Clodronate inhibits angiogenesis in vitro and in vivo

The effects of amino-bisphosphonate clodronate on endothelial cell functions involved in angiogenesis, namely proliferation and morphogenesis on matrigel were tested in vitro, whereas its effects on angiogenesis were studied in vivo. This was performed by using the chick embryo chorioallantoic membrane (CAM) assay. In vitro, clodronate inhibited the endothelial cell proliferation in a dose-dependent fashion, peaking at 30 microM. At the same concentration, clodronate inhibited the fibroblast growth factor-2 (FGF-2)-induced capillary-like tube formation in the morphogenesis assay on matrigel. In vivo, when tested with the CAM assay, clodronate again displayed the capability to inhibit FGF-2-induced angiogenesis. Overall, these results suggest that antiangiogenesis by clodronate can be used to treat a wide spectrum of angiogenesis-dependent diseases, including certain chronic inflammatory diseases and cancer.     

Capsaicin inhibits in vitro and in vivo angiogenesis

Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide), a natural product of Capsicum species, is known to induce excitation of nociceptive terminals involved in pain perception. Recent studies have also shown that capsaicin not only has chemopreventive properties against certain carcinogens and mutagens but also exerts anticancer activity. Here, we demonstrated the antiangiogenic activity of capsaicin using in vitro and in vivo assay systems. In vitro, capsaicin inhibited vascular endothelial growth factor (VEGF) -induced proliferation, DNA synthesis, chemotactic motility, and capillary-like tube formation of primary cultured human endothelial cells. Capsaicin inhibited both VEGF-induced vessel sprouting in rat aortic ring assay and VEGF-induced vessel formation in the mouse Matrigel plug assay. Moreover, capsaicin was able to suppress tumor-induced angiogenesis in chick chorioallantoic membrane assay. Capsaicin caused G(1) arrest in endothelial cells. This effect correlated with the down-regulation of the expression of cyclin D1 that led to inhibition of cyclin-dependent kinase 4-mediated phosphorylation of retinoblastoma protein. Signaling experiments show that capsaicin inhibits VEGF-induced p38 mitogen-activated protein kinase, p125(FAK), and AKT activation, but its molecular target is distinct from the VEGF receptor KDR/Flk-1. Taken together, these results demonstrate that capsaicin is a novel inhibitor of angiogenesis and suggest that it may be valuable to develop pharmaceutical drugs for treatment of angiogenesis-dependent human diseases such as tumors.     

Ellagitannin-rich pomegranate extract inhibits angiogenesis in prostate cancer in vitro and in vivo

Angiogenesis is critical to tumor growth and is stimulated by tissue hypoxia due to poor oxygen delivery. In turn, cellular hypoxia leads to angiogenesis via the induction of hypoxia-inducible factor-1alpha (HIF-1alpha) and vascular endothelial growth factor (VEGF) at a cellular level. Pomegranate juice and extracts, which are rich sources of ellagitannins, have been shown to have chemopreventive potential against prostate cancer, but there have been no studies on the effects of an ellagitannin-rich pomegranate extract on angiogenesis. Human prostate cancer cells (LNCaP) and human umbilical vein endothelial cells (HUVEC) were incubated with a pomegranate extract standardized to ellagitannin content (POMx), under normoxic and hypoxic conditions in vitro. Human prostate cancer cells (LAPC4) were injected subcutaneously into severe combined immunodeficient (SCID) mice and the effects of oral administration of POMx on tumor growth, microvessel density, and HIF-1alpha and VEGF expression were determined after 4 weeks of treatment. POMx inhibited the proliferation of LNCaP and HUVEC cells significantly under both normoxic and hypoxic conditions. HIF-1alpha and VEGF protein levels were also reduced by POMx under hypoxic conditions. POMx decreased prostate cancer xenograft size, tumor vessel density, VEGF peptide levels and HIF-1alpha expression after 4 weeks of treatment in SCID mice. These results demonstrate that an ellagitannin-rich pomegranate extract can inhibit tumor-associated angiogenesis as one of several potential mechanisms for slowing the growth of prostate cancer in chemopreventive applications. Further studies in humans are needed to confirm that angiogenesis can be inhibited by an ellagitannin-rich pomegranate extract administered orally as a dietary supplement.     

