Nuclear orphan receptor TR3/Nur77 mediates melanoma cell apoptosis

TR3 was originally recognized for its role in the regulation of cell survival and differentiation, however, it was recently found to be a potent pro-apoptotic protein. In order to characterize the role of TR3 in melanoma cell apoptosis, we studied expression of TR3 in melanoma cell lines and tissues, its subcellular distribution and function during apoptosis using various expression and RNA interference vectors. We found that TR3 was constitutively expressed in both cultured melanoma cells and melanoma tissues. TR3 expression was significantly decreased in advanced melanomas comparing to benign nevi. Over-expression of wild type TR3 or mutant TR3 lacking the DNA binding domain resulted in massive apoptosis in melanoma cells, whereas stable knockdown of TR3 using RNA interference resulted in melanoma cell resistance to apoptosis induced by chemotherapeutic agents ATRA and cisplatin. We further demonstrated that apoptosis in melanoma cells was mediated, at least partially, through TR3 mitochondrial translocation but not alteration in TR3 expression levels. Our results suggest that TR3 is an important apoptosis inducing factor in melanoma cells. Decreased expression of TR3 in metastatic melanoma cells may contribute to their reduced apoptotic potential and increased resistance to chemotherapy.     

Effector mechanisms of norcantharidin-induced mitotic arrest and apoptosis in human hepatoma cells

NCTD is a demethylated form of cantharidin with antitumor properties, which is now in use as a routine anticancer drug against hepatoma. However, there is limited information on the effect of NCTD on human cancer cells. In the present study, NCTD inhibited proliferation, caused mitotic arrest, then progressed to apoptosis within 96 hr in 3 human hepatoma cell lines: HepG2, Hep3B and Huh-7. NCTD treatment (5 microg/ml) enhanced the expression of Cdc25C and p21(Cip1/Waf1), increasing the phosphorylation of these 2 proteins. In addition, NCTD treatment induced an earlier increase in cyclin B1-associated histone H1 kinase activity within 48 hr, but an approximately 70% reduction of both protein level and kinase activity of cyclin B1 was observed at 72 hr. Treatment with NCTD significantly decreased the expression of p53 protein but did not affect the expression of Cdk1 and p27(Kip1). Moreover, NCTD treatment also increased the phosphorylation of Bcl-2 and Bcl-X(L) but did not affect the expression of Bax or Bad. Bcl-2 phosphorylation appears to inhibit its binding to Bax since less Bax was detected in immunocomplex with Bcl-2 in NCTD-treated HepG2 cells. In addition, NCTD treatment caused activation of caspase-9 and caspase-3, preceding DNA fragmentation and morphologic features of apoptosis. Pretreatment with the broad-spectrum caspase inhibitor z-VAD-fmk markedly inhibited NCTD-induced caspase-3 activity and cell death. These results suggest that phosphorylation of p21(Cip1/Waf1) and Cdc25C and biphasic regulation of cyclin B1-associated kinase activity may contribute to NCTD-induced M-phase cell-cycle arrest. Furthermore, the increase of p21(Cip1/Waf1), phosphorylation of Bcl-2 and Bcl-X(L), activation of caspase-9 and caspase-3 may be the molecular mechanism through which NCTD induces apoptosis.     

Molecular mechanism of black tea polyphenols induced apoptosis in human skin cancer cells: involvement of Bax translocation and mitochondria mediated death cascade

