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.     

6-(1-Oxobutyl)-5,8-dimethoxy-1,4-naphthoquinone inhibits lewis lung cancer by antiangiogenesis and apoptosis

Lung cancer is a leading cause of cancer mortality worldwide. Novel and nontoxic agents targeting angiogenesis and tumor cell proliferation and survival are desirable for lung cancer chemoprevention and treatment. Previously we have reported that 6-(1-oxobutyl)-5,8-dimethoxy-1,4-naphthoquinone (OXO) exhibits anti-tumor activity against S-180 sarcoma in vitro and in vivo. Here we studied the anti-angiogenic and apoptogenic attributes of OXO in vitro and in vivo targeting lung cancer. In human umbilical vein endothelial cells (HUVECs), we show that OXO more potently inhibited VEGF-stimulated than basic bFGF-stimulated HUVEC proliferation and capillary differentiation. In Lewis lung carcinoma (LLC) cells, OXO not only induces S-phase arrest and mitochondria/caspase-9 pathway mediated apoptosis, but also effectively down-regulated the hypoxia-induced expression of HIF-1alpha and VEGF at mRNA and protein levels in LLC and decreased VEGF secretion into conditioned culture media. OXO significantly reduced in vivo functional angiogenesis in the mouse Matrigel plug assay. Furthermore, OXO potently inhibited the growth of LLC cells inoculated on the flank of syngenic mice at dosages that did not affect their body weight. The in vivo anti-cancer effect was associated with decreased HIF-1alpha and VEGF expression, decreased microvessel density as well as a reduction of tumor cell proliferation and increased tumor cell apoptosis. Taken together, these results demonstrate that OXO exerts anti-cancer activity through anti-angiogenesis and tumor cell cycle arrest and apoptosis. These findings warrant additional studies of OXO as a novel agent for the chemoprevention and treatment of lung cancer.     

Everolimus (RAD001) and anti-angiogenic cyclophosphamide show long-term control of gastric cancer growth in vivo

Metronomic dosing of cytotoxic drugs such as cyclophosphamide has shown anti-angiogenic activity, most likely by inducing hypoxia in tumors. Hypoxia leads to activation of escape mechanisms allowing tumor cell survival. This potentially limits the activity of anti-angiogenic strategies. We hypothesized that mTORC1 inhibition by everolimus (RAD001) leads to suppression of HIF-1alpha and VEGF resulting in synergistic anti-tumor activity in combination with anti-angiogenically dosed cyclophosphamide. In vitro, effects of everolimus on mTORC1 signaling, proliferation, cell cycle, HIF-1alpha expression and VEGF secretion were evaluated in two gastric cancer cell lines. In vivo, anti-tumor activity of everolimus in combination with metronomic cyclophosphamide was studied in a NCI-N87 human gastric cancer SCID mouse xenograft model. Expression of Ki-67 and HIF-1alpha, activated caspase 3, microvascular density (MVD) and tumor necrotic area assessed. Everolimus decreased proliferation and attenuated production of HIF-1alpha as well as VEGF in gastric cancer cells in vitro. In vivo, everolimus significantly inhibited tumor growth. This anti-tumor activity was linked to a significant increase in tumor necrotic area (p < 0.02) and trends for decreased proliferation, increased apoptosis, decreased HIF-1alpha and lower tumor MVD (p = n.s.). The combination of everolimus and cyclophosphamide resulted in a striking and highly significant long-term tumor growth control compared to monotherapy (p < 0.001), which was associated with a sharp increase in central tumor necrosis (p < 0.001). In conclusion, the combination of everolimus and metronomic cyclophosphamide showed synergistic anti-tumor activity. Depriving cancer cells by everolimus of factors necessary for their survival under hypoxia induced by anti-angiogenic chemotherapy appears to be a promising approach for treatment of gastric cancer.     

酪丝缬肽对裸小鼠人肝癌BEL-7402移植瘤的抑制作用

目的观察酪丝缬肽对裸小鼠人肝癌BEL-7402移植瘤的抑瘤作用,并初步探讨其可能的作用机制。方法通过动物模型观察酪丝缬肽对裸小鼠人肝癌BEL-7402移植瘤肿瘤重量及体积的影响;以电镜观察肿瘤细胞超微结构的改变;以免疫组化法观察瘤组织中增殖细胞核抗原(PCNA)的表达;以利用TUNEL法检测瘤组织细胞的凋亡情况。结果给药剂量为80μg·kg~(-1)·d~(-1)和160μg·kg~(-1)·d~(-1)时,酪丝缬肽可以显著抑制裸小鼠BEL-7402移植瘤的生长,抑瘤率分别为64．02%和66．27%,与生理盐水及相应氨基酸混合物组比较,差异均有统计学意义(P＜0．05)。酪丝缬肽可引起肿瘤细胞超微结构改变,导致肿瘤细胞广泛坏死和凋亡。酪丝缬肽给药组与生理盐水组比较,肿瘤组织细胞的PCNA表达明显降低,凋亡细胞明显增多。结论酪丝缬肽可显著抑制人肝癌BEL-7402细胞在裸小鼠体内的生长,抑制肿瘤细胞增殖,促进瘤细胞凋亡。     

联合反义缺氧诱导因子-1α与B7-1基因治疗肿瘤的实验研究

目的探讨联合应用反义缺氧诱导因子1α(HIF1α)与B71基因治疗肿瘤的疗效。方法构建反义HIF1α和B71表达载体,用阳离子脂质体辅助,联合或单独导入肿瘤,观察肿瘤生长情况。采用免疫组化、蛋白印迹等方法检测基因表达,并进行血管密度评估。结果单独转染反义HIF1α的表达质粒可以阻断肿瘤缺氧诱导反应,下调血管内皮细胞生长因子的表达,抑制新生血管形成和肿瘤生长,肿瘤的血管密度由对照组的19.5±1.8降至12.4±2.3。联合应用反义HIF1α转染和B71可根除大的肿瘤。结论反义HIF1α基因治疗可以提高B71介导的抗肿瘤免疫治疗的疗效。     