Downregulation of heparanase by RNA interference inhibits invasion and tumorigenesis of hepatocellular cancer cells in vitro and in vivo

Heparanase is an endoglycosidase that degrades heparan sulfate, the main polysaccharide constituent of the extracellular matrix and basement membrane. The expression of heparanase is associated with invasion, as well as the angiogenic and metastatic potential of diverse malignant tumors. We used RNA interference strategies to evaluate the role of human heparanase in a liver cancer cell line and to explore the therapeutic potential of its specific targeting. Using an online siRNA tool, we designed three small interfering RNA sequences to target the heparanase coding region and cloned them into the pGenesil-1 vector. The siRNA vectors were transfected into HepG2 liver cancer cells. Heparanase expression was measured by real-time RT-PCR and Western blotting. Cell proliferation was detected by MTT staining and plate colony formation. Cell cycle analysis was performed by flow cytometry. In vitro invasion was measured by Matrigel invasion assay. We also analyzed tumorigenicity in heparanase-suppressed HepG2 cells in nude mice. We found that siRNA-1 (1214-1232) and siRNA-3 (611-629) targeting heparanase significantly downregulated the expression of heparanase in HepG2 liver cancer cells. Compared with its controls, siRNA-1 or siRNA-3 vectors efficiently inhibi-ted the proliferation and invasion of HepG2 liver cancer cells in?vitro and tumorigenesis in vivo. These results suggest that heparanase-specific RNA interference has potential value as a novel therapeutic agent for human liver cancer.     

Stable RNA interference of hexokinase II gene inhibits human colon cancer LoVo cell growth in vitro and in vivo

The purpose of this study is to investigate the effect of silencing hexokinase II (HK II) gene with RNA interference (RNAi) technique on colon cancer LoVo cell proliferation in vitro and in vivo. A short hairpin RNA (shRNA) eukaryotic expression vector against HK II gene was constructed, named as plasmid pGenesil-1-HK II, and transfected into LoVo cells. The expression of HK II gene was detected by RT-PCR and Western blot analysis respectively. Then, tumor colony formation was observed, and cell cycle was also assessed by flow cytometry and the contents of intracellular adenosine triphosphate (ATP) by high performance liquid chromatography (HPLC). Furthermore, LoVo cells were injected subcutaneously into nude mice. After a 4-week follow-up period, the sizes and weights of tumors were measured. Moreover, the expression of Ki67 protein was observed by immunohistochemical technique, and cell apoptosis by terminal deoxynucleotidyl transferase mediated nick end labeling (TUNEL). Consequently, the expression of HK II gene was efficiently blocked by RNAi. Downregulation of HK II gene expression significantly suppressed cloning efficiency and cell cycle of LoVo cell in vitro and tumor growth in vivo. Compared with untransfected LoVo cells, LoVo cells transfected with pGenesil-1-HK II plasmids showed significant decrease in the cellular ATP contents and Ki67 expression, and obvious increase in the apoptosis indexes. Our results suggest that HK II gene can act as a crucial therapeutic target for slowing colon cancer growth.     

Knockdown of radixin by RNA interference suppresses the growth of human pancreatic cancer cells in vitro and in vivo

Radixin, encoded by a gene on chromosome 11, plays important roles in cell motility, invasion and tumor progression. However, its function in pancreatic cancer remains elusive. In this study, radixin gene expression was suppressed with a lentivirus-mediated short-hairpin RNA (shRNA) method. We found that radixin shRNA caused down-regulation of radixin in PANC-1 cells, associated with inhibition of pancreatic cancer cell proliferation, survival, adhesion and invasive potential in vitro. When radixin-silenced cells were implanted in nude mice, tumor growth and microvessel density were significantly inhibited as compared to blank control cells or nonsense shRNA control cells. Thrombospondin-1 (TSP-1) and E-cadherin were up-regulated in radixin-silenced PANC-1 cells. Our results suggest that radixin might play a critical role in pancreatic cancer progression, possibly through involvement of down-regulation of TSP-1 and E-cadherin expression.     

