J Gastric Cancer.  2011 Mar;11(1):16-22.

Eupatilin Inhibits Gastric Cancer Cell Growth by Blocking STAT3-Mediated VEGF Expression

• Cheong JH
• Hong SY
• Zheng Y
• Noh SH

Abstract

PURPOSE
Eupatilin is an antioxidative flavone and a phytopharmaceutical derived from Artemisia asiatica. It has been reported to possess anti-tumor activity in some types of cancer including gastric cancer. Eupatilin may modulate the angiogenesis pathway which is part of anti-inflammatory effect demonstrated in gastric mucosal injury models. Here we investigated the anti-tumor effects of eupatilin on gastric cancer cells and elucidated the potential underlying mechanism whereby eupatilin suppresses angiogenesis and tumor growth.
MATERIALS AND METHODS
The impact of eupatilin on the expression of angiogenesis pathway proteins was assessed using western blots in MKN45 cells. Using a chromatin immunoprecipitation assay, we tested whether eupatilin affects the recruitment of signal transducer and activator of transcription 3 (STAT3), aryl hydrocarbon receptor nuclear translocator (ARNT) and hypoxia-inducible factor-1alpha (HIF-1alpha) to the human VEGF promoter. To investigate the effect of eupatilin on vasculogenesis, tube formation assays were conducted using human umbilical vein endothelial cells (HUVECs). The effect of eupatilin on tumor suppression in mouse xenografts was assessed.
RESULTS
Eupatilin significantly reduced VEGF, ARNT and STAT3 expression prominently under hypoxic conditions. The recruitment of STAT3, ARNT and HIF-1alpha to the VEGF promoter was inhibited by eupatilin treatment. HUVECs produced much foreshortened and severely broken tubes with eupatilin treatment. In addition, eupatilin effectively reduced tumor growth in a mouse xenograft model.
CONCLUSIONS
Our results indicate that eupatilin inhibits angiogenesis in gastric cancer cells by blocking STAT3 and VEGF expression, suggesting its therapeutic potential in the treatment of gastric cancer.

Keyword

Eupatilin; Stomach neoplasms; Angiogenesis; STAT3; Vascular endothelial growth factor A

MeSH Terms

Animals
Artemisia
Aryl Hydrocarbon Receptor Nuclear Translocator
Blotting, Western
Chromatin Immunoprecipitation
Flavones
Flavonoids
Human Umbilical Vein Endothelial Cells
Humans
Mice
Proteins
STAT3 Transcription Factor
Stomach Neoplasms
Transplantation, Heterologous
Vascular Endothelial Growth Factor A
Aryl Hydrocarbon Receptor Nuclear Translocator
Flavones
Flavonoids
Proteins
STAT3 Transcription Factor
Vascular Endothelial Growth Factor A

Figure

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  Fig. 1 Eupatilin inhibits the expression of angiogenesis gene products, HIF-1α, ARNT, STAT3 and VEGF. MKN45 cells were treated with indicated concentration of eupatilin before being cultured for 6 hr under normoxic (20% O2 v/v) or hypoxic (1% O2 v/v) conditions. Expression levels of HIF-1α, ARNT, STAT3, phospho-STAT3 were analyzed by immunoblotting. β-actin was used for loading control. Proteins were visualized by enhanced chemiluminescence. HIF-1α = hypoxia-inducible factor-1α; ARNT = aryl hydrocarbon receptor nuclear translocator; STAT3 = signal transducer and activator of transcription 3; VEGF = vascular endothelial growth factor.

• 

  Fig. 2 STAT3 interacts with HIF-1 α and eupatilin inhibits STAT3 recruitment to the VEGF promoter. (A) MKN45 cells were incubated under normoxic or hypoxic conditions and cell lysates were immunoprecipitated with an anti-HIF-1α antibody, followed by Western blotting with an anti-ARNT, STAT3, and VEGF antibodies. STAT3, ARNT and VEGF co-precipitated with HIF-1α in hypoxic cells. (B) HIF-1α, STAT3 and ARNT are recruited to the VEGF promoter. Cross-linked, sheared chromatin was prepared from MKN45 cells grown in the absence or presence of eupatilin overnight. Chromatin samples were then immunoprecipitated with the antibodies indicated on the right. The precipitates were subjected to PCR analysis using primer pairs spanning the human VEGF promoter. The control was the PCR product of chromatin obtained before immunoprecipitation. The recruitment of HIF-1α, STAT3 and ARNT was greater under hypoxic conditions. Eupatilin treatment significantly inhibited the recruitment of STAT3, ARNT, and HIF-1α to the VEGF promoter region. IP = immunoprecipitation; HIF-1α = hypoxia-inducible factor-1α; ARNT = aryl hydrocarbon receptor nuclear translocator; STAT3 = signal transducer and activator of transcription 3; VEGF = vascular endothelial growth factor.

• 

  Fig. 3 Eupatilin inhibits in vitro capillary tube formation of HUVECs. HUVECs were seeded into 24-well plates coated with Matrigel at a density of 4×104 per well and cultured in the conditioned media either untreated or treated with eupatilin (50 or 100 µM). Aft er 16-h of incubation, photography was taken. (A) The representative photographs were shown (original magnification, ×40). (B) Summary of in vitro vasculogenesis assay reveals that eupatilin reduced the hypoxia-induced vascular formation in dose dependent manner. Each value represents mean±SD of 3 independent experiments (*P<0.001). All experiments were performed in triplicates. HUVEcs = human umbilical vein endothelial cells.

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  Fig. 4 The effect of eupatilin on the growth of xenografted human gastric cancer. MKN45 xenograft s on the flanks of mice treated with vehicle only (T) or with eupatilin (EPT) for 2 weeks. EPT (10 mg/kg) in 200 µl HBSS or 200 µl of HBSS only were administered three times a week by intraperitoneal injection. (A) Representative photograph of mice treated with either vehicle only (T) or EPT. The arrows indicate MKN45 tumors on mouse flank. (B) Ex vivo tumor weight of tumors from 4A. Each value represents mean±SD (*P<0.05).

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