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Department of Experimental Research, Cancer Center, Sun Yat-sen University, Guangzhou, China (X.-F.Z., B.-F.X., J.-M.Z., G.-K.F., Z.-C.L., Y.-X.Z.)
Southern China Institute of Botany, the Chinese Academy of Sciences, Guangzhou, China (X.-Y.W., F.-X.Z., M.-F.L.)
Abstract
Antiangiogenesis is a promising strategy of cancer treatment. Vascular endothelial growth factor receptor [fetal liver kinase/kinase-inserting domain-containing receptor (KDR)] is a tyrosine kinase receptor and has been strongly implicated in tumor angiogenesis. In this study, we report that 2',4'-dihydroxy-6'-methoxy-3',5'-dimethylchalcone (ON-III), extracted from the dried flower Cleistocalyx operculatus, used in traditional Chinese medicine, reversibly inhibited KDR tyrosine kinase phosphorylation, but epidermal growth factor receptor tyrosine kinase phosphorylation was unaffected under the same concentrations of ON-III. ON-III also inhibited mitogen-activated protein kinase (MAPK) and AKT activation of KDR signal transduction in downstream molecules without reduced total MAPK and AKT. The results in vitro showed that ON-III inhibited growth of human vascular endothelial HDMEC cells in the presence of VEGF preferentially, compared with epidermal growth factor. Systemic administration of ON-III at nontoxic doses in nude mice resulted in inhibition of subcutaneous tumor growth of human hepatocarcinoma Bel7402 and lung cancer GLC-82 xenografts. The tumor vessel density decreased, as determined by immunohistochemical staining, for CD31 after ON-III treatment. These results indicated that ON-III inhibited KDR tyrosine kinase, shut down KDR-mediated signal transduction, and inhibited tumor growth of human xenografts in vivo.
Angiogenesis, the formation of new blood vessels by sprouting from pre-existing endothelium, is a significant component of a wide variety of biological processes, including embryonic vascular development and differentiation, wound healing, organ regeneration, and pathological processes including tumorigenesis (Folkman, 1990; Eberhard et al., 2000). Many growth factors involve this process. Of the numerous growth factors and cytokines that have been shown to have angiogenic effects, VEGF seems to be a pivotal factor in pathological situations that involve neovascularization (Smith et al., 2000; Foekens et al., 2001). VEGF is produced by normal and transformed cells and plays a key role in the physiology of normal vasculature and in tumor-induced angiogenesis, which makes it important to understand the mechanisms through which this mitogen promotes cell proliferation (Nakopoulou et al., 2002).
VEGF first binds to either of two tyrosine kinase receptors, Flk1/KDR or Flt1. Signaling by such receptors facilitates activation of the intrinsic tyrosine kinase followed by autophosphorylation of tyrosine residues in the cytoplasmic signaling molecules that connect the activated receptor to transduction cascades and promote cellular responses (Geng et al., 2001; Colavitti et al., 2002; Dias et al., 2002). KDR receptor possesses intrinsic tyrosine kinase activity that is stimulated after ligand binding and receptor dimerization and is mandatory for transmission of a cytoplasmic signaling response (Binetruy-Tournaire et al., 2000; Zhu et al., 2001). The activation of KDR has also been shown to correlate with tumor growth, lymph node metastasis, and resistance to chemotherapy in many kinds of tumors (Thakker et al., 1999; Baek et al., 2000). Binding of VEGF to KDR receptor on the surface of endothelial cells facilitates its autophosphorylation of the protein tyrosine kianse domain (Takahashi et al., 1999; Ichikura et al., 2001; Zhang et al., 2002). Activation of KDR by VEGF results in activation of phosphatidyl inositol 3-kinase/AKT, MAPK, and protein kinase C; the ultimate cellular response is DNA synthesis and cell proliferation. Vascular endothelial growth factor receptors (VEGFRs) are up-regulated on endothelium at sites of active angiogenesis, providing an opportunity for selective therapeutic intervention (Hanahan, 1997; Zhang et al., 2002).
To exploit targeting at KDR tyrosine kinase for cancer treatment, we have been actively pursuing small molecule therapeutic strategies targeted at KDR receptor-mediated signal transduction pathway (Spiekermann et al., 2002; Wedge et al., 2002). In this report, we describe that ON-III, which is a chalcone derivative from the dried flower Cleistocalyx operculatus, used in traditional Chinese medicine, could inhibit tyrosine phosphorylation of KDR and growth of human cancer xenografts. Although some synthetic molecules have been reported to inhibit the VEGFR kinase, this is a low molecular weight inhibitor of the VEGF/VEGF receptor system reported to be active at inhibiting VEGF-mediated processes from traditional Chinese medicine.