Phenethyl isothiocyanate inhibits angiogenesis in vitro and ex vivo

Previous studies, including those from our laboratory, have revealed that phenethyl isothiocyanate (PEITC), a constituent of many edible cruciferous vegetables, not only affords significant protection against chemically induced cancer in animal models but also inhibits growth of cancer cells in culture and in vivo by causing cell cycle arrest and apoptosis induction. We now report a novel response to PEITC involving inhibition of angiogenesis in vitro and ex vivo at pharmacologically achievable concentrations. The PEITC treatment caused a decrease in survival of human umbilical vein endothelial cells (HUVEC) in a concentration- and time-dependent manner. The capillary-like tube structure formation (in vitro neovascularization) and migration (invasion potential) by HUVEC was also inhibited significantly in the presence of PEITC at pharmacologically relevant concentrations (<1 mumol/L). The PEITC-mediated inhibition of angiogenic features of HUVEC in vitro was associated with suppression of vascular endothelial growth factor (VEGF) secretion, down-regulation of VEGF receptor 2 protein levels, and inactivation of prosurvival serine-threonine kinase Akt. The PEITC treatment reduced migration by PC-3 human prostate cancer cells, which correlated with inactivation of Akt and suppression of VEGF, epidermal growth factor (EGF), and granulocyte colony-stimulating factor (G-CSF) secretion. The PEITC-mediated inhibition of PC-3 cell migration was statistically significantly attenuated by ectopic expression of constitutively active Akt. Most importantly, PEITC treatment inhibited ex vivo angiogenesis as revealed by chicken egg chorioallantoic membrane assay. In conclusion, the present study suggests that inhibition of angiogenesis may be an important mechanism in cancer chemoprevention by PEITC.     

RNAi沉默rictor基因表达抑制人膀胱癌T24细胞体内外生长

背景与目的：膀胱癌是泌尿系统高发肿瘤，浅表性膀胱癌发展成为浸润性膀胱癌后，预后较差。该研究利用RNA干扰（RNA interference，RNAi）技术沉默rictor基因后，观察其对人膀胱癌T24细胞在体内外生长的影响。方法：构建3个rictor-RNAi表达载体（1＃、2＃和3＃）和1个阴性对照载体。采用瞬时转染和稳定转染技术筛选出干扰效果明显的单克隆稳定细胞株。应用细胞计数试剂盒（cell counting kit-8，CCK-8）实验、细胞划痕试验、流式细胞术（flow cytometry，FCM）、裸鼠皮下移植瘤模型研究抑制rictor基因后对T24细胞生长的影响。结果：成功构建rictor-RNAi表达载体和阴性对照载体，瞬时转染T24细胞后，2＃蛋白表达量下降最明显。利用2＃载体稳定转染T24细胞，筛选2＃-1、2＃-2两株单克隆细胞。2＃-1组和2＃-2组细胞较空白组细胞生长减慢（P<0.05）。细胞划痕实验表明，2＃-1组和2＃-2组细胞的迁移速度也较空白组明显降低（P<0.05）。与空白组和阴性对照组相比，2＃-1组和2＃-2组G₁期细胞比例增加（P<0.05），细胞死亡率增加（P<0.05）。裸鼠皮下移植瘤模型显示，移植瘤的平均体积，2#-1组和2#-2组明显低于空白组和阴性对照组（P<0.001）。结论：成功构建rictor-RNAi稳定表达载体，可有效抑制rictor基因表达。抑制rictor基因表达能显著抑制T24细胞增殖，促进肿瘤细胞死亡，诱使细胞停滞在G₁期，并能有效抑制裸鼠移植瘤的生长。RNAi抑制rictor基因的治疗策略可能为膀胱癌的治疗提供新的思路。     

