瘤模型,研究lhFⅦ-LDM对MDA-MB-231肿瘤生长的抑制作用。结果显示,lhFⅦ-LDM强化融合蛋白在体外能很好的诱导MDA-MB-231细胞凋亡,动物实验结果表明,强化融合蛋白对MDA-MB-231肿瘤的生长具有显著的抑制作用。     

Synthesis of novel chromeno-annulated cis-fused pyrano[3,4-c]benzopyran and naphtho pyran derivatives via domino aldol-type/hetero Diels–Alder reaction and their cytotoxicity evaluation

New chromeno-annulated cis-fused pyrano[3,4-c]benzopyran and naphtho pyran derivatives have been synthesized by domino aldol-type reaction/hetero Diels–Alder reaction generated from o-quinone methide in situ from 7-O-prenyl derivatives of 8-formyl-2,3-disubstituted chromenones with resorcinols/naphthols in the presence of 20 mol % ethylenediamine diacetate (EDDA), triethylamine (2 mL) as co-catalyst in CH₃CN under reflux conditions in good yields. The structures were established based on spectroscopic data, and further confirmed by X-ray diffraction analysis. The results showed that compounds 4h and 4j exhibited very potent cytotoxicity against human cervical cancer cell line (HeLa). Compound 4h displayed good inhibitory activity against both breast cancer cell lines, MDA-MB-231 and MCF-7. Further, the compound 4i exhibited good cytotoxicity against only MDA-MB-231, and compound 4j showed promising activity against human lung cancer cell line, A549 with IC₅₀ value of 2.53 ± 0.07 μM, which was comparable to the standard doxorubicin (IC₅₀ = 1.21 ± 0.1 μM).     

The role of GABARAPL1/GEC1 in autophagic flux and mitochondrial quality control in MDA-MB-436 breast cancer cells

GABARAPL1/GEC1 is an early estrogen-induced gene which encodes a protein highly conserved from C. elegans to humans. Overexpressed GABARAPL1 interacts with GABA_A or kappa opioid receptors, associates with autophagic vesicles, and inhibits breast cancer cell proliferation. However, the function of endogenous GABARAPL1 has not been extensively studied. We hypothesized that GABARAPL1 is required for maintaining normal autophagic flux, and plays an important role in regulating cellular bioenergetics and metabolism. To test this hypothesis, we knocked down GABARAPL1 expression in the breast cancer MDA-MB-436 cell line by shRNA. Decreased expression of GABARAPL1 activated procancer responses of the MDA-MB-436 cells including increased proliferation, colony formation, and invasion. In addition, cells with decreased expression of GABARAPL1 exhibited attenuated autophagic flux and a decreased number of lysosomes. Moreover, decreased GABARAPL1 expression led to cellular bioenergetic changes including increased basal oxygen consumption rate, increased intracellular ATP, increased total glutathione, and an accumulation of damaged mitochondria. Taken together, our results demonstrate that GABARAPL1 plays an important role in cell proliferation, invasion, and autophagic flux, as well as in mitochondrial homeostasis and cellular metabolic programs.     

The Role of Kif4A in Doxorubicin-Induced Apoptosis in Breast Cancer Cells

This study was to investigate the mechanism and role of Kif4A in doxorubicin-induced apoptosis in breast cancer. Using two human breast cancer cell lines MCF-7 (with wild-type p53) and MDA-MB-231 (with mutant p53), we quantitated the expression levels of kinesin super-family protein 4A (Kif4A) and poly (ADP-ribose) Polymerase-1 (PARP-1) by Western blot after doxorubicin treatment and examined the apoptosis by flow cytometry after treatment with doxorubicin and PARP-1 inhibitor, 3-Aminobenzamide (3-ABA). Our results showed that doxorubicin treatment could induce the apoptosis of MCF-7 and MDA-MB-231 cells, the down-regulation of Kif4A and upregulation of poly(ADP-ribose) (PAR). The activity of PARP-1 or PARP-1 activation was significantly elevated by doxorubicin treatment in dose- and time-dependent manners (P < 0.05), while doxorubicin treatment only slightly elevated the level of cleaved fragments of PARP-1 (P > 0.05). We further demonstrated that overexpression of Kif4A could reduce the level of PAR and significantly increase apoptosis. The effect of doxorubicin on apoptosis was more profound in MCF-7 cells compared with MDA-MB-231 cells (P < 0.05). Taken together, our results suggest that the novel role of Kif4A in doxorubicin-induced apoptosis in breast cancer cells is achieved by inhibiting the activity of PARP-1.     

广西弄岗黑叶猴栖息地选择与利用

2005年9月至2006年8月对广西弄岗国家自然保护区内一群黑叶猴进行行为观察,采用瞬时扫描法观察并收集猴群的栖息地利用数据,分析黑叶猴对喀斯特石山栖息地的选择与利用规律。结果表明,黑叶猴对不同山体部位的利用有显著性差异(χ~2=43.063,df=4,P0.001)。黑叶猴对崖壁的利用频率最高(占总记录的36.67%±9.44%),其次是山坡(32.30%±9.57%),对山脚(14.15%±5.01%)、山顶(11.24%±8.42%)和平地(5.63%±2.92%)的利用较少。分析发现,黑叶猴对山体部位的利用没有显著的季节性差异(山顶:Z=-0.160,P=0.837;崖壁:Z=-0.320,P=0.749;山坡:Z=-0.480,P=0.631;山脚:Z=-1.601,P=0.109;平地:Z=0,P=1)。黑叶猴将崖壁作为主要的休息场所,山坡和山脚为主要的移动和觅食场所。黑叶猴对栖息地的利用受食物可获得性的影响。当食物中花的可获得性降低时,猴群增加对山脚的利用;当嫩叶可获得性降低时,猴群增加在崖壁移动的频率;当果实的可获得性升高时,猴群增加在平地觅食的频率。食物组成与黑叶猴栖息地利用也有关系。总体来看,黑叶猴对山顶的利用频率与花的觅食比例呈显著正相关;对山脚的利用频率与果实+种子的觅食比例呈显著正相关。猴群在山顶休息的频率随花和成熟叶的觅食比例的上升而上升;在山脚休息的频率随果实+种子的觅食比例的上升而上升。猴群在山坡觅食的频率随果实+种子的觅食比例的下降而上升;在山脚觅食的频率随嫩叶的觅食比例的下降而上升。另外,平均最低温度与猴群在平地觅食的频率呈负相关关系。分析表明食物的分布和数量对黑叶猴栖息地利用有重要影响,黑叶猴对栖息地的选择是在觅食利益与风险之间进行权衡的结果。