An Chi Tsai

Moscatilin, a bibenzyl derivative from the India orchid Dendrobrium loddigesii, suppresses tumor angiogenesis and growth in vitro and in vivo

Attacking angiogenesis is considered an effective strategy for controls the expansion and metastasis of tumors and other related-diseases. The aim of this study was to assess the effects of moscatilin, a bibenzyl derivative, on VEGF and bFGF-induced angiogenesis in cultured human umbilical vein endothelial cells (HUVECs) in vitro and in vivo. Moscatilin significantly inhibited growth of lung cancer cell line A549 (NSCLC) and suppressed growth factor-induced neovascularization. In addition, VEGF- and bFGF-induced cell proliferation, migration, and tube formation of HUVECs was markedly inhibited by moscatilin. Western blotting analysis of cell signaling molecules indicated that moscatilin inhibited ERK1/2, Akt, and eNOS signaling pathways in HUVECs. These results suggest that inhibition of angiogenesis by moscatilin may be a major mechanism in cancer therapy.

Dehydrocostuslactone Suppresses Angiogenesis In Vitro and In Vivo through Inhibition of Akt/GSK-3β and mTOR Signaling Pathways

The traditional Chinese medicine component dehydrocostuslactone (DHC) isolated from Saussurea costus (Falc.) Lipschitz, has been shown to have anti-cancer activity. Angiogenesis is an essential process in the growth and progression of cancer. In this study, we demonstrated, for the first time, the anti-angiogenic mechanism of action of DHC to be via the induction of cell cycle progression at the G0/G1 phase due to abrogation of the Akt/glycogen synthase kinase-3β (GSK-3β)/cyclin D1 and mTOR signaling pathway. First, we demonstrated that DHC has an anti-angiogenic effect in the matrigel-plug nude mice model and an inhibitory effect on human umbilical vein endothelial cell (HUVEC) proliferation and capillary-like tube formation in vitro. DHC caused G0/G1 cell cycle arrest, which was associated with the down-regulation of cyclin D1 expression, leading to the suppression of retinoblastoma protein phosphorylation and subsequent inhibition of cyclin A and cdk2 expression. With respect to the molecular mechanisms underlying the DHC-induced cyclin D1 down-regulation, this study demonstrated that DHC significantly inhibits Akt expression, resulting in the suppression of GSK-3β phosphorylation and mTOR expression. These effects are capable of regulating cyclin D1 degradation, but they were significantly reversed by constitutively active myristoylated (myr)-Akt. Furthermore, the abrogation of tube formation induced by DHC was also reversed by overexpression of Akt. And the co-treatment with LiCl and DHC significantly reversed the growth inhibition induced by DHC. Taken together, our study has identified Akt/GSK-3β and mTOR as important targets of DHC and has thus highlighted its potential application in angiogenesis-related diseases, such as cancer.

Activated PAR-2 Regulates Pancreatic Cancer Progression through ILK/HIF-α–Induced TGF-α Expression and MEK/VEGF-A–Mediated Angiogenesis

Tissue factor initiates the process of thrombosis and activates cell signaling through protease-activated receptor-2 (PAR-2). The aim of this study was to investigate the pathological role of PAR-2 signaling in pancreatic cancer. We first demonstrated that activated PAR-2 up-regulated the protein expression of both hypoxia-inducible factor-1α (HIF-1α) and HIF-2α, resulting in enhanced transcription of transforming growth factor-α (TGF-α). Down-regulation of HIFs-α by siRNA or YC-1, an HIF inhibitor, resulted in depleted levels of TGF-α protein. Furthermore, PAR-2, through integrin-linked kinase (ILK) signaling, including the p-AKT, promoted HIF protein expression. Diminishing ILK by siRNA decreased the levels of PAR-2-induced p-AKT, HIFs-α, and TGF-α; our results suggest that ILK is involved in the PAR-2-mediated TGF-α via an HIF-α-dependent pathway. Furthermore, the culture medium from PAR-2-treated pancreatic cancer cells enhanced human umbilical vein endothelial cell proliferation and tube formation, which was blocked by the MEK inhibitor, PD98059. We also found that activated PAR-2 enhanced tumor angiogenesis through the release of vascular endothelial growth factor-A (VEGF-A) from cancer cells, independent of the ILK/HIFs-α pathways. Consistent with microarray analysis, activated PAR-2 induced TGF-A and VEGF-A gene expression. In conclusion, the activation of PAR-2 signaling induced human pancreatic cancer progression through the induction of TGF-α expression by ILK/HIFs-α, as well as through MEK/VEGF-A-mediated angiogenesis, and it plays a role in the interaction between cancer progression and cancer-related thrombosis.