Materials and Methods
Cell Culture and Reagents. The human dermal microvessel endothelial cells (HDMEC) were prepared as described perviously (Qian et al., 1999). HDMEC cells, human lung adenocarcinoma GLC-82 cells, and hepatocarcinoma cell line Bel7402 cells were cultivated in RMPI 1640 medium (Invitrogen, Carlsbad, CA) (Zhu et al., 2000). Cells were grown in an incubator at 37°C under 5% CO2 in air. KDR, anti-EGFR, and anti-tyrosine antibodies (PY99) were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-Akt, phospho-Akt, MAPK, and phospho-MAPK antibody were purchased from Cell Signaling (Beverly, MA).
Extraction and Isolation. ON-III was extracted and isolated from the flower C. operculatus used in traditional Chinese medicine. Its structure is shown in Fig. 1. The following is the steps of extraction and isolation of ON-III. The fresh buds (5.0 kg) of C. operculatus, collected at South China Botanical Garden, Guangzhou, China, were extracted with 95% EtOH three times at room temperature. The EtOH extract, after concentration in vacuo, was suspended in water and the aqueous suspension was sequentially extracted three times each with ether, EtOAc, and n-BuOH. The combined EtOAc solution, upon evaporation, yielded a brown syrup (13.0 g). This syrup was subjected to silica gel column chromatography, eluted with CHCl3-MeOH mixtures of increasing polarities (99.5:0.5 to 97:3). The fractions obtained on elution with CHCl3-MeOH (99.5:0.5) were combined and concentrated to afford a brown residue. This residue, upon recrystallization in benzene, yielded compound (690 mg): orange needles, melting point 124 to 126°C. The compound was identified by coeCthin-layer chromatography with an authentic sample and by direct comparison of its spectral data with those reported previously (Zhang et al., 1990).
Immunoblot Analysis. After the different treatments, cells were placed on ice and washed twice with ice-cold PBS. Cell lysates were harvested in lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, and 1 mM EGTA) with protein inhibitors (10 e/ml trypsin, 10 e/ml aprotinin, 10 e/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 200 e Na3VO4, and 100 mM NaF). After 10 min of centrifugation at 14,000g, the supernatant was transferred to clean microcentrifuge tubes, protein was determined by bicinchoninic acid protein assay (Pierce, Rockford, IL). Equal amounts of protein were then boiled in 5x sample buffer for 5 min at 90°C and resolved on 10% Tris-glycine gels (Novex, San Diego, CA). After electrophoresis, protein was electrotransferred to nitrocelluose membrane (Amersham Biosciences, Piscataway, NJ). Membrane was blocked with 5% milk in TBST (10 mM Tris, pH 7.4, 150 mM NaCl, and 0.05% Tween 20) for 40 min at room temperature and then incubated with a 1:2000 dilution of primary antibody in 1% milk in TBST overnight at 4°C. After washing with TBST three times, the membrane was probed with a 1:2000 dilution of anti-mouse or anti-rabbit antibody conjugated with horseradish peroxidase (Santa Cruz Biotechnology, San Francisco, CA) for 30 min at room temperature. Washing with TBST three times again, the membrane was developed by enhanced chemiluminescence (ECL; Amersham Biosciences).
Immunoprecipitation. Cells were washed twice with ice-cold PBS and then lysed with lysis buffer for 20 min at 4°C, and the protein content of the supernatants was determined by the bicinchoninic acid method (Pierce). Five hundred micrograms of protein was immunoprecipitated at 4°C overnight using the KDR or EGFR antibody (10 e) and 40 e of agarose-protein G (Roche Molecular Biochemicals, Mannheim, Germany). After being washed twice with lysis buffer, the KDR or EGFR immune complex was resolved by SDS-PAGE, and immunoblot assay was performed as described above.
Phosphorylation Assays. Cells were seeded in 24-well plate in 10% fetal bovine serum/RMPI 1640 medium. On the next day, the cells were washed twice with serum-free RMPI 1640 medium and starved overnight (14 h). On the third day, cells were treated by different concentrations of compounds for 30 min at 37°C before the cells were stimulated by VEGF for 5 min. Then, cell lysates were collected. Immunoprecipitation was conducted using anti-KDR or anti-EGFR antibodies; KDR, EGFR, phosphorylated KDR, or phosphorylated EGFR was examined using anti-PTY or anti-KDR or anti-EGFR antibodies through immunoblot analysis as shown under Immunoblot Analysis.
