Antiagiogenesis represents a promising approach to cancer therapy. We have previously demonstrated that the antitumor effects of endostatin, one of the most potent angiostatic agents, could be enhanced when combined with immunotherapy. Our current study evaluated whether anti-tumor immune response could also potentiate the therapuetic efficacy of another antiangiogenic drug, angiostatin. METHODS AND RESULTS: Using Matrigel assay, we showed that our preparation of recombinant angiostatin possessed potent anti-angiogenic activity in vivo. The antitumor effects of recombinant angiostatin were tested against weakly-immunogenic 3LL Lewis lung carcinoma versus its highly immunogenic variant 3LL-C75. We showed that angiostatin inhibited the growth of 3LL-C75 more potently than that of 3LL tumor, suggesting that the host's immune response potentiates the antitumor effects of angiostatin. This conclusion was further supported by the finding that the antitumor activity of angiostatin against 3LL-C75 tumor was lower in immunodeficient nude mice in comparison with immunocompetent mice. Immunization of C57BL/6 mice with 3LL-C75 cells stimulated the antitumor immunity and inhibited the growth of parental 3LL tumor. Angiostatin treatment of immunized mice further enhanced the antitumor effect of tumor vaccination. CONCLUSION: Antitumor immune response could complement the therapeutic efficacy of angiostatin.

Protocatechualdehyde (PA, a dihydroxybenzene derivative) has previously been shown to induce apoptotic cell death in cytotoxic T cells (CTLL-2). However, the molecular mechanisms by which PA regulates apoptosis are still unclear. In this study, the possible roles of ornithine decarboxylase activity (ODC) and mitogen-activated protein kinases (MAPKs) in the PA-induced apoptosis process were further investigated. We demonstrated that PA inhibited ODC activity induced by IL2 in a time- and dose-dependent manner. Furthermore, the expression of ODC mRNA stimulated by IL2 was also effectively suppressed. 0.12mM PA inhibited the activation of ERK1/2 induced by IL2 and enhanced the activation of JNK, which was abrogated by IL2. No alteration in the effect of p38 MAPK on the apoptosis process was observed in the CTLL-2 cells. PD98059 (a specific ERK1/2 inhibitor) inhibited cell growth, led to cell apoptotic death and effectively decreased ODC activity and suppressed ERK1/2 activation induced by IL2. These data indicate that PA induced apoptosis in CTLL-2 cells by two mechanisms; either via inhibiting ODC induction or interfering with MAPK signaling pathways.

OBJECTIVE: To purify Methamidophos (Met) monoclonal antibodies with two methods and compare immune activity of purified antibodies. METHOD: Caprylic acid ammonium sulphate precipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method were used to purify Met monoclonal antibodies, UV spectrum scanning was used to determine protein content and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linked immunosorbent assay (ELISA) was used to determine immune activity of purified antibodies. RESULTS: Antibody protein content and recovery rate with CAASP method were 7.62mg/mL and 8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45mg/mL and 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASP method. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was 181.26 microg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01 microg/mL and 1.03 microg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82 microg/mL, and the linear working range and LOD were 10.91-11412.29 microg/mL and 3.42 microg/mL, respectively. CONCLUSION: Antibodies purified by SPA method are better than those by CAASP method, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelled testing paper for analyzing Met residue in vegetable and drink water.

AIM: To evaluate the effects of 3,3'-diethyl-9-methylthia-carbocyanine iodide (DMTCCI) on DNA primase activity and on apoptosis of human hepatocellular carcinoma BEL-7402 cells. METHODS: DNA primase assay was used to investigate DNA primase activity. MTT assay was applied to determine cell proliferation. Flow cytometric analysis, transmission electron microscopy, DNA fragmentation assay were performed to detect DMTCCI-induced apoptosis. Expression levels of p53, Bcl-2, Bcl-xL, Bad, Bax, survivin, Caspase-3 and poly (ADP-ribose) polymerase (PARP) were evaluated by immunoblot analysis. Caspase-3 activity was assessed with ApoAlert Caspase-3 colorimetric assay kit. RESULTS: DMTCCI had inhibitory effects on eukaryotic DNA primase activity with IC50 value of 162.2 nmol/L. It also inhibited proliferation of human hepatocellular carcinoma BEL-7402 cells with IC50 value of 2.09 μmol/L. Furthermore, DMTCCI-induced BEL-7402 cell apoptosis was confirmed by DNA fragmentation (DNA ladders and sub-G1 formation) and transmission electron microscopy (apoptotic bodies formation). During the induction of apoptosis, expression of Bcl-2, Bcl-xL and survivin was decreased, and that of p53, Bad and Bax was increased. Caspase-3 was activated and poly (ADP-ribose) polymerase (PARP) was cleaved in BEL-7402 cells treated with DMTCCI. CONCLUSION: The present data suggest that DMTCCI has inhibitory effects on eukaryotic DNA primase and can induce apoptosis of BEL-7402 cells. The modulation of expression of p53 and Bcl-2 family proteins, and activation of Caspase-3 might be involved in the induction of apoptosis.