CHM-1, a new vascular targeting agent, induces apoptosis of human umbilical vein endothelial cells via p53-mediated death receptor 5 up-regulation.

CHM-1 (2′-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone) has been identified as a potent antitumor agent in human hepatocellular carcinoma; however, its role in tumor angiogenesis is unclear. This study investigated the effects of CHM-1 and the mechanisms by which it exerts its antiangiogenic and vascular disrupting properties. Using a xenograft model antitumor assay, we found that CHM-1 significantly inhibits tumor growth and microvessel formation. Flow cytometry, immunofluorescence microscopy, and cell death enzyme-linked immunosorbent assay kit revealed that CHM-1 inhibits growth of human umbilical vein endothelial cells (HUVEC) by induction of apoptotic cell death in a concentration-dependent manner. CHM-1 also suppresses HUVEC migration and capillary-like tube formation. We were able to correlate CHM-1-induced apoptosis in HUVEC with the cleavage of procaspase-3, -7, and -8, as well as with the cleavage of poly(ADP-ribose) polymerase by Western blotting assay. Such sensitization was achieved through up-regulation of death receptor 5 (DR5) but not DR4 or Fas. CHM-1 was also capable of increasing the expression level of p53, and most importantly, the induction of DR5 by CHM-1 was abolished by p53 small interfering RNA. Taken together, the results of this study indicate that CHM-1 exhibits vascular targeting activity associated with the induction of DR5-mediated endothelial cell apoptosis through p53 up-regulation, which suggests its potential as an antivascular and antitumor therapeutic agent.

Aciculatin induces p53-dependent apoptosis via MDM2 depletion in human cancer cells in vitro and in vivo

Aciculatin, a natural compound extracted from the medicinal herb Chrysopogon aciculatus, shows potent anti-cancer potency. This study is the first to prove that aciculatin induces cell death in human cancer cells and HCT116 mouse xenografts due to G1 arrest and subsequent apoptosis. The primary reason for cell cycle arrest and cell death was p53 accumulation followed by increased p21 level, dephosphorylation of Rb protein, PUMA expression, and induction of apoptotic signals such as cleavage of caspase-9, caspase-3, and PARP. We demonstrated that p53 allele-null (−/−) (p53-KO) HCT116 cells were more resistant to aciculatin than cells with wild-type p53 (+/+). The same result was achieved by knocking down p53 with siRNA in p53 wild-type cells, indicating that p53 plays a crucial role in aciculatin-induced apoptosis. Although DNA damage is the most common event leading to p53 activation, we found only weak evidence of DNA damage after aciculatin treatment. Interestingly, the aciculatin-induced downregulation of MDM2, an important negative regulator of p53, contributed to p53 accumulation. The anti-cancer activity and importance of p53 after aciculatin treatment were also confirmed in the HCT116 xenograft models. Collectively, these results indicate that aciculatin treatment induces cell cycle arrest and apoptosis via inhibition of MDM2 expression, thereby inducing p53 accumulation without significant DNA damage and genome toxicity.