Reversibility Test Protocol. We have used the protocol described by Smaill et al. (1999). Treated cells were washed with warm serum-free media, incubated for 30 min, washed again, and incubated for another 8 h. This set of cells was tested for phosphorylation.
Cell Viability Assay. In brief, cells were seeded in 96-well plates (Falcon, Lincoln Park, NJ) at various densities per well. On the next day, compound was prepared at 2-fold dilutions in medium and incubated with the cells until the control cells reached 100% confluence. Cell viability was determined by MTT assay. In brief, MTT (Sigma-Aldrich, St. Louis, MO) was dissolved and sterilized in PBS at 5 mg/ml, and 10 e was added into each well. The plate was incubated in incubator (37°C, 5% CO2) for 4 h, and all the medium was removed. DMSO (100 e) was added to each well to dissolve the dark blue crystal, and the plate was shaken gently for 5 min. The optical density values were read with a test wavelength of 570 nm and a reference wavelength of 650 nm (Zhu et al., 2003).
In Vivo Antitumor Activity. The human liver and lung cancer models were established in nude mice. BALB/c nude mice were obtained from the Experimental Animal Center at Sun Yat-sen University. Female mice were 4 to 6 weeks old. All manipulations were performed under sterile conditions. The procedures involving mice and their care were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and with the UKCCCR (1998). Tumor xenografts were established by 1 x 106 Bel7402 cells injected s.c. into nude mice. Tumors were measured with a caliper in larger diameter and smaller diameter. The tumor volume was calculated by the formula 1/2 x larger diameter x (smaller diameter)2. Treatments were initiated on day 7 after inoculation, by which time the the volume had reached 20 mm3. ON-III was tested alone in mice bearing transplanted Bel7402 cells and GLC-82 cells for determining the efficacious dosage for these tumors. ON-III was given at 5.0, 10.0, and 20.0 mg/kg i.p. five times a week for 4 weeks. Approximate tumor sizes and body weight were measured twice each week. Average volumes and standard deviations were calculated for each group and plotted. Finally, the mice were sacrificed, and the tumor tissues were excised and weighed. The growth inhibitory effect was calculated with the formula: % inhibitory rate = (T control eC T drug)/T control x 100%.
Quantification of Tumor Vessel Counts. To determine whether tumor growth inhibition by ON-III was associated with inhibition of tumor vessel formation, groups of mice bearing the Bel7402 hepatocarcinoma were used for histological examination. The tumor-bearing mice were treated with either 20 mg/kg/day of ON-III or vehicle. Two weeks after starting treatment, the tumor tissues was quickly frozen and stored at eC70°C. Frozen sections were fixed and stained with primary antibodies to CD31. Five random 0.159-mm2 fields at 200x magnification were captured for each tumor by using a Sony three-chip camera mounted on a Zeiss universal microscope (Drevs et al., 2000; Laird et al., 2002).
Results
The Effect of ON-III on Tyrosine Phosphorylation of KDR. Autophosphorylation of KDR increases the velocity of the kinase reaction, and only phosphorylated KDR can provide binding sites for its substrates (such as phospholipase C, Grb-2, Shc, etc.). Thus, we first detected that ON-III inhibited tyrosine phosphorylation of KDR. To examine the relationship of dose-effect of inhibition, HDMEC cells were treated for 30 min in serum-free medium with the concentrations of ON-III indicated in Fig. 2, then stimulated with 10 ng/ml VEGF for 5 min. Cells were lysed and immunoprecipitated using KDR antibody, then resolved in SDS-PAGE. Western blot analysis was conducted with anti-phosphotyrosine antibody and KDR antibody. The results suggested that ON-III significantly inhibited phosphorylation of KDR in a dose-dependent manner at concentrations of 0, 2.5, 5.0, 10.0, and 20.0 e/ml; the IC50 value was 6.20 e/ml in HDMEC cells. But levels of KDR protein were unaffected after ON-III treatment (Fig. 2).
The Selectivity of Inhibition of KDR Phosphorylation by ON-III. ON-III inhibited tyrosine phosphorylation of KDR. It is of interest to investigate the relative selectivity of ON-III toward KDR. To elucidate this issue, human epidermal EGFR-overexpressing A431 cells were employed. A431 cells were treated with 10 and 40 e/ml ON-III for 30 min and then lysed and immunoprecipitated using EGFR antibody and resolved on SDS-PAGE. Western blot analysis was conducted with anti-phosphotyrosine antibody and EGFR antibody. The results showed that ON-III had no inhibitory effect on tyrosine phosphorylation of EGFR after more than 40 e/ml of ON-III treatment for 30 min in A431 cells (Fig. 3).