Theaflavins (TF) and thearubigins (TR) are the most exclusive polyphenols of black tea. Even though few previous reports showed the anticancer effects of TF through apoptosis, the potential effect of TR has not been appraised. This study investigated the induction of apoptosis in human skin cancer cells after treatment of TF and TR. We report that both TF and TR could exert inhibition of A431 (human epidermoid carcinoma) and A375 (human malignant melanoma) cell proliferation without adversely affecting normal human epidermal keratinocyte cells. Growth inhibition of A375 cells occurred through apoptosis, as evident from cell cycle arrest at G(0)/G(1) phase, increase in early apoptotic cells, externalization of phosphatidylserine and DNA fragmentation. In our pursuit to dissect the molecular mechanism of TF- and TR-induced apoptosis in A375 cells, we investigated whether cell death is being mediated by mitochondria. In our system, Bax translocation to mitochondria persuaded depolarization of mitochondrial membrane potential, cytochrome c release in cytosol and induced activation of caspase-9, caspase-3 and poly (ADP-ribose) polymerase cleavage. Our intricate investigations on apoptosis also explained that TF and TR augmented Bax:Bcl2 ratio, up-regulated the expression of p53 as well as p21 and inhibited phosphorylation of the cell survival protein Akt. Furthermore, TF and TR elicited intracellular reactive oxygen species generation in A375 cells. These observations raise speculations that TF as well as TR might exert chemopreventive effect through cell cycle arrest and induction of apoptogenic signals via mitochondrial death cascade in human skin cancer cells.     

Norcantharidin: a potential antiangiogenic agent for gallbladder cancers in vitro and in vivo

Our objective was to explore the antiangiogenic activity of norcantharidin (NCTD) as an angiogenic inhibitor for gallbladder cancers. In vitro and in vivo experiments to determine the effects of NCTD on HUVECs, chicken CAM capillaries and gallbladder cancer xenograft angiogenesis in nude mice were respectively done. The MTT method was used to assay the cytotoxicity of NCTD on HUVECs. Immunofluorescence was used to evaluate HUVEC apoptosis. The scraping line method, matrigel invasion assay and tube formation assay were used to detect the migration, invasion and tube formation of HUVECs. A digital camera was used to observe chicken CAM capillaries. Experiments with NCTD in a xenograft model were used to observe the effect of NCTD on xenograft growth and survival of mice with xenografts. CD?? immunohistochemistry, flow cytometry and micro-MRA were used, respectively, to determine MVD, cell apoptosis and hemodynamic analysis of the xenografts. Immunohistochemistry and RT-PCR were used, respectively, to detect the expression of VEGF, Ang-2, TSP, TIMP-2 proteins/mRNAs of the xenografts. The xenograft MVD associated with tumor volume, the PCNA/apoptosis ratio and related-protein expression was evaluated simultaneously. We found that NCTD effectively inhibited the proliferation, migration, invasion and capillary-like tube formation of HUVECs in vitro; it reduced angiogenesis and directly destroyed the formed CAM capillaries in vivo. In the experiments in mice, NCTD not only inhibited significantly xenograft proliferation and growth, prolonged survival time of mice with xenografts, decreased the xenograft MVD and vascular perfusion, but also, similarly to ES, decreased significantly the expression of VEGF or Ang-2 protein/mRNA, increased the expression of TSP or TIMP-2 protein/mRNA. Moreover, the xenograft MVD was positively related with tumor volume, PCNA/apoptosis ratio, and VEGF or Ang-2 expression, respectively (all P<0.05), but negatively correlated with TSP or TIMP-2 expression (both P<0.05). These data showed that NCTD could serve as a potential antiangiogenic agent for gallbladder cancers.     

Norcantharidin inhibits proliferation and promotes apoptosis via c‐Met/Akt/mTOR pathway in human osteosarcoma cells

Osteosarcoma (OS) is the most common malignant bone tumor and frequently affects adolescents. Norcantharidin (NCTD), a demethylated derivative of cantharidin, has been reported to exhibit anticancer activity against various types of tumors but not human OS. The aim of the present study was to evaluate the effects of NCTD on OS cell lines (MG63 and HOS) and to explore the underlying mechanisms. In the present study, the proliferation of OS cells decreased significantly, while the apoptosis was accelerated significantly after exposure to NCTD. Meanwhile, our results also indicated that NCTD could suppress the migration and invasion, decrease the colony‐forming ability and induce S phase cell cycle arrest of OS cells in a dose‐dependent manner. Moreover, our results revealed that the anticancer effects induced by NCTD on OS cells involved autophagy, mitophagy, endoplasmic reticulum stress and c‐Met pathway. Furthermore, the results of animal experiments showed that NCTD inhibited tumor growth in a xenograft model of human OS. These results provide important new insight into the possible molecular mechanisms of NCTD and highlight its potential use as an antitumor drug for human OS.     

