Suppression of hypoxia inducible factor-1alpha (HIF-1alpha) by YC-1 is dependent on murine double minute 2 (Mdm2)

Inhibition of HIF-1alpha activity provides an important strategy for the treatment of cancer. Recently, 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) has been identified as an anti-HIF-1alpha drug in cancer therapy with unclear molecular mechanism. In the present study, we aimed to investigate the effect and mechanism of YC-1 on HIF-1alpha in a hepatocellular carcinoma cell line under hypoxic condition, which was generated by incubating cells with 0.1% O(2). The phenotypic and molecular changes of cells were determined by cell proliferation assay, apoptosis assay, luciferase promoter assay, and Western blot analysis. YC-1 arrested tumor cell growth in a dose-dependent manner, whereas it did not induce cell apoptosis. Hypoxia-induced upregulation of HIF-1alpha was suppressed by YC-1 administration. YC-1 inhibited HIF-1alpha protein synthesis under normoxia and affected protein stability under hypoxia. YC-1 suppressed the expression of total and phosphorylated forms of murine double minute 2 (Mdm2), whereas this inhibitory effect was blocked by overexpression of Mdm2. In conclusion, YC-1 suppressed both protein synthesis and stability of HIF-1alpha in HCC cells, and its inhibitory effects on HIF-1alpha were dependent on Mdm2.     

Costunolide Induces Apoptosis through Generation of ROS and Activation of P53 in Human Esophageal Cancer Eca‐109 Cells

Costunolide is a sesquiterpene lactone, which possesses potent anti‐cancer properties. However, there is little report about its effects on esophageal cancer. In our study, we investigated the effects of costunolide on the cell viability, cell cycle, and apoptosis in human esophageal cancer Eca‐109 cells. It was found that costunolide inhibited the growth of Eca‐109 cells in a dose‐dependent manner, which was associated with the loss of mitochondrial membrane potential (Δψ_m) and the production of ROS. Costunolide induced apoptosis of Eca‐109 cells as well as cell cycle arrest in G1/S phase by upregulation of P53 and P21. Costunolide triggered apoptosis in esophageal cancer cells via the upregulation of Bax, downregulation of Bcl‐2, and significant activation of caspase‐3 and poly ADP‐ribose polymerase. These effects were markedly abrogated when cells were pretreated with N‐acetylcysteine, a specific reactive oxygen specie inhibitor. These results suggest that costunolide is a potential candidate for the treatment of esophageal cancer.     

Differential effects of HIF-1 inhibition by YC-1 on the overall outcome and blood-brain barrier damage in a rat model of ischemic stroke

Hypoxia-inducible factor 1 (HIF-1) is a master regulator of cellular adaptation to hypoxia and has been suggested as a potent therapeutic target in cerebral ischemia. Here we show in an ischemic stroke model of rats that inhibiting HIF-1 and its downstream genes by 3-(5'-hydroxymethyl-2'-furyl)-1-benzylindazole (YC-1) significantly increases mortality and enlarges infarct volume evaluated by MRI and histological staining. Interestingly, the HIF-1 inhibition remarkably ameliorates ischemia-induced blood-brain barrier (BBB) disruption determined by Evans blue leakage although it does not affect brain edema. The result demonstrates that HIF-1 inhibition has differential effects on ischemic outcomes and BBB permeability. It indicates that HIF-1 may have different functions in different brain cells. Further analyses show that ischemia upregulates HIF-1 and its downstream genes erythropoietin (EPO), vascular endothelial growth factor (VEGF), and glucose transporter (Glut) in neurons and brain endothelial cells and that YC-1 inhibits their expression. We postulate that HIF-1-induced VEGF increases BBB permeability while certain other proteins coded by HIF-1's downstream genes such as epo and glut provide neuroprotection in an ischemic brain. The results indicate that YC-1 lacks the potential as a cerebral ischemic treatment although it confers certain protection to the cerebral vascular system.     