Previous studies have demonstrated that c-Jun NH2-terminal protein kinase (JNK) plays a crucial role in neuronal apoptosis. Here, we report that indirubin-3'-oxime, a known effective inhibitor of cyclin-dependent kinases (CDKs) and glycogen synthase kinase 3-beta (GSK-3β), has a significant inhibitory effect on JNK. Kinase assay showed that indirubin-3'-oxime directly inhibited the activity of all three isoforms of JNK (JNK1, and JNK3) in vitro, with half inhibition dose (IC50) of 0.8μM, 1.4μM, and 1.0μM, respectively. In cerebellar granule neurons (CGNs), indirubin-3'-oxime blocked c-Jun phosphorylation induced by potassium withdrawal and prevented CGNs from apoptosis in a dose dependent manner. However, inhibitors of CDKs and GSK-3β were ineffective in reducing c-Jun phosphorylation both in vitro and in vivo, suggesting that indirubin-3'-oxime prevents c-Jun phosphorylation independent of its inhibition on CDKs and GSK-3β. Our studies give further supports for JNK-targeting strategy in preventing neuronal apoptosis.

Increasing evidence suggests that c-Jun N-terminal kinase (JNK) is an important kinase mediating neuronal apoptosis in Parkinson's disease (PD) model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In order to study roles of JNK activity in neuronal apoptosis in this model, we blocked JNK activity in vivo using a specific inhibitor of JNK, SP600125. Our data showed that MPTP-induced phospho-c-Jun of substantial nigral neurons, caused apoptosis of dopaminergic neurons, and decreased the dopamine level in striatal area. We found that inhibiting JNK with SP600125 reduced the levels of c-Jun phosphorylation, protected dopaminergic neurons from apoptosis, and partly restored the level of dopamine in MPTP-induced PD in C57BL/6N mice. These results indicate that JNK pathway is the major mediator of the neurotoxic effects of MPTP in vivo and inhibiting JNK activity may represent a new and effective strategy to treat PD.

Anoikis is a form of apoptosis induced in normal cells as a result of loss of their adhesion to substrate. In the present study, we have tested whether tumor cells are also sensitive to anoikis and whether selection of tumor cells for resistance to anoikis could increase their metastatic ability. In vitro cultured Cloudman S91 melanoma cells are strongly adherent to the plastic. Prevention of their adherence by rocking or by covering culture plates with polyhydroxyethylmethacrylate resulted in induction of anoikis and death of almost all cells. Their death was prevented in the presence of caspase inhibitor Z-Val-Ala-Asp-fluoromethyl ketone. To select anoikis-resistant cells, S91 cells floating in the culture medium were sequentially isolated and transferred for seven generations. As a result, a new subline of S91 cells capable of growing in free cell suspension was selected. These S91 nonadherent (S91Nadh) cells were completely resistant to anoikis and manifested higher metastatic ability than S91Adh cells. Anoikis resistance of S91Nadh cells was not attributable to their resistance to other apoptotic signals in vitro, and they showed no increase in their survival in vivo in the lungs after i.v. inoculation. Increased metastatic potential of the anoikis-resistant S91Nadh cells was associated with various phenotypic changes, including increased proliferation and loss of VLA-4 integrin expression because of down-regulation of the VLA-49 (CD49d) gene. In parallel, they showed a reduction in homotypic aggregation and binding to endothelial cells, increased Matrigel invasiveness, and decreased matrix metalloproteinase-2 and matrix metalloproteinase-9 activity that paralleled up-regulation of the TIMP-1 gene. S91Nadh cells also manifested changes in cell surface carbohydrates, such as appearance of -galactosyl epitopes as a result of up-regulation of the 1,3-galactosyltransferase gene and concomitant reduction in cell membrane sialylation. Thus, selection of S91 melanoma cells for anoikis resistance resulted in an increase in their metastatic potential in parallel with multiple alterations in their phenotypic properties.

