沙蟾毒精抑制肝癌HepG2细胞黏附、迁移和侵袭的作用

目的研究沙蟾毒精(Arenobufagin)对人肝癌细胞HepG2黏附、迁移和侵袭的抑制作用。方法 MTT法检测沙蟾毒精对HepG2细胞活力和细胞黏附能力的影响,划痕实验观察沙蟾毒精对HepG2细胞运动能力的影响,Transwell小室模型研究沙蟾毒精对HepG2细胞的迁移和侵袭的抑制作用,Western blot检测基质金属蛋白酶MMP 2和MMP 9的表达水平变化。结果与空白组相比,沙蟾毒精可降低HepG2细胞的黏附能力,使Transwell小室膜上的肝癌细胞明显减少,并且呈剂量依赖性。Western blot结果显示沙蟾毒精可以明显抑制基质金属蛋白酶MMP 2和MMP 9的表达。结论沙蟾毒精能抑制人肝癌细胞HepG2的黏附、迁移和侵袭,其机制可能与抑制MMP 2和MMP 9的表达有关。     

沙蟾毒精与牛血清蛋白的结合及分子对接研究

目的:研究沙蟾毒精(arenobufagin)与牛血清蛋白的相互作用。方法:采用平衡透析法,高效液相色谱测定沙蟾毒精与小牛血清蛋白的结合率。荧光光谱法研究药物与牛血清白蛋白(BSA)的相互作用。采用同源建模和分子对接技术分析药物相互作用模式。结果:沙蟾毒精(1μg.mL-1)与小牛血清蛋白的最佳平衡透析时间为30 h,血清蛋白结合率为(57.9±0.7)%,荧光光谱测得沙蟾毒精与BSA的结合常数为1.02×103L.moL-1。BSA同源建模和分子对接结果显示:沙蟾毒精能够结合到BSA的Site I。结论:沙蟾毒精与小牛血清发生中等强度的结合,白蛋白是沙蟾毒精的载药蛋白。     

沙蟾毒精酯类衍生物的合成和抗肿瘤活性研究

目的 基于活性天然产物沙蟾毒精骨架设计3-酯类衍生物,并测试其抗肿瘤活性.方法 通过缩合剂催化下将酸和沙蟾毒精进行酯化反应,得到3-酯类沙蟾毒精衍生物,采用Cell Titer法测试体外抗肿瘤活性.结果 3-沙蟾毒精酯类衍生物对所有的肿瘤细胞株显示出优异的体外抗肿瘤活性,其IC50值均在1μmol/L以下.结论 化合物...     

蛇毒金属蛋白酶抑制剂重组蛋白抗肿瘤血管生成作用及其机制

目的研究蛇毒金属蛋白酶抑制剂重组蛋白(recombinant snake venom metalloproteinase inhibitor,r SVMPI)对血管生成的影响及其分子机制。方法应用鸡胚绒毛尿囊膜(chicken chorioallantoic membrane,CAM)血管生成模型观察SVMPI重组蛋白对血管生成的影响;采用Alamar Blue分析方法检测人脐静脉内皮细胞(human umbilical veins endothelial cells,HUVECs)增殖能力、Annexin V-FITC双标记流式细胞术检测细胞凋亡、划痕标记法检测细胞体外迁移能力、Boyden小室分析方法检测细胞体外趋化能力及管腔形成法检测体外血管新生能力;通过real-time PCR及Western blot检测重组蛋白处理后的HUVECs KDR、FGFR-1表达。结果r SVMPI减少鸡胚尿囊膜新生血管密度指数,减弱由VEGF诱导的HUVECs趋化能力,抑制HUVECs体外新生小管的形成,降低HUVECs细胞KDR和FGFR-1的表达水平。结论r SVMPI可能通过阻断VEGF-KDR或b FGF-FGFR信号转导途径,发挥抑制血管生成的作用。     