Norcantharidin inhibits tumor angiogenesis via blocking VEGFR2/MEK/ERK signaling pathways

Norcantharidin (NCTD), the demethylated form of Cantharidin, a reagent isolated from blister beetles, has been shown to be an anti‐tumor agent capable of inhibiting proliferation as well as inducing apoptosis in many cancer cell lines. However, little is known about the effect of NCTD in tumor angiogenesis. In this study, we demonstrated that NCTD inhibited vascular endothelial growth factor (VEGF)‐induced cell proliferation, migration, invasion, and capillary tube formation of primary human umbilical vein endothelial cells (HUVECs) in a dose‐dependent manner. Furthermore, we showed NCTD inhibited tumor growth and angiogenesis of colon cancer cells (LOVO) in vivo. We then mechanistically described that NCTD specifically abrogated the phosphorylation/activation of vascular endothelial growth factor receptor‐2 (VEGFR2)/MEK/ERK pathway kinases, with little effect on the phosphorylation of p38 MAPK and Akt, and on Cox‐2 expression. In summary, our results indicate that NCTD is a potential inhibitor of tumor angiogenesis by blocking VEGFR2/MEK/ERK signaling.     

Evodiamine,a constituent of Evodiae Fructus,induces anti-proliferating effects in tumor cells

We found that evodiamine, a major alkaloidal component of Evodiae Fructus (Goshuyu in Japan), inhibited proliferation of several tumor cell lines, but had less effect on human peripheral blood mononuclear cells (PBMC). We used human cervical cancer cells, HeLa, as a model to elucidate the molecular mechanisms of evodiamine-induced tumor cell death. The results showed that evodiamine induced oligonucleosomal fragmentation of DNA in HeLa cells and increased the activity of caspase-3, but not that of caspase-1, in vitro . Both evodiamine-induced DNA fragmentation and caspase-3 activity were effectively inhibited by a caspase-3 inhibitor, z-DEVD-fmk (z-Asp-Glu-Val-Asp-fmk). In addition, evodiamine increased the expression of the apoptosis inducer Bax, but decreased the expression of the apoptosis suppressor Bcl-2 in mitochondria. Taken together, our data indicated that evodiamine alters the balance of Bcl-2 and Bax gene expression and induces apoptosis through the caspase pathway in HeLa cells. (Cancer Sci 2003; 94: 92–98)     

Tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis of human melanoma is regulated by smac/DIABLO release from mitochondria

In previous studies we have shown that the sensitivity of melanoma cell lines to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis was determined largely by the level of expression of death receptor TRAIL receptor 2 on the cells. However, approximately one-third of melanoma cell lines were resistant to TRAIL, despite expression of high levels of TRAIL receptor 2. The present studies show that these cell lines had similar levels of TRAIL-induced activated caspase-3 as the TRAIL-sensitive lines, but the activated caspase-3 did not degrade substrates downstream of caspase-3 [inhibitor of caspase-activated DNase and poly(ADP-ribose) polymerase]. This appeared to be due to inhibition of caspase-3 by X-linked inhibitor of apoptosis (XIAP) because XIAP was bound to activated caspase-3, and transfection of XIAP into TRAIL-sensitive cell lines resulted in similar inhibition of TRAIL-induced apoptosis. Conversely, reduction of XIAP levels by overexpression of Smac/DIABLO in the TRAIL-resistant melanoma cells was associated with the appearance of catalytic activity by caspase-3 and increased TRAIL-induced apoptosis. TRAIL was shown to cause release of Smac/DIABLO from mitochondria, but this release was greater in TRAIL-sensitive cell lines than in TRAIL-resistant cell lines and was associated with down-regulation of XIAP levels. Furthermore, inhibition of Smac/DIABLO release by overexpression of Bcl-2 inhibited down-regulation of XIAP levels. These results suggest that Smac/DIABLO release from mitochondria and its binding to XIAP are an alternative pathway by which TRAIL induces apoptosis of melanoma, and this pathway is dependent on the release of activated caspase-3 from inhibition by XIAP and possibly other inhibitor of apoptosis family members.     