Reversible Inhibition of KDR Tyrosine Kinase Phosphorylation by ON-III. The results shown above indicate that ON-III selectively inhibits tyrosine phosphorylation of KDR receptor. To investigate whether inhibition of tyrosine phosphorylation of KDR by ON-III is reversible, HDMEC cells were treated with 10 e/ml ON-III in serum-free medium for 30 min, and the cells were washed with serum-free medium twice, then incubated separately for 30 min and 8 h. The tyrosine phosphorylation of KDR was detected. The results showed that the levels of phosphorylation of KDR completely recovered 8 h after ON-III was washed out (Fig. 4).
Effect of ON-III on the Activation of MAPK and AKT, Downstream Targets of the KDR Signal Pathway. Activation of KDR leads to endothelial cell proliferation, presumably by inducing activation of extracellular signal-regulated kinase 1/2 and phosphatidyl inositol 3-kinase/AKT pathway. To examine whether ON-III can inhibit activation of MAPK and AKT, HDMEC cells were treated with varying concentrations of ON-III for 30 min, and phospho-MAPK and MAPK were detected with anti-phospho-MAPK and anti-MAPK antibody in HDMEC cells. Phospho-Akt and Akt were detected with anti-phospho-AKT and anti-AKT antibody. Phospho-MAPK and phospho-Akt were the active types of MAPK and Akt, respectively. The results showed that phospho-MAPK and phospho-Akt decreased significantly after treatment of HDMEC cells with ON-III (Fig. 5), but total MAPK and Akt were unaffected. However, phospho-MAPK and phospho-AKT in A431 were unaffected by ON-III (data not shown). This suggested that ON-III has potent inhibitory effect on activation of MAPK and Akt in a dose-dependent manner in VEGF-stimulating HDMEC cells after treatment with ON-III for 30 min.
Effect of ON-III on the Growth of Human Endothelial Cells in the Presence of VEGF. The results described above suggested that ON-III inhibited tyrosine phosphorylation of KDR in HDMEC cells. Can ON-III inhibit cell growth more potently in the presence of VEGF To elucidate this issue, HDMEC cells and MTT analysis were employed. The results shown in Fig. 6 indicate that ON-III had more potential inhibitory effect on HDMEC cells in the presence of VEGF. It indicated that ON-III inhibited VEGF-mediated cell growth in HDMEC cells.
Antitumor Activity of ON-III in Vivo. ON-III inhibited KDR tyrosine kinase phosphorylation and KDR-mediated signal transduction. This suggested that ON-III might inhibit the tumor growth in vivo. Therefore, we established human liver cancer and lung adenocarcinoma xenografts to detect antitumor activity of ON-III in vivo. ON-III administration was initiated on day 7 after implantation, when the tumor size was approximately 20 mm3. ON-III was alone given by i.p. injection every day at doses of 5, 10, or 20 mg/kg, and tumor growth was inhibited with 38%, 45%, and 65%, respectively, after 4 weeks of treatment. There were significant differences (P < 0.05) compared with control (Fig. 7a). No obvious toxicity was observed in mice receiving the dosage treatment. ON-III had no effect on body weight of nude mice bearing hepatocarcinoma cell Bel7402 (Table 1). In addition, ON-III inhibited human lung cancer GLC-82 xenografts in nude mice. The inhibitory rates were 21.8% (P > 0.05) and 40% (P < 0.01) after 4 weeks treatment at dose of 5 and 20 mg/kg ON-III, respectively (Fig. 7b).
The treatment of ON-III has no significant effect on body weight of nude mice compared with DMSO group, P > 0.05.
Effect of ON-III on Tumor Vascularization. ON-III can inhibit VEGFR tyrosine kinase phosphorylation. Thus, inhibition of tumor growth in vivo by ON-III may occur by inhibiting tumor angiogenesis. Therefore, we employed immunohistochemical staining for CD31 to detect vessels in tumor tissues. The results revealed a significant decrease in tumor vessel counts in the ON-III groups compared with those in the control group (Fig. 8).