从赤魟组织中分离到的福安肽的抗血管生成作用

目的研究从赤魟组织中分离到的福安肽(FAT)对鸡胚绒毛尿囊膜(CAM)血管生成、人低分化鼻咽癌细胞(CNE-2Z)诱导的CAM血管生成和小鼠肝癌组织血管生成的影响。方法用CAM法检测FAT对CAM血管生成和CNE-2Z细胞诱导的CAM血管生成的影响;用免疫组化法检查FAT对小鼠肝癌组织内微血管密度(MVD)的影响。结果FAT显著抑制CAM血管生成,当其用量为30,60,120μg/(胚.d)×3时,其抑制率分别为30.8%,50.1%,64.0%;CNE-2Z细胞明显诱导CAM血管生成,当CNE-2Z细胞的接种量为1.9×106细胞/胚时,其血管生成诱导率为30.1%;FAT明显抑制CNE-2Z细胞诱导的CAM血管生成,当其用量为30,60,120μg/(胚.d)×4时,其抑制率分别为35.8%,43.1%,51.3%。FAT明显降低小鼠肝癌组织的MVD,FAT平均微血管数为(8.2±1.3)与对照组的(21.3±2.8)相比,差异显著(P<0.05)。结论FAT有显著的抗血管生成作用,这可能是其抑制小鼠肿瘤生长的重要原因。     

HPLC法同时测定蟾酥中11个蟾毒配基成分的含量

目的:建立HPLC法同时测定蟾酥中11个蟾毒配基成分(伪异沙蟾毒精、日蟾毒它灵、沙蟾毒精、蟾蜍它里定、远华蟾蜍精、蟾毒它灵、脂蟾毒精、南美蟾毒精、蟾毒灵、脂蟾毒配基和华蟾酥毒基)的含量。方法:采用Agilent InfinityLab Poroshell 120 EC-C₁₈色谱柱(150 mm×3.0 mm 2.7μm),以0.3%乙酸水溶液-乙腈为流动相,梯度洗脱,流速为0.5 mL·min^-1,柱温为30℃,检测波长为296 nm。结果:11个蟾毒配基成分的线性范围分别为2.024～20.24μg·mL^-1(r=0.999 8)、18.44～184.4μg·mL^-1(r=0.999 9)、12.40～124.0μg·mL^-1(r=0.999 5)、3.720～37.20μg·mL^-1(r=0.999 5)、10.28～102.8μg·mL^-1(r=0.999 8)、22.16～221.6μg·mL^-1(r=...     

BNIP3介导锰经线粒体凋亡途径诱导SH-SY5Y细胞凋亡的研究

目的探讨线粒体蛋白BNIP3在锰诱导线粒体依赖的SH-SY5Y细胞凋亡中的作用。方法取对数生长期的人神经母细胞瘤SH-SY5Y细胞,经MnCl_2处理24 h后应用透射电子显微镜直接观察MnCl_2作用下SH-SY5Y细胞的凋亡情况,使用流式细胞仪检测SH-SY5Y细胞线粒体膜电位(Δψm),通过蛋白免疫印记法(Western blot)检测BNIP3及凋亡相关蛋白Caspase-3的表达。并使用shRNA将SH-SY5Y细胞中的BNIP3沉默,观察BNIP3对MnCl_2诱导的SH-SY5Y细胞凋亡、线粒体膜电位丢失(Δψm)及凋亡相关蛋白Caspase-3表达的影响。结果 MnCl_2可使线粒体膜电位逐渐丢失(F=49.061,P0.01)、并使线粒体蛋白BNIP3及凋亡相关蛋白Caspase-3的表达随着MnCl_2浓度的提高而上升(F=334.832,F=299.902,均P0.01)、明显增加SH-SY5Y细胞的凋亡小体个数,上述差异均有统计学意义。沉默BNIP3(F=1.301,P=0.318,t=32.647,P0.01)可以减少MnCl_2诱导线粒体膜电位的丢失(F=1.786,P=0.252;t=20.290,P0.01),减少凋亡相关蛋白Caspase-3的表达(F=2.816,P=0.168;t=22.073,P0.01)并使SH-SY5Y凋亡小体的个数减少,上述差异均有统计学意义。结论在MnCl_2通过线粒体凋亡途径诱导SH-SY5Y细胞凋亡的过程中BNIP3起到了重要作用,这种调控机制可能是锰产生神经毒性的作用机制之一。     

