The antitumor thioredoxin-1 inhibitor PX-12 (1-methylpropyl 2-imidazolyl disulfide) decreases thioredoxin-1 and VEGF levels in cancer patient plasma

Thioredoxin-1 (Trx-1) is a small redox protein that is overexpressed in many human tumors, where it is associated with aggressive tumor growth and decreased patient survival. Trx-1 is secreted by tumor cells and is present at increased levels in the plasma of cancer patients. PX-12 is an irreversible inhibitor of Trx-1 currently in clinical development as an antitumor agent. We have used SELDI-TOF mass spectroscopy to measure plasma Trx-1 from patients treated with PX-12 during a phase I study. Mean plasma Trx-1 levels at pretreatment were significantly elevated in the cancer patients at 182.0 ng/mL compared with 27.1 ng/mL in plasma from healthy volunteers. PX-12 treatment significantly lowered plasma Trx-1 in cancer patients having the highest plasma Trx-1 pretreatment levels. High-plasma vascular endothelial growth factor (VEGF) levels have been correlated to decreased patient survival. PX-12 treatment also significantly lowered plasma VEGF levels in cancer patients with high pretreatment VEGF levels. SELDI-TOF mass spectrometry identified seven additional plasma proteins whose levels decreased after PX-12 administration, one of which was identified as a truncated form of transthyretin. The results of this study suggest that the lowering of elevated levels of plasma Trx-1 in cancer patients may provide a surrogate for the inhibition of tumor Trx-1 by PX-12. Furthermore, PX-12 decreases plasma VEGF levels that may contribute to the antitumor activity of PX-12.     

Molecular mechanisms for the activity of PX-478, an antitumor inhibitor of the hypoxia-inducible factor-1alpha

We have reported previously that PX-478 (S-2-amino-3-[4'-N,N,-bis(chloroethyl)amino]phenyl propionic acid N-oxide dihydrochloride) has potent antitumor activity against a variety of human tumor xenografts associated with the levels of the hypoxia-inducible factor-1alpha (HIF-1alpha) within the tumor. We now report that PX-478 inhibits HIF-1alpha protein levels and transactivation in a variety of cancer cell lines. Hypoxia-induced vascular endothelial growth factor formation was inhibited by PX-478, whereas baseline levels of vascular endothelial growth factor in normoxia were unaffected. Studies of the mechanism of PX-478 action showed that HIF-1alpha inhibition occurs in both normoxia and hypoxia and does not require pVHL or p53. In addition, PX-478 decreases levels of HIF-1alpha mRNA and inhibits translation as determined by 35S labeling experiments and reporter assays using the 5' untranslated region of HIF-1alpha. Moreover, to a lesser extent, PX-478 also inhibits HIF-1alpha deubiquitination resulting in increased levels of polyubiquitinated HIF-1alpha. The inhibitory effect of PX-478 on HIF-1alpha levels is primarily due to its inhibition of translation because HIF-1alpha translation continues in hypoxia when translation of most proteins is decreased. We conclude that PX-478 inhibits HIF-1alpha at multiple levels that together or individually may contribute to its antitumor activity against HIF-1alpha-expressing tumors.     

Fasudil-induced hypoxia-inducible factor-1alpha degradation disrupts a hypoxia-driven vascular endothelial growth factor autocrine mechanism in endothelial cells

Hypoxic response of endothelial cells (EC) is an important component of tumor angiogenesis. Especially, hypoxia-inducible factor-1 (HIF-1)-dependent EC-specific mechanism is an essential component of tumor angiogenesis. Recently, the Rho/Rho-associated kinase (ROCK) signaling has been shown to play a key role in HIF-1alpha induction in renal cell carcinoma and trophoblast. The present study was designed to investigate whether low oxygen conditions might modulate HIF-1alpha expression through the Rho/ROCK signaling in human umbilical vascular ECs (HUVEC). Pull-down assay showed that hypoxia stimulated RhoA activity. Under hypoxic conditions, HUVECs transfected with small interfering RNA of RhoA and ROCK2 exhibited decreased levels of HIF-1alpha protein compared with nontargeted small interfering RNA transfectants, whereas HIF-1alpha mRNA levels were not altered. One of ROCK inhibitors, fasudil, inhibited hypoxia-induced HIF-1alpha expression without altering HIF-1alpha mRNA expression. Furthermore, proteasome inhibitor prevented the effect of fasudil on HIF-1alpha expression, and polyubiquitination was enhanced by fasudil. These results suggested that hypoxia-induced HIF-1alpha expression is through preventing HIF-1alpha degradation by activating the Rho/ROCK signaling in ECs. Furthermore, hypoxia induced both vascular endothelial growth factor (VEGF) and VEGF receptor-2 expression through the Rho/ROCK/HIF-1alpha signaling in HUVECs. Thus, augmented VEGF/VEGF receptor-2 autocrine mechanism stimulated HUVEC migration under hypoxic conditions. In summary, the Rho/ROCK/HIF-1alpha signaling is an essential mechanism for hypoxia-driven, VEGF-mediated autocrine loop in ECs. Therefore, fasudil might have the antimigratory effect against ECs in tumor angiogenesis.     