Knockdown of STAT3 expression by RNA interference inhibits the induction of breast tumors in immunocompetent mice

Constitutively activated STAT3 is involved in the formation of multiple types of tumors including breast cancer. We examined the effects of Stat3 protein knockdown by RNA interference using a dicistronic lentivirus small hairpin (shRNA) delivery system on the growth of mammary tumors in BALB/c mice induced by the 4T1 cell line. A single exposure of 4T1 cells to shRNA/STAT3 lentivirus transduced 75% of the cells with green fluorescent protein (GFP) within 96 hours. In cells selected for GFP expression, neither Stat3 protein nor phosphotyrosine Stat3 was detected. Tumor formation induced by injecting 4T1 cells into the mammary fat pad was blocked by expression of the shRNA for STAT3 whereas all mice injected with 4T1 cells expressing only GFP efficiently formed tumors. c-Myc expression was reduced 75% in cells expressing greatly reduced levels of Stat3 compared with the GFP control. Of interest, the level of activated Src, which is known to activate Stat3, was virtually eliminated but the level of the Src protein itself remained the same. Importantly, expression of Twist protein, a metastatic regulator, was eliminated in STAT3 knockdown cells. Invasion activity of STAT3 knockdown cells was strongly inhibited. However, the proliferation rate of cells in Stat3 knockdown cells was similar to that of the GFP control; the cell cycle was also not affected. We conclude from these studies that activated Stat3 protein plays a critical role in the induction of breast tumors induced by 4T1 cells by enhancing the expression of several important genes including c-Myc and the metastatic regulator Twist. These studies suggest that stable expression of small interfering RNA for STAT3 has potential as a therapeutic strategy for breast cancer.     

ShRNA silencing glycogen synthase kinase-3 beta inhibits tumor growth and angiogenesis in pancreatic cancer

Glycogen synthase kinase-3 beta (GSK-3β), a serine/threonine protein kinase, plays a vital role in the tumorigenesis of many cancers, but its role in pancreatic cancer remains unknown. In this study, we showed that GSK-3β was aberrantly activated in pancreatic cancer. GSK-3β knockdown resulted in arrested proliferation and increased apoptosis in pancreatic cancer cell lines. Expression of Bcl-2 and vascular endothelial growth factor (VEGF) decreased significantly in a GSK-3β knockdown group. In a xenograft tumor model, GSK-3β knockdown inhibited tumor growth and angiogenesis. Our study showed that GSK-3β may become a promising therapeutic target for human pancreatic cancer.     

Knockdown of RAGE expression inhibits colorectal cancer cell invasion and suppresses angiogenesis in vitro and in vivo

The receptor for advanced glycation end-products (RAGE) is a transmembrane receptor in cells, and the interaction of RAGE with ligands results in pro-inflammatory gene activation. Aberrant RAGE activation was reported to promote the pathogenesis of colorectal cancer. This study aimed to investigate the effects of RAGE on the regulation of cell viability, invasion, and angiogenesis, as well as the underlying molecular mechanisms regulating these interactions in colorectal cancer cells.The RAGE mRNA and protein were evaluated in five colorectal cancer cell lines and in 45 cases of colorectal cancer tissue specimens (using immuohistochemistry). RAGE expression was then knockdown using RAGE shRNA for assessing cell viability and invasion assays as well as for tube formation and CAM assays in human umbilical vein endothelial cells and chick embryos, respectively.RAGE was highly expressed in colorectal cancer tissues, and was associated with increased microvessel density. Two of the four RAGE shRNA constructs were able to significantly knockdown RAGE expression in SW480 cells. RAGE knockdown inhibited invasion capacity of SW480 cells, but did not significantly affect cell viability. Furthermore, the conditioned growth medium from stable RAGE shRNA-transfected cells suppressed tube formation of human umbilical vein endothelial cells and angiogenesis of chicken embryos. Knockdown of RAGE inhibited expression of VEGF and SP1 protein in colorectal cancer cells.In summary, these data suggest that silence of RAGE expression could effectively inhibit colorectal cancer angiogenesis in vitro and in vivo.     

