Original Paper Acta Haematol 2014;131:102–111 DOI: 10.1159/000354770
Received: February 27, 2013 Accepted after revision: July 8, 2013 Published online: October 18, 2013
Celastrol Inhibits Lipopolysaccharide-Induced Angiogenesis by Suppressing TLR4-Triggered Nuclear Factor-Kappa B Activation Haiwen Ni a Wanzhou Zhao b Xiangtu Kong a Haitao Li c Jian Ouyang d a
Affiliated Hospital of Nanjing University of TCM, b Sino-EU Biomedical Innovation Center (SEBIC), OG Pharma Corporation, c Nanjing University of Chinese Medicine, and d Department of Hematology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, China
Key Words Celastrol · Human multiple myeloma · Angiogenesis · Toll-like receptor 4 · Nuclear factor-kappa B
Abstract Celastrol is an active compound extracted from the root bark of the traditional Chinese medicine Tripterygium wilfordii Hook F. In this study, we investigated the effect of celastrol on lipopolysaccharide (LPS)-activated LP-1 human multiple myeloma cell-induced angiogenesis, and identified its molecular mechanism of action. Migration of human umbilical vein endothelial cells (HUVECs) was tested using a wound-healing assay. HUVEC invasion was assayed using a Transwell chamber. Cell surface expression of Toll-like receptor 4 (TLR4) was analyzed by flow cytometry. Angiogenic factor vascular endothelial growth factor (VEGF) level was quantified by LUMINEX and protein expression was analyzed by Western blot. Translocation of nuclear factor-kappa B (NF-κB) was observed by fluorescence microscopy. Celastrol inhibited LPS-stimulated LP-1 human multiple myeloma-induced HUVEC migration and invasion in a concentration-dependent manner. Wound diameters increased by 72.9, 165.4 and 246.2% at 0.025, 0.05 and 0.1 μM, respectively, compared to LPS alone. A 45–74% inhibition of LPS-dependent cell invasion was achieved in the presence of 0.025–0.1 μM celastrol. Celastrol significantly downregulated LPS-induced TLR4 expression and inhibited
© 2013 S. Karger AG, Basel 0001–5792/14/1312–0102$39.50/0 E-Mail [email protected] www.karger.com/aha
LPS-induced VEGF secretion in LP-1 cells. VEGF levels decreased by 64.8, 84.4 and 92.9% after coexposure to celastrol at 0.025, 0.05 and 0.1 μM, respectively, compared to LPS alone. Celastrol also inhibited the IκB kinase (IKK)/NF-κB pathway induced by LPS. Protein levels of NF-κB p65, IKKα and IκB-α decreased in a dose-dependent manner after coexposure to celastrol. Celastrol also blocked nuclear translocation of the p65 subunit. These results suggest that celastrol inhibits LPSinduced angiogenesis by suppressing TLR4-triggered NF-κB activation. © 2013 S. Karger AG, Basel
Introduction
Multiple myeloma (MM) is a malignant B cell neoplasia caused by the proliferation of clonal plasma cells, primarily in the bone marrow (BM) [1]. Previous studies indicate that inflammation is involved in its pathogenesis [2, 3]. In support of this hypothesis, autoimmune diseases as well as infectious and inflammatory disorders have all been associated with an elevated risk of MM [4]. Chronic infection leading to unresolved inflammation is an important contributor to cellular transformation, tumorigenesis and tumor progression in MM [5, 6]. The hallmarks of cancer-related inflammation include the presence of inflammatory cells and inflammatory mediaJian Ouyang Department of Hematology, Nanjing Drum Tower Hospital The Affiliated Hospital of Nanjing University Medical School 321 Zhongshan Road, Nanjing 210008 (China) E-Mail ouyangjiangulou @ gmail.com
Celastrol Inhibits Lipopolysaccharide
Color version available online
tors such as chemokines, cytokines, prostaglandins and cell adhesion molecules in tumor tissues. Tissue remodeling and angiogenesis occur during the chronic inflammatory response in an attempt to repair damaged tissue [7, 8]. Angiogenesis is a constant hallmark of MM progression that can markedly affect disease prognosis [9, 10]. The pathophysiology of MM-induced angiogenesis is complex. It involves the production of angiogenic cytokines by plasma cells and their subsequent interaction within the inflammatory microenvironment [11, 12]. Previous studies have shown that chronic inflammation is linked to angiogenesis [13]. It has also been proposed that Toll-like receptors (TLRs) may play an important role in tumor development and progression caused by chronic inflammation [14]. Lipopolysaccharide (LPS) is an important structural