蛇床子素体外对人前列腺癌DU145细胞增殖的抑制作用及机制

目的观察天然化合物蛇床子素对人前列腺癌DU145细胞的增殖抑制和诱导凋亡作用,为临床应用提供实验基础。方法用不同浓度的蛇床子素作用于DU145细胞,用四甲基偶氮噻蓝(MTT)法检测细胞增殖;用流式细胞仪AnnexinV/PI双染法检测蛇床子素诱导DU145细胞凋亡;用荧光显微镜观察蛇床子素作用于DU145细胞后细胞核的形态变化。结果 MTT法显示,蛇床子素对DU145细胞的增殖有明显的抑制作用(P<0.05),并呈现明显的时间浓度依赖性;流式细胞仪法显示,蛇床子素可诱导DU145细胞凋亡,且呈浓度依赖性;荧光显微镜下观察到蛇床子素处理组可见核固缩、核碎裂等典型的凋亡改变。结论蛇床子素对人前列腺癌DU145细胞的增殖具有抑制作用,机制与其诱导细胞凋亡有关。     

姜黄素对人鼻咽癌株CNE-2Z细胞凋亡的影响及机制研究

目的探讨姜黄素(CU)对人鼻咽癌株CNE-2Z细胞凋亡的影响及机制。方法体外培养CNE-2Z细胞在梯度浓度CU(0、20、40、80μmol/L)干预48 h后,采用MTT法检测其对CNE-2Z抑制增殖的作用,ELISA法检测8-羟基-2-脱氧鸟苷(8-OHdG)水平,Hoechst33342染色检测细胞形态学的变化,Western blotting法检测PI3K和Akt磷酸化水平。结果与空白对照组(0μmol/L)比较,CU干预后明显抑制CNE-2Z的细胞增殖(P<0.01),同时减少内源性的8-OHdG生成(P<0.01)。此外,CU干预可有效促进癌细胞凋亡,下调p-PI3K和p-Akt的表达。结论姜黄素具有良好的抗肿瘤作用,其机制与抑制癌细胞增殖、诱导细胞凋亡及抑制PI3K/Akt通路有关。     

6'-羟基爵床素A诱导人肝癌HepG2细胞凋亡及其机制

目的探讨6'-羟基爵床素A(JR6)对人肝癌Hep G2细胞凋亡的诱导作用及其机制。方法体外培养人肝癌Hep G2细胞,采用MTT法观察不同浓度的JR6和5-氟尿嘧啶(5-FU)对Hep G2细胞存活的作用,荧光显微镜观察Hoechst33258染色后细胞核形态改变,用流式细胞仪检测AnnexinⅤ-FITC/PI双染后Hep G2细胞的凋亡率,JC-1荧光染色观察药物对细胞线粒体膜电位的影响,Western蛋白质印迹法检测细胞凋亡相关蛋白细胞色素c、Bax和Bcl-2蛋白的表达。结果 JR6 6.1~196μmol·L-1和5-FU 3.4~192μmol·L-1分别作用Hep G2细胞48 h,对Hep G2细胞存活具有抑制作用,IC50分别为74.90和49.75μmol·L-1。JR6 12.3,49和196μmol·L-1作用Hep G2细胞48 h,细胞凋亡率明显增加,由正常对照组的(6.9±2.0)%增加至(13.8±2.0)%,(18.6±4.3)%和(32.4±3.2)%(P<0.05,P<0.01)。Hoechst33258染色结果显示,JR6 49和196μmol·L-1作用Hep G2细胞48 h,部分细胞出现细胞核固缩和染色质凝集等凋亡变化。线粒体膜电位检测结果发现,JR6 49和196μmol·L-1作用48 h能使Hep G2细胞线粒体膜电位水平明显降低(P<0.05,P<0.01)。线粒体凋亡相关蛋白的检测结果表明,JR6 12.3,49和196μmol·L-1作用Hep G2细胞48 h,胞浆细胞色素c表达增加,促凋亡蛋白Bax表达增加,抗凋亡蛋白Bcl-2表达降低,且Bax/Bcl-2比值增加(P<0.05,P<0.01)。JR6 49μmol·L-1和5-FU组上述蛋白表达无明显差异。结论 JR6可能通过促进细胞色素c释放、打破Bcl-2/Bax平衡诱导Hep G2细胞凋亡。     