Inhibiting systemic autophagy during interleukin 2 immunotherapy promotes long-term tumor regression

Administration of high-dose interleukin-2 (HDIL-2) has durable antitumor effects in 5% to 10% of patients with melanoma and renal cell carcinoma. However, treatment is often limited by side effects, including reversible, multiorgan dysfunction characterized by a cytokine-induced systemic autophagic syndrome. Here, we hypothesized that the autophagy inhibitor chloroquine would enhance IL-2 immunotherapeutic efficacy and limit toxicity. In an advanced murine metastatic liver tumor model, IL-2 inhibited tumor growth in a dose-dependent fashion. These antitumor effects were significantly enhanced upon addition of chloroquine. The combination of IL-2 with chloroquine increased long-term survival, decreased toxicity associated with vascular leakage, and enhanced immune cell proliferation and infiltration in the liver and spleen. HDIL-2 alone increased serum levels of HMGB1, IFN-γ, IL-6, and IL-18 and also induced autophagy within the liver and translocation of HMGB1 from the nucleus to the cytosol in hepatocytes, effects that were inhibited by combined administration with chloroquine. In tumor cells, chloroquine increased autophagic vacuoles and LC3-II levels inhibited oxidative phosphorylation and ATP production and promoted apoptosis, which was associated with increased Annexin-V(+)/propidium iodide (PI)(-) cells, cleaved PARP, cleaved caspase-3, and cytochrome c release from mitochondria. Taken together, our findings provide a novel clinical strategy to enhance the efficacy of HDIL-2 immunotherapy for patients with cancer.     

Norcantharidin-induced apoptosis in oral cancer cells is associated with an increase of proapoptotic to antiapoptotic protein ratio

Norcantharidin (NCTD), the demethylated analogue of cantharidin, has been used to treat human cancers in China since 1984. It was recently found to be capable of inducing apoptosis in human colon carcinoma, hepatoma and glioblastoma cells by way of an elusive mechanism. In this study, we demonstrated that NCTD also induces apoptosis in human oral cancer cell lines SAS (p53 wild-type phenotype) and Ca9-22 (p53 mutant) as evidenced by nuclear condensation, TUNEL labeling, DNA fragmentation and cleavage of PARP. Apoptosis induced by NCTD was both dose- and time-dependent. We found NCTD did not induce Fas and FasL, implying that it activated other apoptosis pathways. Our data showed that NCTD caused accumulation of cytosolic cytochrome c and activation of caspase-9, suggesting that apoptosis occurred via the mitochondria mediated pathway. NCTD enhanced the expression of Bax in SAS cells consistent with their p53 status. Moreover, we showed that NCTD downregulated the expression of Bcl-2 in Ca9-22 and Bcl-XL in SAS. Our results suggest that NCTD-induced apoptosis in oral cancer cells may be mediated by an increase in the ratios of proapoptotic to antiapoptotic proteins. Since oral cancer cells with mutant p53 or elevated Bcl-XL levels showed resistance to multiple chemotherapeutic agents, NCTD may overcome the chemoresistance of these cells and provide potential new avenues for treatment.     

去甲斑蝥素诱导小鼠肺纤维瘤L929细胞凋亡

目的：研究去甲斑蝥素(norcantharidin，NCTD)抑制小鼠肺纤维瘤(L929)细胞的作用机制。方法：用MTT法测定细胞生长抑制率。采用Hoechst 33258染色、DNA凝胶电泳和乳酸脱氢酶(LDH)活力检测细胞凋亡。用免疫印迹法(Western blot)分析了细胞外信号调节激酶(extracellular signal-regulated kinase，ERK)和磷酸化ERK、c-Jun N-末端激酶(C-Jun N-terminal kinase，JNK)和磷酸化JNK蛋白表达的变化。结果：NCTD诱导L929细胞凋亡。JNK抑制剂(SP600125)与ERK抑制剂(PD98059)，可明显抑制NCTD对细胞的杀伤作用。同时磷酸化ERK和磷酸化JNK表达增加。结论：NCTD对L929细胞的细胞毒作用是通过诱导其凋亡而发生的，且呈时间剂量依赖性。这种作用可能与JNK，ERK通路激活有关。     