ODC和AdoMetDC双反义腺病毒载体对食管癌细胞凋亡影响的研究

为探讨ODC和AdoMetDC双反义腺病毒载体(Ad-ODC-AdoMetDCas)对食管癌Eca109细胞凋亡作用的影响,应用MTT法观察Ad-ODC-AdoMetDCas对食管癌Eca109细胞生长增殖的影响,采用Western blot和HPLC的方法分别检测腺病毒载体对食管癌Eca109细胞中ODC和AdoMetDC蛋白表达以及胞内多胺含量的抑制作用,同时应用原位末端标记(TUNEL) 法观察Ad-ODC-AdoMetDCas对食管癌Eca109细胞凋亡作用的影响, 透射电镜进一步观察细胞超微结构的改变． 实验结果显示,应用MTT法观察发现Ad-ODC-AdoMetDCas对食管癌Eca109细胞生长增殖有显著抑制作用． 以Ad-ODC- AdoMetDCas感染食管癌Eca109细胞,可明显抑制食管癌Eca109细胞中ODC和AdoMetDC基因表达． HPLC结果显示,食管癌Eca109细胞感染Ad-ODC-AdoMetDCas后,细胞内3种多胺含量都明显降低． TUNEL标记检测结果显示Ad-ODC-AdoMetDCas可明显引起食管癌Eca109细胞凋亡．透射电镜观察到典型的细胞凋亡特征(表现细胞体积缩小,核皱缩、碎裂,染色质呈块状边集等)． 实验表明,ODC和AdoMetDC双反义腺病毒载体(Ad-ODC-AdoMetDCas)具有显著抑制食管癌细胞生长增殖,降低细胞多胺合成,促进细胞凋亡,为探讨食管癌基因治疗的可行性提供实验依据．     

Activation of the AMP-activated protein kinase (AMPK) by nitrated lipids in endothelial cells

The AMP-activated protein kinase (AMPK) is an important regulator of endothelial metabolic and functional homeostasis. Here, we examined the regulation of AMPK by nitrated oleic acid (OA-NO(2)) and investigated the implications in endothelial function. Treatment of bovine aortic endothelial cells (BAECs) with OA-NO(2) induced a significant increase in both AMPK-Thr172 phosphorylation and AMPK activity as well as upregulation of heme oxygenase (HO)-1 and hypoxia-inducible factor (HIF)-1α. Pharmacologic inhibition or genetic ablation of HO-1 or HIF-1α abolished OA-NO(2)-induced AMPK phosphorylation. OA-NO(2) induced a dramatic increase in extracellular signal-regulated kinase (ERK)1/2 phosphorylation that was abrogated by the HO-1 inhibitor, zinc deuteroporphyrin IX 2,4-bis-ethylene glycol (ZnBG). Inhibition of ERK1/2 using UO126 or PD98059 reduced but did not abolish OA-NO(2)-induced HIF-1α upregulation, suggesting that OA-NO(2)/HO-1-initiated HIF-1α induction is partially dependent on ERK1/2 activity. In addition, OA-NO(2) enhanced endothelial intracellular Ca(2+), an effect that was inhibited by the HIF-1α inhibitor, YC-1, and by HIF-1α siRNA. These results implicate the involvement of HIF-1α. Experiments using the Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibitor STO-609, the selective CaMKII inhibitor KN-93, and an isoform-specific siRNA demonstrated that OA-NO(2)-induced AMPK phosphorylation was dependent on CaMKKβ. Together, these results demonstrate that OA-NO(2) activates AMPK in endothelial cells via an HO-1-dependent mechanism that increases HIF-1α protein expression and Ca(2+)/CaMKKβ activation.     

LncRNA ZNF667-AS1靶向miR-31-5p对食管癌细胞增殖、迁移和凋亡的机制研究

目的 探究长链非编码RNA(lncRNA)ZNF667-AS1通过靶向miR-31-5p对食管癌细胞增殖和迁移的影响及潜在的机制.方法 采用实时荧光定量PCR(qPCR)技术检测ZNF667-AS1在食管癌细胞Eca109、EC1、TE1和正常食管上皮细胞Het-1A的表达水平,并选择表达差异最大的细胞株进行后续实验....     