Effects of extracellular nucleotides and nucleosides on prostate carcinoma cells

1. The purpose of this work was to characterize the receptors involved in the action of nucleotides on the human prostate carcinoma cell lines LNCaP, PC-3 and DU145. 2. Northern blotting revealed the presence of P2Y(2), P2Y(6) and P2Y(11) messengers in the three cell lines. P2Y(1) mRNA was only observed in the DU145 cells. In both PC-3 and DU145 cells, ATP and UTP stimulated inositol phosphate accumulation in an equipotent, equiactive and non-additive way, suggesting the involvement of P2Y(2) receptors. ATP also increased cyclic AMP, but this effect is likely to result from degradation into adenosine and activation of A(2) receptor. A(2) receptor activation led to a synergistic enhancement of prostate-specific antigen secretion induced by vasoactive intestinal peptide. 3. RT - PCR experiments detected the expression of the P2X(4) and P2X(5) receptors in the DU145 cells and the P2X(4), P2X(5) and P2X(7) receptors in the PC-3 cells. The calcium influx induced by BzATP confirmed the functional expression of P2X receptors. 4. ATP inhibited the growth of PC-3 and DU145 cells. This effect was mimicked neither by UTP nor by adenosine, indicating that it does not result from phospholipase C or adenylyl cyclase activation. On the contrary, in PC-3 cells, BzATP reproduced the effect of ATP, which was associated to a moderate decrease of proliferation and an increase of apoptosis. In DU145 cells, ATP was more potent than BzATP and growth inhibition was mainly associated with necrosis. We suggest that P2X receptors might be involved in the inhibition by nucleotides of prostate carcinoma cell growth.     

Effect of lipidated gonadotropin-releasing hormone peptides on receptor mediated binding and uptake into prostate cancer cells in vitro

Gonadotropin-releasing hormone (GnRH) receptors are overexpressed on many cancer cells but not on primary cell lines. This study was designed to investigate the targeting ability and uptake of dendritic lipidated [Gln¹]-GnRH peptide analogues on receptor-positive prostate cancer PC-3 cells relative to receptor-negative ovarian carcinoma SKOV-3 cells for potential application in drug delivery. Direct antiproliferative effect of these was investigated on three GnRH-receptor positive cancer cells, PC-3, LNCaP and DU145. A significant dose dependent growth inhibitory effect was produced in DU145 cells by 5 dendrimers giving an IC₅₀ value of 22–35 μM. All compounds were non-toxic to the normal peripheral blood mononuclear cells.From the Clinical EditorThis study demonstrates the use of specific dendritic lapidated GnRH analogues in growth inhibition of GnRH receptor positive prostate cancer cell lines, suggesting potential future clinical use of this or similar strategies to address GnRH receptor positive cancer cells.     

Calcitriol inhibits lipopolysaccharide-induced proliferation,migration and invasion of prostate cancer cells through suppressing STAT3 signal activation

Increasing evidence suggests that infection promotes the initiation and progression of prostate cancer. This study investigated the effects of lipopolysaccharide (LPS), a major component of Gram-negative bacilli, on proliferation, migration and invasion of prostate cancer cells and the protective effects of 1α,25(OH)₂D₃ (calcitriol). PC-3 and DU145 cells were stimulated with LPS (2.0 μg/mL) in the presence or absence of 1α,25(OH)₂D₃ (100 nM). Our results shown that 1α,25(OH)₂D₃ reduced the proportion of S phase cells in LPS-stimulated PC-3 and DU145 cells, and down-regulated the nuclear protein levels of Cyclin D1 and PCNA in LPS-stimulated PC-3 cells. In addition, 1α,25(OH)₂D₃ inhibited migration and invasion, as determined by wound healing and transwell assay, in LPS-stimulated PC-3 and DU145 cells. Of interest, we observed that 1α,25(OH)₂D₃ inhibits NF-κB activation and subsequent synthesis and secretion of IL-6 and IL-8 by promoting VDR and NF-κB p65 interaction. Surprisingly, 1α,25(OH)₂D₃ blocks nuclear translocation of pSTAT3 by promoting physical interaction between VDR and pSTAT3 (Tyr705) in LPS-stimulated PC-3 and DU145 cells. These results suggest that 1α,25(OH)₂D₃ inhibits LPS-induced proliferation, migration and invasion in prostate cancer cells by directly and indirectly blocking STAT3 signal transduction.     

