INTERNATIONAL JOURNAL OF ONCOLOGY 42: 651-656, 2013 Hedyotis diffusa Willd extract suppresses Sonic hedgehog signaling leading to the inhibition of colorectal cancer angiogenesis JIUMAO LIN1,2, LIHUI WEI1,2, ALING SHEN1, QIAOYAN CAI1,2, WEI XU3, HUANG LI3, YOUZHI ZHAN1,2, ZHENFENG HONG1 and JUN PENG1,2 1Academy of Integrative Medicine Biomedical Research Center, 2Fujian Key Laboratory of Integrative Medicine on Geriatrics, 3Department of Pharmacology, Fujian University of Traditional Chinese Medicine, Minhou Shangjie, Fuzhou, Fujian 350122, P.R. China Received November 3, 2012; Accepted December 7, 2012 DOI: 10.3892/ijo.2012.1753 Abstract. Sonic hedgehog (SHH) signaling pathway promotes Introduction the process of angiogenesis, contributing to the growth and progression of many human malignancies including colorectal Colorectal cancer (CRC) is one of the most common forms of cancer (CRC), which therefore has become a promising target cancer and a leading cause of cancer-related deaths around the for cancer chemotherapy. Hedyotis diffusa Willd (HDW), as world (1). To date, chemotherapy is one of the main therapeutic a well-known traditional Chinese herbal medicine, has long approaches for patients with advanced CRC (2,3). However, both been used in China for the clinic treatment of various cancers. drug resistance and toxicity limit the effectiveness of current Recently, we reported that HDW can inhibit colorectal cancer CRC chemotherapy, thus there is a need for the development growth in vivo and in vitro via suppression of the STAT3 of new therapeutic agents. Recently, natural products received pathway. In addition, we demonstrated the anti-angiogenic great interest since they have relatively fewer side-effects and activity of HDW in vitro. To further elucidate the mecha- have been used clinically to treat various kinds of diseases nism of the tumoricidal activity of HDW, by using a CRC including cancer for thousands of years. Numerous plants mouse xenograft model we evaluated the in vivo effect of the and their constituents have been shown to possess beneficial ethanol extract of HDW (EEHDW) on tumor angiogenesis, therapeutic effects for cancer (4,5). Therefore, discovering and investigated the underlying molecular mechanisms. naturally occurring agents is a promising approach for cancer We found that EEHDW could significantly reduce intratu- chemotherapy. moral microvessel density (MVD), indicating its activity Angiogenesis plays a critical role in the growth and devel- of antitumor angiogenesis in vivo. EEHDW suppressed the opment of cancer, suggesting that anti-angiogenic therapy is activation of SHH signaling in CRC xenograft tumors since effective in inhibiting tumor progression (6-8). Tumor angio- it significantly decreased the expression of key mediators of genesis is regulated by multiple cellular signaling pathways SHH pathway. EEHDW treatment inhibited the expression of including hedgehog (HH) transduction cascade. HH pathway is the critical SHH signaling target gene VEGF-A as well as its important for embryonic development (9) and its aberrant acti- specific receptor VEGFR2. Taken together, we propose for vation is implicated as an initiating or maintaining factor in the the first time that Hedyotis diffusa Willd inhibits colorectal progression of various human cancers (9-17). Mammals have cancer growth in vivo via inhibition of SHH-mediated tumor three hedgehog homologues (Sonic hedgehog, Indian hedgehog angiogenesis. and Desert hedgehog), of which Sonic hedgehog (SHH) is the best studied. Activation of HH signaling is initiated by binding of HH to the transmembrane receptor Patched (Ptch), resulting in the release of Ptch-mediated suppression of Smoothened (Smo), Correspondence to: Dr Jun Peng, Academy of Integrative Medicine a G-protein-coupled receptor. Released Smo subsequently Biomedical Research Center, Fujian University of Traditional Chinese activates the Gli family of transcription factors that regulate Medicine, 1 Huatuo Road, Minhou Shangjie, Fuzhou, Fujian 350122, the expression of HH target genes (9,17-19), including vascular P. R. Ch i na endothelial growth factor-A (VEGF-A). VEGF-A is considered E-mail: [email protected] as one of the most potent angiogenic factors overexpressed in many human cancers (20-24). VEGF-A secreted from tumor Abbreviations: EEHDW, ethanol extract of Hedyotis diffusa Willd; CRC, colorectal cancer; IHS, immunohistochemical staining; cells primarily binds to specific receptors such as VEGFR-2 VEGF-A, vascular endothelial growth factor-A; MVD, microvessel that are located on vascular endothelial cells (EC) (17,25-27). density; SHH, sonic hedgehog signal pathway Binding of VEGF-A to VEGFR-2 in turn triggers a tyrosine kinase signaling cascade that induces EC proliferation, migra- Key words: Hedyotis diffusa Willd, Chinese medicine, colorectal tion, survival, sprouting and tube formation (17,25,28-30). Thus, cancer, angiogenesis, sonic hedgehog signal pathway inhibition of angiogenesis via modulation of SHH signaling could be a major focus for anticancer drug development. 