没食子酸诱导ROS积蓄介导黑色素瘤B16-F10细胞内源性凋亡及周期阻滞北大核心

目的:探究没食子酸诱导活性氧(reactive oxygen species,ROS)积蓄介导黑色素瘤B16-F10细胞凋亡及周期阻滞的作用机制。方法:以梯度浓度的没食子酸作用于黑色素瘤B16-F10细胞,采用MTT法检测没食子酸对细胞生长的影响,平板克隆技术检测细胞克隆形成率,Transwell实验检测细胞迁移及侵袭能力,ROS Assay Kit检测B16-F10细胞内ROS水平,线粒体膜电位检测试剂盒检测细胞膜电位的变化,Hoechst 33258荧光染色法进行细胞形态学检测,流式细胞术检测细胞凋亡及细胞周期阻滞水平,Western blot检测细胞内相关蛋白水平的变化。结果:结果显示,没食子酸明显抑制B16-F10细胞的生长,且具有浓度依赖性。没食子酸作用后,B16-F10细胞增殖、迁移及侵袭能力明显下降,细胞内ROS水平明显升高,线粒体膜电位明显下降;细胞数减少,细胞凋亡率增加,G_(0)/G_(1)期细胞数明显增多;细胞内凋亡相关蛋白Bax、Cytochrome C、Caspase-9以及Caspase-3表达量增加,而Bcl-2表达量减少;周期相关蛋白Chk2、p53、p21表达量明显增加,CyclinE1、CDK2表达量减少。结论:没食子酸作用于黑色素瘤B16-F10细胞后,通过诱导ROS积蓄,引起B16-F10细胞内源性凋亡及周期阻滞。     

Taurolidine induces apoptosis of murine melanoma cells in vitro and in vivo by modulation of the Bcl-2 family proteins

BACKGROUND: This study evaluates whether taurolidine, a novel antibiotic agent, induces murine melanoma cell apoptosis in vitro and in vivo. METHODS: Murine melanoma cells (B16 4A5 and B16 F10) were treated with taurolidine (0-100 microM) for 12 and 24 hr. Cell viability and apoptosis were assessed by MTT assay and FACScan analysis. Expression of the Bcl-2 family proteins was detected by Western blot analysis. In vivo, taurolidine-induced anti-tumor cytotoxicity was assessed in C57BL/6 mice. Therapeutic effectiveness, by intraperitoneal injection of taurolidine (15 mg/mouse) on alternate days for 2 weeks, was evaluated in mice bearing B16 4A5 tumor xenografts. Primary and metastatic tumor growth and intra-tumor apoptotic index were measured. RESULTS: Taurolidine induced cell apoptosis and reduced cell viability in murine melanoma cells. The pro-apoptotic protein Bax was enhanced, whereas the anti-apoptotic protein Bcl-2 was inhibited by taurolidine treatment. In vivo, systemic injection of 15-mg taurolidine was identified as the maximally tolerated dose. Administration of taurolidine at 15 mg/mouse significantly inhibited primary and metastatic tumor growth, which was mirrored by a significantly increased intra-tumor apoptotic index. CONCLUSIONS: These results demonstrate that taurolidine significantly attenuated melanoma tumor growth, which may result from taurolidine-induced apoptosis by modulation of the Bcl-2 family proteins.     