The combination of gamma ionizing radiation and 8-Cl-cAMP induces synergistic cell growth inhibition and induction of apoptosis in human prostate cancer cells

The antiproliferative and cytotoxic potential of the nucleotide analog 8-Cl-cAMP was tested in PC-3 and DU145 metastatic human prostate cancer cells. The drug was examined as the only therapeutic agent and in combination with ionizing irradiation (IR). Highly synergistic effects of IR and 8-Cl-cAMP were observed in both cell lines when examined by the MTT viability and BrdU proliferation assays. The combination of IR and 8-Cl-cAMP at clinically relevant doses exerted substantial growth inhibition. The combination of IR and 8-Cl-cAMP caused a significant disturbance in the distribution of cell cycle phases. Cell cycle arrest in the sub-G0/G1 phase predominated in both cell lines. The most striking observation was a significant increase in apoptotic PC-3 and DU145 cells. The DU145 cells were three times more sensitive to the combined treatment than PC-3 cells. The initial resistance to IR-induced apoptosis in these p53-deficient prostate cancer cell lines was overcome through an alternative proapoptotic pathway induced by 8-Cl-cAMP. Considering the low effective doses of treatments, improved tumor eradication rates and minimal undesirable side effects, the combination of IR and 8-Cl-cAMP could be the therapy of choice in treating prostate cancer.     

灵芝多糖对肿瘤细胞与内皮细胞相互作用的影响

目的研究灵芝多糖(GlPS)抗肿瘤作用及其机制。方法通过相差显微镜和间接免疫荧光的方法鉴定了人脐静脉内皮细胞(HUVECs)。采用MTT法检测GlPS对人前列腺癌细胞(PC-3M)和HUVECs增殖的影响。检测GlPS对PC-3M细胞与HUVECs粘附的影响。采用transwell双层小室观察GlPS对PC-3M迁移穿过单层HUVECs的影响。结果GlPS对PC-3M无直接细胞毒作用,对HUVECs增殖无显著性作用。GlPS能够减少粘附和迁移穿过单层内皮细胞的肿瘤细胞数目。结论灵芝多糖通过抑制肿瘤细胞粘附并迁移穿过内皮细胞发挥其抗肿瘤作用。     

Perturbation of dopamine metabolism by 3-amino-2-(4'-halophenyl)propenes leads to increased oxidative stress and apoptotic SH-SY5Y cell death

We have recently characterized a series of 3-amino-2-phenyl-propene (APP) derivatives as reversible inhibitors for the bovine adrenal chromaffin granule vesicular monoamine transporter (VMAT) that have been previously characterized as potent irreversible dopamine-beta-monooxygenase (DbetaM) and monoamine oxidase (MAO) inhibitors. Halogen substitution on the 4'-position of the aromatic ring gradually increases VMAT inhibition potency from 4'-F to 4'-I, parallel to the hydrophobicity of the halogen. We show that these derivatives are taken up into both neuronal and non-neuronal cells, and into resealed chromaffin granule ghosts efficiently through passive diffusion. Uptake rates increased according to the hydrophobicity of the 4'-substituent. More importantly, these derivatives are highly toxic to human neuroblastoma SH-SY5Y but not toxic to M-1, Hep G2, or human embryonic kidney 293 non-neuronal cells at similar concentrations. They drastically perturb dopamine (DA) uptake and metabolism in SH-SY5Y cells under sublethal conditions and are able to deplete both vesicular and cytosolic catecholamines in a manner similar to that of amphetamines. In addition, 4'-IAPP treatment significantly increases intracellular reactive oxygen species (ROS) and decreases glutathione (GSH) levels in SH-SY5Y cells, and cell death is significantly attenuated by the common antioxidants alpha-tocopherol, N-acetyl-l-cysteine and GSH, but not by the nonspecific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. DNA fragmentation analysis further supports that cell death is probably due to a caspase-independent ROS-mediated apoptotic pathway. Based on these and other findings, we propose that drastic perturbation of DA metabolism in SH-SY5Y cells by 4'-halo APP derivatives causes increased oxidative stress, leading to apoptotic cell death.     