component of the outer membrane of Gram-negative bacteria. It has been shown to act as an inflammatory mediator which induces angiogenesis in several chronic inflammatory diseases [15]. LPS-induced angiogenesis may also involve TLRs [16]. Other studies suggest that LPS/ TLR-activated signaling pathways may involve nuclear factor-kappa B (NF-κB), which itself has been linked to inflammation, transformation, proliferation, angiogenesis, invasion, metastasis, chemoresistance and radioresistance [17–20]. The tripterine celastrol is a functional ingredient originally identified and extracted from the root bark of Tripterygium wilfordii Hook F, used in traditional Chinese medicine. It has been used in the treatment of chronic inflammation and autoimmune diseases such as arthritis, lupus erythematosus and sclerosis [21]. It has been shown to inhibit cell proliferation and promote cell death in breast [22], colon [23] and prostate cancers [24, 25], oral squamous cell carcinoma [26], glioma [27], melanoma [28] and leukemia [29]. It also inhibits angiogenesis [27, 30–33], possibly by inhibition of the AKT/mammalian target of rapamycin pathway [34] or by interference with the HIF-1α pathway [31], causing suppression of vascular endothelial growth factor (VEGF) [35]. However, it remains unknown whether celastrol is able to suppress LPS-mediated induction of angiogenesis in chronic inflammatory diseases. Its molecular mechanisms also remain poorly understood and warrant further investigation. In this study, we investigated whether celastrol could interfere with LPS-induced signaling in MM cells and prevent angiogenesis. We also investigated its possible mechanism of action.
O CH3 O
CH3
H3 C
H3C
OH
CH3
HO CH3
Fig. 1. Chemical structures of celastrol. Celastrol is a natural trit-
erpenoid quinone methide isolated from the plant genuses of Celastrus, Maytenus and Tripterygium, all of which are indigenous to China.
Material and Methods Reagents A 100-mM solution of celastrol (Sigma, MW450.61; fig. 1) was prepared in dimethyl sulfoxide (DMSO) and stored as small aliquots at –20 ° C. Subsequent dilutions were made in cell culture medium. The same proportion of DMSO/culture medium was added to the controls. The final DMSO content was
Received: February 27, 2013 Accepted after revision: July 8, 2013 Published online: October 18, 2013
Celastrol Inhibits Lipopolysaccharide-Induced Angiogenesis by Suppressing TLR4-Triggered Nuclear Factor-Kappa B Activation Haiwen Ni a Wanzhou Zhao b Xiangtu Kong a Haitao Li c Jian Ouyang d a
Affiliated Hospital of Nanjing University of TCM, b Sino-EU Biomedical Innovation Center (SEBIC), OG Pharma Corporation, c Nanjing University of Chinese Medicine, and d Department of Hematology, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, China
Key Words Celastrol · Human multiple myeloma · Angiogenesis · Toll-like receptor 4 · Nuclear factor-kappa B
Abstract Celastrol is an active compound extracted from the root bark of the traditional Chinese medicine Tripterygium wilfordii Hook F. In this study, we investigated the effect of celastrol on lipopolysaccharide (LPS)-activated LP-1 human multiple myeloma cell-induced angiogenesis, and identified its molecular mechanism of action. Migration of human umbilical vein endothelial cells (HUVECs) was tested using a wound-healing assay. HUVEC invasion was assayed using a Transwell chamber. Cell surface expression of Toll-like receptor 4 (TLR4) was analyzed by flow cytometry. Angiogenic factor vascular endothelial growth factor (VEGF) level was quantified by LUMINEX and protein expression was analyzed by Western blot. Translocation of nuclear factor-kappa B (NF-κB) was observed by fluorescence microscopy. Celastrol inhibited LPS-stimulated LP-1 human multiple myeloma-induced HUVEC migration and invasion in a concentration-dependent manner. Wound diameters increased by 72.9, 165.4 and 246.2% at 0.025, 0.05 and 0.1 μM, respectively, compared to LPS alone. A 45–74% inhibition of LPS-dependent cell invasion was achieved in the presence of 0.025–0.1 μM celastrol. Celastrol significantly downregulated LPS-induced TLR4 expression and inhibited
© 2013 S. Karger AG, Basel 0001–5792/14/1312–0102$39.50/0 E-Mail [email protected] www.karger.com/aha