背景与目的：死亡相关蛋白激酶DAPKl失活是影响非小细胞肺癌患者生存的独立因子，过表达的DAPKl可以诱导肿瘤细胞凋亡，抑制肿瘤转移，但抑制转移的机制尚未完全清楚。本文探讨DAPKl基因抑制高转移性肺癌PGCl3细胞生长及转移的可能机制。方法：利用基因重组技术构建含DAPKl基因开放读码框（ORF）的真核表达载体pcDNA3.1-DAPKl，用脂质体LipofectAMINE2000介导转染PGcL3细胞系。检测转染后PGCL3细胞的生长曲线、体外软琼脂克隆形成率、体外侵袭、运动和粘附能力的变化。同时检测了胶原酶活性，p53，bcl-2基因表达的变化。结果：转染DAPKl基因的细胞生长比空白组及pcDNA3.1转染组减缓；体外软琼脂克隆形成率下降P<0.05；pcrDNA3.1-DAPKl转染组体外侵袭能力是空白组的68.5%。而pcDNA3.1转染组是空白组的88.0%；pcDNA3.1-DAPKl转染组运动能力是空白组的87.3%。pcDNA3.1转染组是空白组的95.7%；pcDNA3.1-DAPKl转染组粘附能力是空白组的62.7%。pcDNA3.1转染组是空白组的91.2%；各组的胶原酶变化不明显；pcDNA3.1-DAPKl转染组p53基因表达升高。bcl-2基因下调。结论：DAPKl基因的过表达可以在-定程度上逆转PGCl3细胞的恶性表型。使PGCl3细胞生长受抑制。体外侵袭、运动和粘附能力下降。p53基因表达的上调及bcl-2基因表达下调。这些变化是DAPKl抑制非小细胞肺癌转移的可能原因。

目的：探索一种检测和纯化单链抗体的新方法。方法：使用不同的酶标记物，观察ELISA法检测培养上清液中单链抗体的相对灵敏度。含单链抗体的培养上清液经超滤、313gPL饱和硫酸铵沉淀及蛋白质L2琼脂糖亲和层析纯化单链抗体。结果：HRP标记蛋白质L可检测到1:16孔的阳性结果，而HRP标记蛋白质A仅检测到1:2孔的阳性结果。用9E10单抗检测培养上清液中的单链抗体时，除加入未经稀释培养上清液孔略有显色外，其他加入稀释后的培养上清液孔均不显色。蛋白质L亲和层析纯化的单链抗体经SDS2PAGE及ELISA鉴定，所得到的单链抗体纯度高、活性保持良好。结论：蛋白质L是一种能灵敏检测和有效纯化单链抗体的结合物，本实验建立的方法是一种有效、简便实用的方法。

目的：用反义核酸技术抑制小鼠B16黑色素瘤细胞酪氨酸酶基因（tyr）的表达。方法：构建tyr反义重组体pcDNA311（-）-tyr，用脂质体法导入B16细胞，用酪氨酸酶活性及黑色素含量测定，多巴染色及透射电镜等检测由反义重组体pcDNA311（-）-tyr转录产生的tyr反义核酸对B16细胞tyr表达的抑制情况。结果：成功构建了反义重组体pcDNA311（-）-tyr，转染了反义重组体的B16细胞其酪氨酸酶活性为0.0498±0.0036，显著低于对未转染组B16细胞的0.0916±0.0132（P<0.01）；转染空质粒pcDNA311（-）及真核表达重组体pcDNA311（+）-tyr组B16细胞的氨酸酶活性分别为0.1015±0.0166 和0.0948±0.0096，两者同对照组相比均无显著差异（P>0.05）。多巴染色及电镜观察结果表明转染了反义重组体的B16细胞其黑色素颗粒的含量明显低于对照组。结论：反义核酸可以显著抑制B16黑色素瘤细胞tyr的表达。