Regression of solid tumors by engineered overexpression of von Hippel-Lindau tumor suppressor protein and antisense hypoxia-inducible factor-1alpha

The von Hippel-Lindau tumor suppressor protein (pVHL) suppresses tumor formation by binding the alpha subunits of hypoxia-inducible factors (HIFs) responsible for stimulating tumor angiogenesis and glycolysis, targeting them for ubiquitination and proteasomal destruction. Loss of pVHL leads to the development of sporadic renal cell carcinomas (RCCs). In the present study, we sought to determine whether engineered overexpression of pVHL in tumors other than RCC can inhibit tumor growth, either as a monotherapy, or in combination with antisense HIF-1alpha therapy. Intratumoral injection of subcutaneous EL-4 thymic lymphomas with an expression plasmid encoding pVHL resulted in the downregulation of HIF-1alpha and vascular endothelial growth factor (VEGF). There was a concomitant reduction in tumor angiogenesis and increased tumor cell apoptosis due in part to downregulation of Bcl-2 expression. VHL therapy resulted in the complete regression of small (0.1 cm diameter) tumors whereas, in contrast, large (0.4 cm diameter) EL-4 tumors were only slowed in their growth. Nevertheless, large tumors completely regressed in response to intratumoral injection of a combination of antisense HIF-1alpha and VHL plasmids. Combination therapy resulted in increased losses of HIF-1alpha, VEGF, and tumor blood vessels, and increased tumor cell apoptosis. These novel results suggest that synergistic therapies that simultaneously block the expression or function of HIF-1alpha, and enhance the expression or function of VHL may be beneficial in the treatment of cancer.     

Green tea extract and (-)-epigallocatechin-3-gallate inhibit hypoxia- and serum-induced HIF-1alpha protein accumulation and VEGF expression in human cervical carcinoma and hepatoma cells

Green tea extract and its major component (-)-epigallocatechin-3-gallate (EGCG) exhibit antiangiogenic activities in various experimental tumor models. A growing body of evidence has established that hypoxia-inducible factor-1alpha (HIF-1alpha) and its downstream target, vascular endothelial growth factor (VEGF), play a critical role in tumor angiogenesis. In this study, we investigated the effect of green tea extract and EGCG on HIF-1alpha and VEGF expression in human cervical carcinoma (HeLa) and hepatoma (HepG2) cells. Our results showed that green tea extract and EGCG significantly inhibited hypoxia- and serum-induced HIF-1alpha protein accumulation in these cancer cells but had no effects on HIF-1alpha mRNA expression. Suppression of HIF-1alpha protein by green tea extract and EGCG also resulted in a drastic decrease in VEGF expression at both mRNA and protein levels. The mechanisms of green tea extract and EGCG inhibition of hypoxia-induced HIF-1alpha protein accumulation seem to involve the blocking of both phosphatidylinositol 3-kinase/Akt and extracellular signal-regulated kinase 1/2 signaling pathways and the enhancing of HIF-1alpha protein degradation through the proteasome system. In addition, green tea extract and EGCG inhibited serum-induced HIF-1alpha protein and VEGF expression by interfering with the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin signaling pathways, which play a crucial role in the protein translational machinery cascade. Functionally, green tea extract and EGCG abolished both chemoattractant- and hypoxia-stimulated HeLa cell migration. Our data suggested that HIF-1alpha/VEGF function as therapeutic target for green tea extract and EGCG in the context of cancer chemoprevention and anticancer therapy.     