Discussion
Tumor growth depends on angiogenesis. This angiogenic switch is characterized by oncogene-driven tumor expression of pro-angiogenic proteins, such as VEGF, basic fibroblast growth factor, interleukin-8, and others. The significance of VEGF/VEGFR signaling in tumor initiation, progression, and metastasis make them important targets for development of specific inhibitors (Blagosklonny, 2004). Activation of KDR by VEGF results in its receptor tyrosine kinase phosphorylation. Phosphorylated KDR receptor provides binding sites for SH-2 domain-containing protein, including the adaptor proteins Grb-2 and Shc. In addition to SH-2 domain, Grb-2 binds to the small nucleotide exchange proteins, such as Sos through its SH-3 domain, then activates ras by exchanging GTP for GDP on ras (Jeong et al., 2002). Activated ras binds to and facilitates raf activation. Activated raf stimulates extracellular signal-regulated kinase kinase activity, which, in turn, activates the MAPK pathway. Inhibiting VEGF production and blocking VEGF/VEGFR signaling can inhibit tumor angiogenesis and tumor growth in vivo (Teicher et al., 2001; Keyes et al., 2004). Although some small molecules targeting KDR tyrosine kinase (e.g., SU5416, SU6668, ZD6474, and PTK787) have been reported, it is very interesting to identify VEGFR tyrosine kinase inhibitors from traditional Chinese medicine because it has usually no or low toxicity to host (Carlomagno et al., 2002; Mendel et al., 2003; O`Farrell et al., 2003).
KDR tyrosine kinase is a potent target for cancer treatment (Huang et al., 2002; Itokawa et al., 2002). In this study, we found that ON-III, which is one of the chalcone derivatives from the dried flower C. operculatus, inhibited tyrosine phosphorylation of KDR. But the expression of KDR protein was unaffected. This indicates that inhibition of phosphorylation of KDR was not via reduced KDR expression. The data demonstrated that inhibition of KDR phosphorylation by ON-III was reversible and relatively selective compared with EGFR tyrosine kinase phosphorylation. Our results demonstrated that ON-III inhibited activation of MAPK and Akt and shut down KDR downstream signaling. ON-III inhibited VEGF-stimulating HDMEC cell proliferation significantly. In comparison, growth inhibition of HDMEC cells by ON-III in the presence of EGF, but without VEGF, was obviously weaker. Moreover, ON-III produced a dose-dependent inhibition of tumor growth in human tumor xenograft models (hepatocarcinoma Bel7402 and lung adenocarcinoma GLC-82), despite their varied histological origin (liver and lung, respectively) and different growth inhibitory rates. In both cases, the percentage of inhibition on tumor growth kept increasing as the duration of drug application was extended, which is indicative of a sustained antitumor effect of ON-III. Furthermore, histological examination showed that tumor vessel counts decreased in tumor tissues in ON-III-treated mice. This may be the result of VEGF signaling blockage. In all of the in vivo experiments described in this report, ON-III was extremely well tolerated, with no obvious effects on body weight or animal behavior, and no target organ toxicity was observed.
From this experiment, we found that efficacy of ON-III after daily i.p. dosing was dependent on the growth rate of the tumors and was more optimal against slowly growing tumors and more variable against quickly growing tumors. Our findings that ON-III is less effective against lung carcinoma GLC-82 than hepatocarcinoma cancer Bel7402 is consistent with studies reported using the tyrosine kinase inhibitors SU5416 and PTK787/ZK222584 and neutralizing antibodies against VEGF (Laird et al., 2000; Mendel et al., 2000). These inhibitors are also less effective against some tumors than others, perhaps because of differences in the requirement of new blood vessels for the growth of particular tumors and the angiogenic factors produced by a particular tumor cell population. Although others have shown that almost all tumor cell lines in vitro and tumors grown in vivo produce VEGF, cytokines or growth factors other than VEGF may also contribute to endothelial cell survival and tumor angiogenesis and even be up-regulated when the effects of VEGF are inhibited (Fong et al., 1999; Buchdunger et al., 2000). We have observed in vitro that ON-III has a low inhibitory effect on endothelial cell growth if EGF is present in the medium. This suggests that several different factors support endothelial survival and tumor vascularization. Anti-VEGF therapy may be more effective against some types of tumors than others, and future therapy may necessitate a combination of antiangiogenic agents with different mechanisms of action, as well as conventional therapies targeting the tumor cells. This may give VEGF inhibitors additional therapeutic applications over antiangiogenic agents with other mechanisms of action. Soluble VEGF receptors and antibodies against VEGF or its receptors have also been proposed as agents to block VEGF; compared with ON-III, however, the antibodies have the disadvantages of large proteins (Klement et al., 2000; Dias et al., 2001).
Taken together, we identified ON-III, a potent inhibitor of VEGF signaling with antitumor effect in vivo. This compound is from the dried flower C. operculatus used in traditional Chinese medicine for treatment of inflammation for many years. No adverse events have been observed clinically. Therefore, ON-III is a promising anticancer agent targeting VEGFR tyrosine kinase.
This study was supported by grants 39900183 and 30171092 from the National Natural Science Foundation of China, and grants 013126, 2003C30101, and 2004Z3-E4021 from the Natural Science Foundation of Guangdong Province.
doi:10.1124/mol.104.009894.
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