Aggregation of human dental pulp cells into 3D spheroids enhances their migration ability after reseeding

Multicellular three-dimensional (3D) spheroids allow intimate cell–cell communication and cell–extracellular matrix interaction. Thus, 3D cell spheroids better mimic microenvironment in vivo than two-dimensional (2D) monolayer cultures. The purpose of this study was to evaluate the behaviors of human dental pulp cells (DPCs) cultured on chitosan and polyvinyl alcohol (PVA) membranes. The protein expression of hypoxia-inducible factor 1-α (HIF-1α) and vascular endothelial growth factor (VEGF), and the migration ability of the DPCs from 2D versus 3D environments were investigated. The results showed that both chitosan and PVA membranes support DPCs aggregation to form multicellular spheroids. In comparison to 2D cultures on tissue culture polystyrene, DPC spheroids exhibited higher protein expression of HIF-1α and VEGF. The treatment with YC-1 (inhibitor to HIF-1α) blocked the upregulation of VEGF, indicating a downstream event to HIF-1α expression. When DPC spheroids were collected and subjected to the transwell assay, the cells growing outward from 3D spheroids showed greater migration ability than those from 2D cultures. Moreover, DPCs aggregation and spheroid formation on chitosan membrane were abolished by Y-27632 (inhibitor to Rho-associated kinases), whereas the inhibitory effect did not exist on PVA membrane. This suggests that the mechanism regulating DPCs aggregation and spheroid formation on chitosan membrane is involved with the Rho-associated kinase signaling pathway. In summary, the multicellular spheroid structure was beneficial to the protein expression of HIF-1α and VEGF in DPCs and enhanced the migration ability of the cells climbing from spheroids. This study showed a new perspective in exploring novel strategies for DPC-based research and application.     

HIF-1α coordinates lymphangiogenesis during wound healing and in response to inflammation

This study aimed to investigate the mechanisms that coordinate lymphangiogenesis. Using mouse models of lymphatic regeneration and inflammatory lymphangiogenesis, we explored the hypothesis that hypoxia inducible factor-α (HIF-1α) is a central regulator of lymphangiogenesis. We show that HIF-1α inhibition by small molecule inhibitors (YC-1 and 2-methyoxyestradiol) results in delayed lymphatic repair, decreased local vascular endothelial growth factor-C (VEGF-C) expression, reduced numbers of VEGF-C(+) cells, and reductions in inflammatory lymphangiogenesis. Using transgenic HIF-1α/luciferase mice to image HIF-1α expression in real time in addition to Western blot analysis and pimonidazole staining for cellular hypoxia, we demonstrate that hypoxia stabilizes HIF-1α during initial stages of wound repair (1-2 wk); whereas inflammation secondary to gradients of lymphatic fluid stasis stabilizes HIF-1α thereafter (3-6 wk). In addition, we show that CD4(+) cell-mediated inflammation is necessary for this response and regulates HIF-1α expression by macrophages, as CD4-deficient or CD4-depleted mice demonstrate 2-fold reductions in HIF-1α expression as compared to wild-types. In summary, we show that HIF-1α is a critical coordinator of lymphangiogenesis by regulating the expression of lymphangiogenic cytokines as part of an early response mechanism to hypoxia, inflammation, and lymphatic fluid stasis.     

MicroRNA-21 promotes the proliferation and inhibits apoptosis in Eca109 via activating ERK1/2/MAPK pathway

The aim of this study was to investigate how miR-21 promotes proliferation and inhibits apoptosis in esophageal squamous cell carcinoma (ESCC). MTT, wound healing assay and cell cycle showed that proliferation and migration of ESCC cell line Eca109 cells were increased in miR-21 mimics group, and decreased in anti-miR-21 Oligonucleotide (AMO) group after transfection into Eca109 cells with miR-21 mimics, AMO and scramble sequence, respectively. Cell apoptosis assay indicated that cell apoptosis can be obviously inhibited by overexpression of miR-21 and promoted by downregulation of miR-21. Meanwhile, western-blot results showed that p-ERK1/2 expression was elevated in miR-21 mimics group, whereas decreased in AMO group. Furthermore, the ERK1/2, a key component of MAPK signaling pathway, was knocked down, and overexpressed successfully using shRNA-ERK1/2 and overexpressing plasmids containing full length cDNA of ERK1/2, respectively. It was observed that shRNA-ERK1/2 can significantly decreased the level of miR-21 expression, while overexpression of ERK1/2 can up-regulate expression of miR-21. As further confirmation, Eca109 cells were treated with gradient concentration of U0126, a kind of MEK inhibitor, and expression of miR-21 was subsequently examined. It was found that U0126 can significantly decreased endogenous expression of miR-21. In parallel, U0126 decreased cell proliferation, migration and increased the apoptosis in Eca109 cells, with the expression of miR-21 being reduced significantly in U0126 group as compared with control groups. Our findings indicated that miR-21 promoted the proliferation, migration and inhibited apoptosis of Eca109 cells through activating ERK1/2/MAPK pathway, and that targeting miR-21 could be a promising therapeutic strategy in ESCC.     