Inhibition of hypoxia-inducible factor-1alpha in RPMI8226 myeloma cells results in reduced tumor growth in nude mice

OBJECTIVE: To explore the influence of inhibition of hypoxia-inducible factor-1alpha (HIF-1alpha) by RNA interference (RNAi) on tumorigenesis of human myeloma cell line (HMCL) RPMI8226 cells in nude mice. METHOD: RNAi vector of HIF-1alpha was constructed with commercial shRNA expression vector pSilencer 2. 1-U6 hygro. RT-PCR and western blot were used to detect HIF-1alpha mRNA and protein expression respectively. Vascular endothelial growth factor (VEGF) secretion and cell cycle changes were detected by ELISA and flow cytometry respectively. Expression of target gene of HIF-1alpha, VEGF and Glut-1 were tested under hypoxia condition. Tumorigenesis was observed after transfected cells were injected subcutaneously in nude mice. RESULTS: After interference, expression of HIF-1alpha decreased significantly at both mRNA and protein level. Under normoxia condition, VEGF concentrations in HIF-la inhibited cells (RPMI8226-il and RPMI8226-i2) and non-inhibited cells (RPMI8226-c and RPMI8226) showed no differences. While under hypoxia condition, VEGF concentration in the above four cells was (506.0 +/- 53.2), (494.7 +/- 63.1), (984.4 +/- 61.9) and (938.2 +/- 62.2) pg/ml, respectively, being significantly lower in RPMI8226-il and RPMI8226-i2 cells than in RPMI8226-c and RPMI8226 cells (P <0.05). HIF-1alpha interference was found to suppress the cells shift from S-phase to G1 induced by hypoxia. VEGF and Glut-1 expressions were markedly attenuated (P <0.05). The growth rate of HIF-1alpha inhibition tumors in subcutaneous xenograft model decreased drastically. CONCLUSIONS: RNAi inhibits HIF-1alpha expression. Reduced tumor growth by HIF-1alpha inhibition may partly through inhibiton of angiogenesis and glycolysis metabolism.     

低氧诱导因子过表达对人外周血内皮祖细胞分化影响的体外研究

为了研究低氧诱导因子（hypoxia inducible factor-1alpha,HIF-1α）在体外对人外周血内皮祖细胞（endothelial progenitor cells,EPC）向血管内皮细胞（endothelial cells,EC）分化的影响,用密度梯度离心法分离人外周血EPC,电穿孔技术转染HIF-1α质粒至EPC,计算转染效率,RT-PCR方法测定HIF-1α、HIF-1β及HIF-1α下游靶基因血管内皮生长因子（vascular endothelial growth factor,VEGF）mRNA在质粒转染前后含量变化,免疫细胞化学方法检测在常氧下HIF-1α蛋白在HIF-1α质粒转染前后不同时点蛋白表达情况,细胞膜表面抗原决定簇流式细胞术测定FITC-CD31^＋EPCs/EC在未转染组、pEGFP空质粒或HIF-1α质粒转染组转染3-10天后的组间差异,光镜下观察转染前后细胞形态及分化程度.结果表明：EPC获得后经电穿孔转染,质粒转染效率约20%;RT-PCR示HIF-1α mRNA在常氧中有表达,并在HIF-1α过表达组合量增加（P＜0.05）;其下游靶基因VEGF在HIF-1α过表达组表达上调（P＜0.05）;HIF-1β表达在各组中无明显差异（P＞0.05）.免疫组织化学显示常氧下HIF-1α蛋白无表达,转染HIF-1α质粒12小时后HIF-1α蛋白表达阳性,转染24小时后蛋白表达阴性.流式细胞仪检测显示电穿孔转染HIF-1α质粒使CD31＋细胞的百分比增加（P＜0.05）.细胞形态学观察显示：未转染及pEGFP空质粒转染组细胞在培养6天后呈集落样散在分布,培养14天后贴壁细胞部分呈梭样;穿孔转染的HIF-1α质粒组细胞于培养6天后集落周边分化出梭样贴壁细胞,培养14天后呈梭样,或铺路石样牢固贴壁.结论：HIF-1α质粒能有效地用于基因干扰治疗,有助于EPC向EC分化,为体内研究奠定了基础,也为进一步体内诱导血管新生、治疗缺血性心脏病提供了更广阔的治疗选择.     