Pyrimethamine sensitizes pituitary adenomas cells to temozolomide through cathepsin B‐dependent and caspase‐dependent apoptotic pathways

Invasive pituitary adenomas (PAs) are generally refractory to conventional therapy and salvage treatment with temozolomide (TMZ). In addition to antiprotozoan effects, pyrimethamine (PYR) has recently shown its strong antitumor activity as an antineoplastic agent or in combination with TMZ in metastatic melanoma cells. In this study, the effects of TMZ, PYR or TMZ/PYR combination on rat/mouse PA cell lines αT3‐1, GH3, MMQ and ATt‐20 as well as GH3 xenograft tumor model were evaluated. TMZ/PYR combination synergistically inhibited proliferation, invasion and induced apoptosis of these PA cell lines in vitro. Strikingly, combination treatment with TMZ and PYR produced synergistic antitumor activity and enhanced the survival rate of GH3 xenograft tumor models without increasing systemic side effects. In addition, TMZ/PYR induced cell cycle arrest, increased DNA damage, upregulated the expression of cathepsin B, BAX, cleaved PARP and phosphorylated histone H2AX as well as elevated caspase3/7, 8 and 9 activities. The decreased expression of Bcl‐2, MMP‐2 and MMP‐9 alone with cytochrome c release from mitochondria into the cytosol was also observed in the TMZ/PYR combination group. The increase in cell apoptosis due to combination with PYR was rescued by leucovorin. These data suggest that PYR may enhance the efficacy of TMZ via triggering both cathepsin B‐dependent and caspase‐dependent apoptotic pathways. Therefore, combination of PYR and TMZ may provide a novel regimen for invasive PAs refractory to standard therapy and TMZ.     

Preferential induction of apoptosis by interferon (IFN)-beta compared with IFN-alpha2: correlation with TRAIL/Apo2L induction in melanoma cell lines.

On the basis of in vitro inhibition of tumor cell growth, IFNs have been generally considered to be antiproliferative proteins. To probe further the potential mechanisms of the antitumor effects of IFNs, we have assessed apoptosis in response to IFN-alpha2 and IFN-beta in cell lines of varied histologies, with a focus on melanomas. Many of the cell lines tested underwent apoptosis in response to IFN-beta, as assessed both by Annexin V and terminal deoxynucleotidyl transferase-mediated nick end labeling staining. In general, IFN-beta had greater growth inhibitory and proapoptotic effects than IFN-alpha2 on all cell lines. The melanoma cell line WM9, sensitive to growth inhibition by IFNs, had a greater degree of apoptosis than A375 melanoma cells, which were largely resistant to antigrowth effects of IFNs. IFN-beta-induced apoptosis was dependent on activation of the caspase cascade with cleavage of caspases 3, 8, and 9 and of the caspase 3 substrate, poly(ADP-ribose) polymerase. Caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl keton or benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethyl keton, inhibited IFN-beta-induced apoptosis. Other changes associated with apoptosis, including the movement of cytochrome c from mitochondria to cytoplasm and DNA fragmentation, were also identified in response to IFN-beta. Apo2L ligand [tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)] was one of the early genes induced by IFN-beta in apoptosis-sensitive WM9 cells. Other sensitive melanoma cell lines had a similar IFN-beta-specific induction of TRAIL. Neutralizing antibody to TRAIL inhibited IFN-beta-induced apoptosis in WM9 cells. In resistant A375 cells, IFN-beta did not induce TRAIL/Apo2L expression. Thus, induction of TRAIL by IFNs in some tumor types may initiate the apoptotic cascade. This study offers another mechanism for the antitumor effects of IFNs.     

RPS3 regulates melanoma cell growth and apoptosis by targeting Cyto C/Ca2+/MICU1 dependent mitochondrial signaling

Melanoma is one of the most aggressive and lethal cancers. Discovery and identification of novel therapeutic targets is urgently needed. In this study, we demonstrated that ribosomal protein S3 (RPS3) was a potential target involved in melanoma growth. Knockdown of RPS3 by siRNA suppressed cell growth and induced apoptosis in melanoma cells. Further mechanism studies showed that RPS3 knockdown in melanoma cells triggered the release of cytochrome C (Cyto C) from mitochondrial, increased the location of BID on mitochondrial membrane and the cleavage of the pro-apoptotic proteins (PARP, caspase-3 and -9), promoted the opening of mitochondrial permeability transition pore and the flooding of calcium ions (Ca²⁺) into the mitochondrial, and decreased the expression of the Ca²⁺ gatekeeper MICU1 and its location on the mitochondrial. We also found that knockdown of RPS3 significantly inhibited tumor growth in a melanoma xenograft mouse model. Furthermore, we showed that RPS3 was highly expressed in melanoma cell lines and melanoma tumor tissues, and overexpression of RPS3 was associated with the poor prognosis of melanoma patients. Our results therefore demonstrate that RPS3 regulates melanoma growth through the modulation of the Cyto C/Ca²⁺/MICU1 dependent mitochondrial signaling and suggest that RPS3 is a potential therapeutic target for melanoma treatment.     