Induction of apoptosis by N-(4-hydroxyphenyl)retinamide and its association with reactive oxygen species, nuclear retinoic acid receptors, and apoptosis-related genes in human prostate carcinoma cells

The synthetic retinoid N-(4-hydroxyphenyl)retinamide (4HPR) has been shown to induce apoptosis in various malignant cells including human prostate carcinoma cells (HPC). We examined several possible mechanisms by which 4HPR could induce apoptosis in HPC cells. 4HPR exhibited concentration- and time-dependent decrease in cell number both in androgen-dependent (LNCaP) and -independent (DU145 and PC-3) cells. The 4HPR concentrations causing 50% decrease in cell number in LNCaP, DU145, and PC-3 cultures were 0.9 +/- 0.16, 4.4 +/- 0.45, and 3.0 +/- 1.0 microM, respectively, indicating that LNCaP cells were more sensitive to 4HPR than the other cells. 4HPR-induced apoptosis in all three cell lines was evidenced by increased enzymatic labeling of DNA breaks and formation of a DNA ladder. 4HPR increased the level of reactive oxygen species, especially in LNCaP cells. 4HPR-induced apoptosis could be suppressed in LNCaP cells by antioxidant and in DU145 cells by a nuclear retinoic acid receptor-specific antagonist, suggesting the involvement of reactive oxygen species or retinoic acid receptors in mediating apoptosis induced by 4HPR in the different HPC cells. Furthermore, 4HPR modulated the expression levels of some apoptosis-related gene (p21, c-myc, and c-jun), which may also contribute to the induction of apoptosis by 4HPR in HPC cells.     

Regression of prostate tumors after intravenous administration of lactoferrin-bearing polypropylenimine dendriplexes encoding TNF-α, TRAIL,and interleukin-12

The possibility of using gene therapy for the treatment of prostate cancer is limited by the lack of intravenously administered delivery systems able to safely and selectively deliver therapeutic genes to tumors. Given that lactoferrin (Lf) receptors are overexpressed on prostate cancer cells, we hypothesized that the conjugation of Lf to generation 3-diaminobutyric polypropylenimine dendrimer would improve its transfection and therapeutic efficacy in prostate cancer cells. In this study, we demonstrated that the intravenous administration of Lf-bearing DAB dendriplexes encoding TNFα resulted in the complete suppression of 70% of PC-3 and 50% of DU145 tumors over one month. Treatment with DAB-Lf dendriplex encoding TRAIL led to tumor suppression of 40% of PC-3 tumors and 20% of DU145 tumors. The treatment was well tolerated by the animals. Lf-bearing generation 3-polypropylenimine dendrimer is therefore a highly promising delivery system for non-viral gene therapy of prostate cancer.     

5-氮-2′-脱氧胞苷对DU145前列腺癌细胞TIMP-3基因去甲基化作用及侵袭能力的影响

目的观察5-氮-2′-脱氧胞苷(5Aza-dc)对DU145前列腺癌细胞金属蛋白酶组织抑制因子3(TI MP-3)启动子去甲基化作用及侵袭能力的影响。方法用5Aza-dc处理DU145前列腺癌细胞,Transwell检测癌细胞的侵袭及运动能力,反转录-聚合酶链反应(RT-PCR)检测TI MP-3 mRNA的表达,Western印迹检测TI MP-3的蛋白表达。结果5Aza-dc作用后的DU145细胞侵袭及运动能力降低;TI MP-3 mRNA及蛋白恢复表达;TI MP-3启动子区去甲基化。结论5Aza-dc可以使DU145前列腺癌细胞TI MP-3启动子区去甲基化,使TI MP-3基因重新表达,恢复其抑制肿瘤侵袭和移动的能力。