缺氧诱导因子-1α、血管内皮生长因子在胃癌中的表达及意义

目的：研究缺氧诱导因子-1α（HIF-1α）和血管内皮生长因子（VEGF）蛋白在胃癌中的表达及意义。方法应用免疫组织化学EnvisionTM二步法检测172例胃癌和30例癌旁正常胃组织（距癌灶5 cm以上）中HIF-1α和VEGF蛋白的表达,应用卡方趋势检验对HIF-1α及VEGF蛋白表达与胃癌临床病理参数进行相关性检验;应用Spearman秩相关检验对HIF-1α和VEGF表达之间相关性进行分析。结果在172例胃癌中,HIF-1α和VEGF蛋白的阳性表达率分别为55.81％（96/172）和63.37％（109/172）,30例癌旁中HIF-1α和VEGF表达均阴性,两组比较,差异均有统计学意义（χ2分别=11.16、13.76,P均＜0.0,5）;卡方趋势检验显示,胃癌组织中HIF-1α表达和VEGF表达与胃癌的临床分期、组织学分级、淋巴结转移和肿瘤坏死呈正相关（χ2分别=25.53、24.99、11.20、30.35;25.43、13.13、11.31、23.29, P均＜0.05）,与患者年龄和术后5年生存率均无关（χ2分别=3.92、4.63、4.79、4.65,P均＞0.05）。Spearman结果显示,胃癌组织中HIF-1与VEGF蛋白表达之间的关系呈正相关（r=0.65,P＜0.05）。结论胃癌组织中HIF-1α及VEGF的蛋白表达水平明显增高,并与胃癌的侵袭性生物学行为有关。     

Hypoxia-inducible factor-1-dependent and -independent regulation of insulin-like growth factor-1-stimulated vascular endothelial growth factor secretion

Hypoxia-induced stress plays a central role in retinal vascular disease and cancer. Increased hypoxia-inducible factor-1 alpha (Hif-1 alpha) expression leads to HIF-1 formation and the production of vascular endothelial growth factor (VEGF). Cytokines, including insulin-like growth factor-1 (IGF-1), also stimulate VEGF secretion. In this study, we examined the relationship between IGF-1 signaling, HIF-1 alpha protein turnover and VEGF secretion in the ARPE-19 retinal pigment epithelial cell line. Northern analysis revealed that IGF-1 stimulated Hif-1 alpha message expression, whereas the hypoxia-mimetic CoCl2 did not. CoCl2 treatment increased Hif-1 alpha protein accumulation to a greater extent than IGF-1 treatment. However, IGF-1 stimulated a more significant increase in VEGF secretion. IGF-1-stimulated VEGF promoter activity was phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR (mammalian target of rapamycin)-dependent, whereas VEGF secretion was only partially reduced by inhibition of PI3K/Akt/mTOR and HIF-1 activities. Analysis of VEGF promoter truncation mutants indicated that sensitivity to CoCl2 was hypoxia response element (HRE)-dependent with the region upstream of the HRE conferring IGF-1 sensitivity. In conclusion, IGF-1 regulates VEGF expression and secretion via HIF-1-dependent and -independent pathways.     

von Hippel-Lindau mutation in mice recapitulates Chuvash polycythemia via hypoxia-inducible factor-2alpha signaling and splenic erythropoiesis

The R200W mutation in the von Hippel-Lindau (VHL) tumor suppressor protein (pVHL) is unique in that it is not associated with tumor development, but rather with Chuvash polycythemia, a heritable disease characterized by elevated hematocrit and increased serum levels of erythropoietin and VEGF. Previous studies have implicated hypoxia-inducible factor-1alpha (HIF-1alpha) signaling in this disorder, although the effects of this mutation on pVHL function are not fully understood. In order to explore the mechanisms underlying the development of this polycythemia, we generated mice homozygous for the R200W mutation (Vhl(R/R)). Vhl(R/R) mice developed polycythemia highly similar to the human disease. The activity of HIF proteins, specifically the HIF-2alpha isoform, was upregulated in ES cells and tissues from Vhl(R/R) mice. Furthermore, we observed a striking phenotype in Vhl(R/R) spleens, with greater numbers of erythroid progenitors and megakaryocytes and increased erythroid differentiation of Vhl(R/R) splenic cells in vitro. These findings suggest that enhanced expression of key HIF-2alpha genes promotes splenic erythropoiesis, resulting in the development of polycythemia in Vhl(R/R) mice. This mouse model is a faithful recapitulation of this VHL-associated syndrome and represents a useful tool for studying polycythemias and investigating potential therapeutics.     