EPOX Inhibits Angiogenesis by Degradation of Mcl-1 through ERK Inactivation

Purpose: Antiangiogenic therapy is considered as an effective strategy for controlling the growth and metastasis of tumors. Among a myriad of biological activities described for xanthone derivatives, the anticancer activity is quite remarkable, but the molecular mechanism is not clearly resolved. In the present study, we investigated the antiangiogenic mechanism of 3,6-di(2,3-epoxypropoxy)xanthone (EPOX), a novel Mcl-1 targeting drug. Experimental Design: To evaluate the antiangiogenic activity of EPOX, we did cell viability, cell cycle, tube formation assay in vitro, and Matrigel plug assay in vivo. To evaluate the effect of EPOX on the endothelial signaling pathway, we did immunoblotting, immunoprecipitation, and immunofluorescence analysis. Intracellular glutathione levels were determined with the use of monochlorobimane, a glutathione-specific probe. Results: EPOX induced endothelial cell apoptosis in association with proteasome-dependent Mcl-1 degradation. Down-regulation of Mcl-1 resulted in an increase in Mcl-1–free Bim, activation of Bax, and then signaling of mitochondria-mediated apoptosis. Additionally, glutathione depletion and extracellular signal-regulated kinase (ERK) inactivation was observed in EPOX-treated cells. Glutathione supplementation reversed the inhibitory effects of EPOX on ERK, which increases the phosphorylation of Mcl-1 at T163. Overexpression of mitogen-activated protein/ERK kinase (MEK) partially reversed the effect of EPOX on Mcl-1 dephosphorylation, ubiquitination, and degradation, further implicating ERK in the regulation of Mcl-1 stability. Conclusions: This study provides evidence that EPOX induces glutathione depletion, ERK inactivation, and Mcl-1 degradation on endothelial cells, which leads to inhibition of angiogenesis. Our results suggest that EPOX is a novel antiangiogenic agent, making it a promising lead compound for further development in the treatment of angiogenesis-related pathologies.

Synergistic Interaction between the HDAC Inhibitor, MPT0E028, and Sorafenib in Liver Cancer Cells In Vitro and In Vivo

Purpose: To investigate the antitumor activities of a histone deacetylase (HDAC) inhibitor, MPT0E028, plus sorafenib in liver cancer cells in vitro and in vivo. Experimental Design: Different liver cancer cell lines were exposed to sorafenib in the presence or absence of MPT0E028, and cell viability was determined by MTT assay. Effects of combined treatment on cell cycle and intracellular signaling pathways were assessed by flow cytometry and Western blot analysis. The Hep3B xenograft model was used to examine the antitumor activity in vivo. Results: Our data indicate that sorafenib and MPT0E028 synergistically reduced cell viability in liver cancer cells, and also markedly induced apoptotic cell death in these cells, as evidenced by the cleavage of caspase-3, PARP, and DNA fragmentation. MPT0E028 altered the global modifications of histone and nonhistone proteins regardless of the presence of sorafenib. However, sorafenib blocked MPT0E028-induced Erk activation and its downstream signaling cascades, such as Stat3 phosphorylation (Ser727) and Mcl-1 upregulation. Ectopic expression of constitutively active Mek successively reversed the apoptosis triggered by the combined treatment. Pharmacologic inhibition of Mek by PD98059 potentiated MPT0E028-induced apoptosis, suggesting that the synergistic interaction between MPT0E028 and sorafenib occurs at least partly through inhibition of Erk signaling. The data demonstrated that transcriptional activation of fibroblast growth factor receptor 3 (FGFR3) contributes to MPT0E028-mediated Erk phosphorylation. Finally, MPT0E028 plus sorafenib significantly improved the tumor growth delay (TGD) in a Hep3B xenograft model. Conclusions: These findings suggest that MPT0E028 in combination with sorafenib has significant anti-hepatocellular carcinoma activity in preclinical models, potentially suggesting a novel therapeutic strategy for patients with advanced hepatocellular carcinoma. Clin Cancer Res; 20(5); 1274–87. ©2014 AACR.

The inhibition of angiogenesis and tumor growth by denbinobin is associated with the blocking of insulin-like growth factor-1 receptor signaling.