LPS-induced VEGF secretion in LP-1 cells. VEGF levels decreased by 64.8, 84.4 and 92.9% after coexposure to celastrol at 0.025, 0.05 and 0.1 μM, respectively, compared to LPS alone. Celastrol also inhibited the IκB kinase (IKK)/NF-κB pathway induced by LPS. Protein levels of NF-κB p65, IKKα and IκB-α decreased in a dose-dependent manner after coexposure to celastrol. Celastrol also blocked nuclear translocation of the p65 subunit. These results suggest that celastrol inhibits LPSinduced angiogenesis by suppressing TLR4-triggered NF-κB activation. © 2013 S. Karger AG, Basel
Introduction
Multiple myeloma (MM) is a malignant B cell neoplasia caused by the proliferation of clonal plasma cells, primarily in the bone marrow (BM) [1]. Previous studies indicate that inflammation is involved in its pathogenesis [2, 3]. In support of this hypothesis, autoimmune diseases as well as infectious and inflammatory disorders have all been associated with an elevated risk of MM [4]. Chronic infection leading to unresolved inflammation is an important contributor to cellular transformation, tumorigenesis and tumor progression in MM [5, 6]. The hallmarks of cancer-related inflammation include the presence of inflammatory cells and inflammatory mediaJian Ouyang Department of Hematology, Nanjing Drum Tower Hospital The Affiliated Hospital of Nanjing University Medical School 321 Zhongshan Road, Nanjing 210008 (China) E-Mail ouyangjiangulou @ gmail.com
Celastrol Inhibits Lipopolysaccharide
Color version available online
tors such as chemokines, cytokines, prostaglandins and cell adhesion molecules in tumor tissues. Tissue remodeling and angiogenesis occur during the chronic inflammatory response in an attempt to repair damaged tissue [7, 8]. Angiogenesis is a constant hallmark of MM progression that can markedly affect disease prognosis [9, 10]. The pathophysiology of MM-induced angiogenesis is complex. It involves the production of angiogenic cytokines by plasma cells and their subsequent interaction within the inflammatory microenvironment [11, 12]. Previous studies have shown that chronic inflammation is linked to angiogenesis [13]. It has also been proposed that Toll-like receptors (TLRs) may play an important role in tumor development and progression caused by chronic inflammation [14]. Lipopolysaccharide (LPS) is an important structural component of the outer membrane of Gram-negative bacteria. It has been shown to act as an inflammatory mediator which induces angiogenesis in several chronic inflammatory diseases [15]. LPS-induced angiogenesis may also involve TLRs [16]. Other studies suggest that LPS/ TLR-activated signaling pathways may involve nuclear factor-kappa B (NF-κB), which itself has been linked to inflammation, transformation, proliferation, angiogenesis, invasion, metastasis, chemoresistance and radioresistance [17–20]. The tripterine celastrol is a functional ingredient originally identified and extracted from the root bark of Tripterygium wilfordii Hook F, used in traditional Chinese medicine. It has been used in the treatment of chronic inflammation and autoimmune diseases such as arthritis, lupus erythematosus and sclerosis [21]. It has been shown to inhibit cell proliferation and promote cell death in breast [22], colon [23] and prostate cancers [24, 25], oral squamous cell carcinoma [26], glioma [27], melanoma [28] and leukemia [29]. It also inhibits angiogenesis [27, 30–33], possibly by inhibition of the AKT/mammalian target of rapamycin pathway [34] or by interference with the HIF-1α pathway [31], causing suppression of vascular endothelial growth factor (VEGF) [35]. However, it remains unknown whether celastrol is able to suppress LPS-mediated induction of angiogenesis in chronic inflammatory diseases. Its molecular mechanisms also remain poorly understood and warrant further investigation. In this study, we investigated whether celastrol could interfere with LPS-induced signaling in MM cells and prevent angiogenesis. We also investigated its possible mechanism of action.
O CH3 O
CH3
H3 C
H3C
OH
CH3
HO CH3
Fig. 1. Chemical structures of celastrol. Celastrol is a natural trit-
erpenoid quinone methide isolated from the plant genuses of Celastrus, Maytenus and Tripterygium, all of which are indigenous to China.
Material and Methods Reagents A 100-mM solution of celastrol (Sigma, MW450.61; fig. 1) was prepared in dimethyl sulfoxide (DMSO) and stored as small aliquots at –20 ° C. Subsequent dilutions were made in cell culture medium. The same proportion of DMSO/culture medium was added to the controls. The final DMSO content was