The quinoline-3-carboxamide Linomide inhibits angiogenesis in vivo

Linomide (Roquinimex) has antitumor activity when given in vivo (but not when applied in vitro) that has been attributed to immune host mechanisms. Recent studies, however, suggest that Linomide may also possess anti-angiogenic properties. The aim of the present study was to evaluate the antiangiogenic effect of Linomide using an intravital microscopic technique. Syngeneic pancreatic islets were isolated and implanted into the dorsal skinfold chamber of Syrian golden hamsters. This model allows detailed repeated in vivo observations and quantitative analysis of revascularization of pancreatic islet grafts. The neovascularization process of the islets is a highly reproducible phenomenon that is completed within about 2 weeks, resulting in a microvascular network very similar to that of islets in situ. The plasma concentration profile of Linomide following a single oral dose of the compound was determined. The elimination of Linomide was fast, the half-life being 2.6±0.2 h. Due to the short half-life, the hamsters were given Linomide twice a day. One group of animals (n=9) was force-fed Linomide (100 mg/kg per day) from the day of implantation throughout the 2-week observation period, and the results were compared with those obtained in a nontreated control group (n=7). At days 6, 10, and 14 after implantation, the neo-vasculature of the islets was examined. In the control group, 91%±4% (mean ± SEM) of the islets showed the first signs of angiogenesis at day 6, whereas in the Linomidetreated group the corresponding value was 48%±12%. At days 10 and 14, the take-rate in the control group increased to 94%±3% for day 0 and to 94%±4% (n=6) for day 14, whereas in the treated group the corresponding take-rate was 67%±11% and 72%±12%, respectively. The functional capillary density in the control group at days 6, 10, and 14 was 223±17, 348±29, and 495±29 cm^–1, respectively, and than in the Linomide treated group was 91±28, 181±43, and 229±47 cm^–1, respectively. These results demonstrate that Linomide suppresses the neovascularization of the islet grafts by both delaying the onset of and reducing the percentage of islets displaying angiogenesis as well as by decreasing the rate of proliferation of capillary endothelium of the revascularized islets.     

RNA interference remarkably suppresses bcl-2 gene expression in cancer cells in vitro and in vivo

Bcl-2 is an anti-apoptotic protein. If the level of Bcl-2 protein can be reduced sufficiently in tumors using RNA interference (RNAi) to target the gene message, the apoptosis of tumor cells may be promoted. In this study, we synthesized 19 nucleotides (nts) small interference RNA (siRNA) constructs suppressing bcl-2 gene expression in human tumor cells (HeLaB2 and BGC-823 cell lines) in vitro. The bcl-2 gene expression levels were significantly reduced when these siRNA were transfected into experimental two tumor cells for 72 hours. The apoptosis process was also examined in the tumor cells. Here we synthesized siRNA from a DNA template under the control of the RNA polymerase III promoter in transfected tumor cells. Using this DNA vector-based approach, we found that the siRNA efficiently and specifically inhibited the synthesis of protein encoded by the bcl-2 gene in HeLaB2 and BGC-823 tumor cells. Tumor growth was inhibited by 66.5% with 2mg/kg pSilencer 3.1H1-bcl-2 in mouse liver tumor-bearing BALB/c mice. This approach may prove to be a valuable clinical technique for the analysis of specific gene functions and gene therapy of malignant tumors that utilize the bcl-2 gene via RNA interference.     