652 LIN et al: Hedyotis diffusa Willd INHIBITS SHH-MEDIATED TUMOR ANGIOGENESIS Hedyotis diffusa Willd (HDW), belonging to the Rubiaceae ethical guidelines and the National Institutes of Health Guide family, is a medicinal herb widely distributed in Northeast concerning the Care and Use of Laboratory Animals, and Asia. As a well known traditional Chinese folk-medicine, the experiments were approved by the Institutional Animal HDW has long been used as a major component in many Care and Use committee of Fujian University of Traditional traditional Chinese medicine (TCM) formulas for the clinical Chinese Medicine. treatment of various kinds of cancer including CRC (31-33). Recently, we reported that HDW can inhibit cancer growth In vivo nude mouse xenograft study. HT-29 cells (5x106) mixed in vivo and in vitro via suppression of STAT3 pathway (3,34). with Matrigel (1:1) were subcutaneously injected in the right In addition, we proposed that HDW displays anti-angiogenic flank area of athymic nude mice to initiate tumor growth. After activity in vitro (25). However, the precise mechanism of its 5 days of xenograft implantation, mice were randomly divided anticancer activity remains largely unclear. To further elucidate into two groups (n=10) and given intra-gastric administration the mechanism of the tumoricidal activity of Hedyotis diffusa with 3 g/kg of EEHDW or saline daily, 6 days a week for Willd, here we evaluated its in vivo effect on colorectal cancer 16 days. Tumor diameters were measured at regular intervals growth and tumor angiogenesis and investigated the underlying with digital calipers and the tumor volume (V) was calculated molecular mechanisms. using the formula: V = (width)2 x length x π/6. At the end of experiment, the animals were anaesthetized and the tumor issue Materials and methods was removed. Blood was obtained aseptically from orbit. Blood- containing tubes were allowed to stand at room temperature for Materials and reagents. Dulbecco's modified Eagle's medium 5 h and serums were obtained by centrifugation at 3, 000 x g (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, for 20 min. Trypsin-EDTA and TRIzol Reagent were purchased from Invitrogen (Carlsbad, CA, USA). SuperScript II reverse tran- RNA extraction and RT-PCR analysis. Total RNA was scriptase was obtained from Promega (Madison, WI, USA). isolated from fresh tumor tissues with TRIzol Reagent. CD31, SHH, PTCH-1, SMO, Gli-1, VEGF-A and VEGFR2 anti- Oligo(dT)-primed RNA (1 µg) was reverse-transcribed with bodies, secondary antibodies were obtained from Santa Cruz SuperScript II reverse transcriptase (Promega) according to Biotechnology (Santa Cruz, CA, USA). All other chemicals, the manufacturer's instructions. The obtained cDNA was unless otherwise stated, were obtained from Sigma Chemicals used to determine the mRNA amount of SHH, PTCH, SMO, (St. Louis, MO, USA). Gli-1, VEGF-A and VEGFR2 by PCR with Taq DNA poly- merase (Fermentas). GAPDH was used as an internal control. Preparation of ethanol extract from Hedyotis diffusa Willd Samples were analyzed by gel electrophoresis (1.5% agarose). (EEHDW). Authentic plant material was purchased from The DNA bands were examined using a Gel Documentation Guo Yi Tang Chinese Herbal medicine store, Fujian, China. System (Bio-Rad, Model Gel Doc 2000, Hercules, CA, USA). The original herb was identified as Hedyotis diffusa Willd (HDW) by Dr Wei Xu at Department of Pharmacology, Immunohistochemical staining. After fixing with Fujian University of Traditional Chinese Medicine, China. 10% formaldehyde for 12 h, tumor samples were processed Ethanol extract of HDW (EEHDW) was prepared as previ- conventionally for 5-µm-thick paraffin-embedded tumor ously described (3). Briefly, 500 g of HDW was extracted slides. The slides were subjected to antigen retrieval and with 5,000 ml of 85% ethanol using a refluxing method and the endogenous peroxidase activity was quenched with filtered. The ethanol solvent was then evaporated on a rotary hydrogen peroxide. After blocking non-specific proteins evaporator (Yarong, Model RE-2000, Shanghai, China). The with normal serum in PBS, slides were incubated with rabbit resultant solution was concentrated to a relative density of 1.05 polyclonal antibodies against CD31, SHH, PTCH-1, SMO, and the dried powder of EEHDW was obtained by a spraying Gli-1, VEGF-A and VEGFR2 (all in 1:100 dilution, Santa desiccation method using a spray dryer (Buchi, Model B-290,