纳米介导的缺氧诱导因子1α小干扰RNA抑制人食管鳞癌TE-1细胞生长

背景:纳米微粒作为一种比较理想的非病毒基因递送载体,具有无免疫原性和致瘤性等优越性.利用纳米技术和基因干扰技术阻断缺氧诱导因子1α(hypoxia-inducible factor 1,HIF-1α)在食管鳞癌组织的表达,降低癌细胞对化疗药物的耐药性,理论上成为能够抑制食管癌细胞生长的有效方法.目的:探讨HIF-1α小干扰RNA纳米微粒对人食管鳞癌TE-1细胞生长的抑制作用.设计、时间及地点:以体外培养的食管鳞癌TE-1细胞为对象,基因水平完全随机对照实验,于2007-01/2008-12在中山大学附属第三医院中心实验室完成.材料:由上海生物工程公司合成HIF-1α小干扰RNA,采用超声乳化法制备纳米微粒.人食管鳞癌TE-1细胞株购自中科院上海细胞库.方法:体外培养的人食管鳞癌TE-1细胞分为4组:分别为生理盐水组,不含基因的纳米微粒组,HIF-1α小干扰RNA组,HIF-1α小干扰RNA纳米微粒组.主要观察指标:RT-PCR检测HIF-1αmRNA的表达,Western-blot法检测HIF-1α蛋白的表达;流式细胞仪检测细胞凋亡情况;MTT法检测细胞增殖情况.结果:处理72 h后HIF-1α小干扰RNA纳米微粒组细胞的HIF-1αmRNA表达和HIF-1α蛋白表达低于其他3组(P＜0.01),细胞凋亡率高于其他3组(P＜0.01).HIF-1α小干扰RNA纳米微粒组细胞增殖受明显抑制,生长缓慢,与其他3组比较,差异有显著性意义(P＜0.01).结论:HIF-1α小干扰RNA纳米微粒能够有效减少食管鳞癌TE-1细胞的HIF-1αmRNA和蛋白的表达,提高肿瘤细胞的凋亡,显著抑制TE-1细胞的生长.     

抑制缺氧诱导因子-1α表达对多发性骨髓瘤RPMI8226细胞裸鼠体内成瘤性的影响

目的 利用RNA干扰(RNAi)技术抑制多发性骨髓瘤(MM)细胞系RPMI8226细胞中缺氧诱导因子-1α(hypoxia-inducible factor,HIF-1α)表达,探讨对RPMI8226细胞在裸鼠体内成瘤性的影响.方法 利用短发夹RNA(shRNA)表达载体pSilencer 2.1-U6 hygro构建RNAi载体,在RPMI8226细胞中抑制HIF-1α的表达.应用RT-PCR技术和Western blot方法 ,鉴定RNAi后HIF-1α的表达.ELISA法测定细胞上清中血管内皮生长因子(VEGF)表达的改变.流式细胞术检测RNAi前后细胞周期的改变.研究缺氧(PO2为1%)条件下,抑制HIF-1α的表达对其靶基因VEGF和葡萄糖转运蛋白(Glut-1)表达的影响.利用异种移植瘤模型观察HIF-1α干扰后对肿瘤生长的影响.结果 RNAi后,HIF-1α的表达在mRNA和蛋白水平均明显降低.常氧条件下,RNAi组(RPMI8226-i1和RPMI8226-i2)和未干扰组(RPMI8226-c和RPMI8226)细胞上清中VEGF浓度无明显差异;缺氧条件下四组细胞上清中VEGF浓度分别为(506.0±53.2)、(494.7±63.1)、(984.4±61.9)和(938.2±62.2)pg/ml.RPMI8226-i1和RPMI8226-i2组较RPMI8226-c和RPMI8226组明显降低(P＜0.05).G0/G1期累积受到抑制,S期细胞比例增加.缺氧条件下VEGF和Glut-1等靶基因的表达明显下调(P＜0.05).裸鼠皮下成瘤性实验表明,抑制HIF-1α的表达,能够明显抑制肿瘤的生长.结论 RNAi抑制RPMI8226细胞HIF-1α表达,可能通过抑制肿瘤的血管新生和糖代谢过程来发挥肿瘤抑制作用.