Plumbagin inhibits tumorigenesis and angiogenesis of ovarian cancer cells in vivo

Angiogenesis is a hallmark of tumor development and metastatic progression, and anti‐angiogenic drugs targeting the VEGF pathway have shown to decrease the disease progression in cancer patients. In this study, we have analyzed the anti‐proliferative and anti‐angiogenic property of plumbagin in cisplatin sensitive, BRCA2 deficient, PEO‐1 and cisplatin resistant, BRCA2 proficient PEO‐4 ovarian cancer cells. Both PEO‐1 and PEO‐4 ovarian cancer cells are sensitive to plumbagin irrespective of BRCA2 status in both normoxia and hypoxia. Importantly, plumbagin treatment effectively inhibits VEGF‐A and Glut‐1 in PEO‐1 and PEO‐4 ovarian cancer cells. We have also analyzed the p53 mutant, cisplatin resistant, and BRCA2 proficient OVCAR‐5 cells. Plumbagin challenge also restricts the VEGF induced pro‐angiogenic signaling in HUVECs and subsequently endothelial cell proliferation. In addition, we observe a significant effect on tumor regression among OVCAR‐5 tumor‐bearing mice treated with plumbagin, which is associated with significant inhibition of Ki67 and vWF expressions. Plumbagin also significantly reduces CD31 expression in an ear angiogenesis assay. Collectively, our studies indicate that plumbagin, as an anti‐cancer agent disrupts growth of ovarian cancer cells through the inhibition of proliferation as well as angiogenesis.     

Foretinib inhibits angiogenesis,lymphangiogenesis and tumor growth of pancreatic cancer in vivo by decreasing VEGFR-2/3 and TIE-2 signaling

Foretinib, a multiple kinase inhibitor undergoing clinical trials, could suppress the activity of hepatocyte growth factor (HGF) receptor c-MET and vascular endothelial growth factor receptor-2 (VEGFR-2). In addition, Foretinib may inhibit two critical lymphangiogenic signaling receptors VEGFR-3 and TIE-2. However, the effect of Foretinib on lymphatic endothelial cells (LECs) in vitro and lymphangiogenesis in vivo is still unknown. We found Foretinib decreased basal- and HGF-induced c-MET activity at low concentrations. However, Foretinib only reduced the proliferation of pancreatic cancer cells at high concentration reflecting the intrinsic chemoresistance of pancreatic cancer cells. Foretinib inhibited VEGF-A, VEGF-C and Angiopoetin-2 (ANG-2)-stimulated tube formation and sprouting of LECs by reducing VEGFR-2, VEGFR-3 and TIE-2 activation and increased apoptosis of LECs. In xenograft animal study, Foretinib suppressed tumor growth by inhibiting proliferation, angiogenesis and lymphangiogenesis. Additionally, Foretinib inhibited angiogenesis and lymphangiogenesis more significantly and exhibited low detrimental effect in orthotopic animal study. Collectively, we suggested that Foretinib simultaneously inhibits cancer cells and LECs to reduce pancreatic tumor growth in vivo and demonstrated for the first time that Foretinib suppresses angiogenesis and lymphangiogenesis by blocking VEGFR-2/3 and TIE-2 signaling.     

小RNA干扰STAT3基因表达对胃癌细胞增殖和侵袭的影响

目的:构建针对STAT3基因的短发夹状小干扰RNA重组质粒,探讨应用RNAi技术沉默STAT3基因对胃癌细胞BGC-823增殖和侵袭的影响。方法:应用DNA重组技术,构建针对STAT3基因的干扰RNA重组质粒(pSilencer-STAT3),经脂质体转染BGC-823胃癌细胞,逆转录聚合酶链反应和Western blot免疫印迹法检测STAT3的表达水平;MTT法检测RNA干扰后细胞增殖情况;Transwell细胞侵袭实验检测细胞侵袭能力的变化。结果:酶切鉴定和测序证实了重组质粒构建成功。与未转染和转染阴性对照质粒组相比,pSilencer-STAT 3转染组胃癌细胞BGC-823中STAT 3mRNA和STAT 3蛋白的表达水平均明显降低,F=39.424,P=0.000和F=31.911,P=0.001。pSilencer-STAT3转染组胃癌细胞BGC-823的生长受到明显抑制,抑制率达47.3%,与阴性对照质粒组的8.1%抑制率相比,差异具有统计学意义,F=40.835,P=0.000。侵袭实验显示,pSilencer-STAT3转染组胃癌细胞BGC-823穿膜细胞数,显著低于阴性对照质粒组和未转染...