Denbinobin, which is a phenanthraquinone derivative present in the stems of Ephemerantha lonchophylla, has been demonstrated to display antitumor activity. Recent reports suggest that the enhanced activity of insulin-like growth factor-1 receptor (IGF-1R) is closely associated with tumor angiogenesis and growth. This study aims at investigating the roles of denbinobin in suppressing these effects and at further elucidating the underlying molecular mechanisms. In the present study, we used an in vivo xenograft model antitumor and the Matrigel implant assays to show that denbinobin suppresses lung adenocarcinoma A549 growth and microvessel formation. Additionally, crystal violet and capillary-like tube formation assays indicated that denbinobin selectively inhibits insulin-like growth factor-1 (IGF-1)-induced proliferation (GI50=1.3×10⁻⁸ M) and tube formation of human umbilical vascular endothelial cells (HUVECs) without influencing the effect of epidermal growth factor; vascular endothelial growth factor and basic fibroblast growth factor. Furthermore, denbinobin inhibited the IGF-1-induced migration of HUVECs in a concentration-dependent fashion. Western blotting and immunoprecipitation demonstrated that denbinobin causes more efficient inhibition of IGF-1-induced activation of IGF-1R and its downstream signaling targets, including , extracellular signal-regulated kinase, Akt, mTOR, p70S6K, 4EBP and cyclin D1. All of our results provide evidences that denbinobin suppresses the activation of IGF-1R and its downstream signaling pathway, which leads to the inhibition of angiogenesis. Our findings suggest that denbinobin may be a novel IGF-1R kinase inhibitor and has potential therapeutic abilities for angiogenesis-related diseases such as cancer.

The indazole derivative YD-3 specifically inhibits thrombin-induced angiogenesis in vitro and in vivo

Angiogenesis is a process that involves endothelial cell proliferation, migration, invasion, and tube formation, and the inhibition of these processes has implications for angiogenesis-mediated disorders. The purpose of this study was to examine the antiangiogenic efficacy of YD-3 [1-benzyl-3(ethoxycarbonylphenyl)-indazole], a selective thrombin inhibitor, on thrombin-induced endothelial cell proliferation and neoangiogenesis in a murine Matrigel model. First, the effect of YD-3 on angiogenesis was evaluated in vivo using the mouse Matrigel implant model. Plugs treated with 1 and 10 &mgr;M of YD-3 inhibited neovascularization induced by thrombin, protease-activated receptor (PAR) 1, and PAR-4, but not by vascular endothelial growth factor, in a concentration-dependent manner over 7 days. These results indicate that YD-3 has specific antiangiogenic activity on thrombin. YD-3 also inhibited (in a concentration-dependent manner) the ability of thrombin, PAR-1, and PAR-4, but not PAR-2, to induce the proliferation of human umbilical vascular endothelial cells, using a [3H]thymidine incorporation assay. YD-3 predominantly inhibited thrombin-induced vascular endothelial growth factor receptor 2 (Flk-1) expression, but not extracellular signal-regulated kinase 1/2 phosphorylation, using Western blot analysis. YD-3 may have benefit in elucidating pathophysiology induced by thrombin-induced angiogenesis.ABBREVIATIONS-YD-3-[1-benzyl-3(ethoxycarbonylphenyl)-indazole]; PARs - protease-activated receptors; VEGF - vascular endothelial growth factor; ERK1/2 - extracellular signal-regulated kinase 1/2; HUVECs - human umbilical vein endothelial cells; ECGs - endothelial cell growth supplements; PKC - protein kinase C; SLIGKV - PAR-2-activating peptide (SER-LEU-ILE-GLY-LYS-VAL)

In vitro and in vivo anti-tumour effects of MPT0B014, a novel derivative aroylquinoline, and in combination with erlotinib in human non-small-cell lung cancer cells

The purpose of the current study was to assess a novel anti‐cancer drug, MPT0B014, which is not a substrate for the P‐glycoprotein (P‐gp) transporter, alone and in combination with erlotinib, against human non‐small cell lung cancer (NSCLC).