A novel benzimidazole analogue inhibits the hypoxia-inducible factor (HIF)-1 pathway

Hypoxia-inducible factor (HIF)-1 is a therapeutic target in solid tumors. We report the novel benzimidazole analogue AC1-004, obtained from a chemical library using an HRE-dependent cell-based assay in colorectal carcinoma HCT-116 cells. The accumulation of hypoxia-induced HIF-1α was inhibited by compound AC1-004 in various cancer cells, including HCT-116, MDA-MB435, SK-HEP1, and Caki-1. Further, AC1-004 down-regulated VEGF and EPO, target genes of HIF-1, and inhibited in vitro tube formation of HUVEC, suggesting its potential inhibitory activity on angiogenesis. Importantly, AC1-004 was found to regulate the stability of HIF-1α through the Hsp90-Akt pathway, leading to the degradation of HIF-1α. An in vivo antitumor study demonstrated that AC1-004 reduced tumor size significantly (i.e., by 58.6%), without severe side effects. These results suggest the benzimidazole analogue AC1-004 is a novel HIF inhibitor that targets HIF-1α via the Hsp90-Akt pathway, and that it can be used as a new lead in developing anticancer drugs.     

shRNA interference for extracellular signal-regulated kinase 2 can inhibit the growth of esophageal cancer cell line Eca109

Background: Esophageal squamous cell carcinoma is one of the most common digestive tract cancers with 5-year survival rate less than 10% owing to its poor prognosis. Mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway has been mainly involved in the pathogenesis of various cancers. In present study, we investigated the role of ERK2 in human esophageal cancer cell line Eca109.

Methods: Short-hairpin RNA (shRNA) interference vector targeted ERK2 was constructed using pGeneclip U1 hairpin cloning systems, then transfected into Eca109 cell line. The transfection efficiency was observed by fluorescence microscope and cell growth after transfection with shRNA-ERK2 vector was determined by methylthiazolyl blue tetrazolium (MTT) assay. The ERK2 expression after transfection was detected by western-blotting. The cell apoptosis and cell-cycle was analyzed by flow cytometry. The role of p-ERK2 was confirmed by immunohistochemistry and soft agar colony formation assay.

Results: The growth of Eca109 transfected with shRNA-ERK2 vector was obviously inhibited compared to control group via MTT analysis. The inhibition rate after transfection with shRNA-ERK2 for 96?h was 10.45%, the expression of ERK2 was obviously reduced compared to the control analyzed by western-blot, cell apoptosis was 9.7% (compared to control, P?<?0.05), and cell-cycle was arrested at G1 phase.

Conclusions: In present study we demonstrated for the first time that transfection with shRNA-ERK2 targeted ERK2 into Eca109 cells can inhibit growth of Eca109, inducing cell apoptosis and influencing cell-cycle. Together, these results we obtained suggested that ERK2 plays an important role in cell growth of Eca109.     

Coumestrol suppresses hypoxia inducible factor 1α by inhibiting ROS mediated sphingosine kinase 1 in hypoxic PC-3 prostate cancer cells

Among many signals to regulate hypoxia inducible factor 1α (HIF-1α), sphingosine kinase 1 (SPHK1) is also involved in various biological activities such as cell growth, survival, invasion, angiogenesis, and carcinogenesis. Thus, in the present study, molecular mechanisms of coumestrol were investigated on the SPHK1 and HIF-1α signaling pathway in hypoxic PC-3 prostate cancer cells. Coumestrol significantly suppressed SPHK1 activity and accumulation of HIF-1α in a time- and concentration-dependent manner in hypoxic PC-3 cells. In addition, coumestrol inhibited the phosphorylation status of AKT and glycogen synthase kinase-3β (GSK 3β) signaling involved in cancer metabolism. Furthermore, SPHK1 siRNA transfection, sphigosine kinase inhibitor (SKI), reactive oxygen species (ROS) enhanced the inhibitory effect of coumestrol on the accumulation of HIF-1α and the expression of pAKT and pGSK 3β in hypoxic PC-3 cells by combination index. Overall, our findings suggest that coumestrol suppresses the accumulation of HIF-1α via suppression of SPHK1 pathway in hypoxic PC-3 cells.