缺氧条件下YC-1对人胰腺癌细胞VEGF和GPI基因的抑制效应

目的探讨缺氧条件下3-(5’-hydroxymethyl-2’-furyl)-1-benzylindazole(YC-1)对人胰腺癌细胞血管内皮生长因子(VEGF)和磷酸葡萄糖异构酶(GPI)基因的调控作用及其机制。方法缺氧条件下体外培养胰腺癌PC-3细胞株,免疫细胞化学染色法检测常氧和缺氧条件下缺氧诱导因子- 1α(HIF-1α)的表达。半定量RT-PCR检测YC-1对PC-3细胞VEGF、GPI、HIF-1αmRNA表达的调节作用。Western blot检测YC-1对PC-3细胞HIF-1α蛋白表达的调节作用。MTT法检测YC-1对缺氧PC-3细胞恶性增殖的影响。结果缺氧条件下,HIF-1α主要表达于PC-3细胞胞核内。随着YC-1浓度升高,VEGF和GPI mRNA表达、HIF-1α蛋白表达逐渐受抑,并呈明显的剂量依赖性;实验组HIF- 1αmRNA均呈强表达,其表达水平不随YC-1作用浓度的升高而降低。缺氧条件下,实验组细胞增殖抑制率均增高,100μmol／L YC-1组细胞增殖抑制率达73．28%±2．01％。结论缺氧状态下,YC-1抑制人胰腺癌PC-3细胞VEGF和GPI基因转录与其抑制HIF-1α蛋白的表达有关,YC-1能够抑制PC-3细胞生长和增殖。     

肝癌缺氧诱导因子-1α异常表达与靶向干预的临床价值研究

Objective: The aim of this study was to analyze the expression features of hypoxia inducible factor-1α (HIF-1α) in hepatocellular carcinoma (HCC) and effects of HIF-1α silencing on HepG2 cells.Methods: HIF-1α expression was analyzed in the self-control HCC specimens by immunohistochemistry.After HepG2 cells with miRNA transfection, the expression of HIF-1α was determined at mRNA or protein level by real-time polymerase chain reaction (PCR) or Western blotting.Vascular endothelial growth factor (VEGF) and angiopoietin-2 (ANG-2) were determined by ELISA.Alterations of cell cycles and apoptosis of HepG2 cells were measured using a flow cytometer.Results: Positive HIF-1α was brown and granule-like in the cytoplasm or nucleus.Significant difference was found between HCC (80%) and its surrounding tissues (100%, χ2 = 22.35, P < 0.001) and HIF-1α expression related to tumor size.At 72 h after miRNA transfection, the expression of HIF-1α in HepG2 cells was down-regulated by 87% at mRNA or 65% at protein level, with VEGF and ANG-2 decreased to 54% and 36%, respectively.After RNA interference combined with anti-cancer drug, the apoptotic rate of HepG2 cells was increasing from 22.46% ± 0.61% to 36.99% ± 0.88%, with up-regulation of G1 phase (65.68% ± 0.91%) and down-regulation of S phase (19.47 ± 1.34 %).Conclusion: Abnormal expression of HIF-1α is associated with development of HCC, and HIF-1α gene silencing can effectively inhibit HepG2 cell proliferation.     

Ａｄ-ＩＮＧ４抑制裸鼠人肝癌移植瘤生长的体内实验研究

目的　研究携带ＩＮＧ４基因的重组腺病毒（Ａｄ-ＩＮＧ４）对人肝癌细胞株ＳＭＭＣ-７７２１移植瘤生长的抑制作用及其潜在的分子机制。方法　将扩增的Ａｄ-ＩＮＧ４感染ＳＭＭＣ-７７２１细胞,Ｗｅｓｔｅｒｎｂｌｏｔ法检测Ａｄ-ＩＮＧ４在ＳＭＭＣ-７７２１细胞中的转录。用ＳＭＭＣ-７７２１细胞建立裸鼠人肝癌移植瘤模型,予Ａｄ-ＩＮＧ４（１×１０^９ｐｆｕ／ｍｌ）５０μｌ对移植瘤瘤体注射治疗,观察肿瘤生长变化;３周后处死裸鼠,摘除瘤体,称瘤重;免疫组化法检测ｐ２１、ＣＯＸ-２、Ｆａｓ、Ｂｃｌ-２、Ｂａx、Ｃａｓｐａｓｅ-３、ＶＥＧＦ、ＣＤ３４等因子的表达。结果　Ａｄ-ＩＮＧ４在ＳＭＭＣ-７７２１细胞中成功表达;Ａｄ-ＩＮＧ４对裸鼠人肝癌移植瘤有明显的生长抑制作用,Ａｄ-ＩＮＧ４组瘤体显著减小（Ｐ＜０.０１）,抑瘤率达４１.６４％;免疫组化检测显示,Ａｄ-ＩＮＧ４抑癌的分子机制可能与上调ｐ２１、Ｆａｓ、Ｂａｘ和Ｃａｓｐａｓｅ-３的表达及下调ＣＯＸ-２、Ｂｃｌ-２、ＶＥＧＦ和ＣＤ３４的表达有关。结论　Ａｄ-ＩＮＧ４能有效抑制ＳＭＭＣ-７７２１裸鼠人肝癌移植瘤的生长,其机制可能通过抑制肿瘤细胞生长、血管形成和激活细胞凋亡等多个途径共同发挥抑癌效应。