ACTIBIND, a T2 RNase, competes with angiogenin and inhibits human melanoma growth, angiogenesis, and metastasis

Melanoma is a very aggressive and highly angiogenic tumor in which standard treatments have had only limited success. Patients with advanced disease have a 5-year survival rate of 5%. In search for alternatives, we identified a natural product extracted from the fungus Aspergillus niger, termed ACTIBIND, that inhibits tumor growth and metastasis of melanoma in vivo. ACTIBIND, a T2 RNase, exerts antitumorigenic and antiangiogenic activities by competing with the angiogenic factor angiogenin (itself an RNase homologue). Thus, there was decreased expression and activity of the matrix metalloproteinase 2 in melanoma and vascular endothelial cells, decreased vascularization, and increased tumor cell apoptosis in vivo. ACTIBIND significantly inhibited angiogenesis in an in vivo angiogenesis assay with sponges containing angiogenin. In vitro, ACTIBIND was internalized by both melanoma and human umbilical vein endothelial cells, reached the cell nuclei, and inhibited the activity of angiogenin response elements in a dose-dependent manner. Collectively, our data indicate that ACTIBIND should be tested for its potential as a new antiangiogenic modality for the treatment of melanoma.     

去甲斑蝥素对人类胃癌SGC-7901细胞凋亡相关基因表达的影响

 目的 探讨去甲斑蝥素（NCTD）对人类胃癌SGC-7901细胞凋亡相关基因表达的影响。方法 实验分对照组、5-Fu组、NCTD 组和5-Fu+NCTD 组，分别应用MTT、流式细胞术、免疫组化SABC法检测NCTD 对体外培养胃癌SGC-7901细胞的杀伤抑制率、凋亡率和survivin、bcl-2、caspase-3的表达。结果　NCTD对人类胃癌SGC-7901 细胞生长有抑制作用，呈剂量- 时间效应关系；凋亡率由（8.30±1.49）%上升到（20.56±1.32）%；survivin基因表达由（86.57±4.39）%下降到（26.11±2.27）%；bcl-2基因表达由（85.35±3.25）%下降到（30.26±1.83）%；caspase-3基因表达由（54.49±3.07）%上升到（92.78±2.47）%；与5-Fu联合应用上述作用更明显。结论 NCTD能抑制人类胃癌SGC-7901细胞的生长；与5-Fu具有协同作用，其机制可能与其诱导细胞凋亡，影响凋亡相关基因表达有关。     

RNAi silencing of the WT1 gene inhibits cell proliferation and induces apoptosis in the B16F10 murine melanoma cell line

The Wilms' tumor protein 1 (WT1) is essential for tumor cell proliferation and is highly expressed in various hematological and solid malignancies including human malignant melanoma. We investigated whether WT1 expression is essential for growth in the B16F10 murine melanoma cell line. Toward this end, we examined WT1 protein expression and WT1 isoforms (17AA+/17AA-, KTS+/KTS-) in this cell line. WT1 was silenced by two RNA interference constructs, designated WT1-1 and WT1-2. RNA interference-mediated reduction of WT1 protein expression significantly inhibited B16F10 cell viability. Loss of WT1 also increased caspase-3 and poly-ADP-ribose polymerase activation, as well as apoptotic body formation, chromatin condensation, and DNA fragmentation. Together, these findings implicate decreased WT1 protein levels in the induction of apoptosis. These results imply that WT1 plays a distinct role in B16F10 melanoma growth.