Antitumor Activity of Taspine by Modulating the EGFR Signaling Pathway of Erk1/2 and Akt In Vitro and In Vivo

• Abstract
• Abbreviations
• Introduction
• Materials and Methods
• Materials
• Cell lines
• Animals
• Extraction and isolation of taspine
• Cell viability assay
• Migration assay
• EGF secretion in vitro
• Western blot analysis
• RNA isolation and real-time RT-PCR
• Docking study
• Antitumor effect of taspine on A431 cell lines xenografted in athymic mice
• Statistic analysis
• Results
• Discussion
• Acknowledgements
• References

Abstract

EGFR, as a critical signaling pathway in many human tumors, has become an important target of cancer drug design. Taspine has shown meaningful angiogenesis activity in previous studies. This paper is to investigate the antitumor action of taspine by modulating the EGFR signaling pathway. The study determined the expression of key signaling molecules of EGFR (EGFR, Akt, p-Akt, Erk, and p-Erk) by Western blot and real-time PCR and analyzed their correlations with subsequent reactions. In addition, the cell proliferation, migration, and EGF production were examined by MTT, transwell system, and ELISA. The antitumor activity in vivo was carried out by xenograft in athymic mice. The results showed that taspine could inhibit A431 and Hek293/EGFR cell proliferation and A431 cell migration as well as EGF production. Compared to the negative control, EGFR, Akt, and phosphorylation of Akt were significantly inhibited by taspine treatment in A431 and HEK293/EGFR cells. Consistent with the inhibition of Akt activity, Erk1/2 and its phosphorylation were reduced. Moreover, taspine inhibited A431 xenograft tumor growth. These results suggest that EGFR activated by EGF and its downstream signaling pathways proteins could be downregulated by taspine in a dose-dependent manner. The antitumor mechanism of taspine through the EGFR pathway lies in the ability to inhibit A431 cell proliferation and migration by reducing EGF secretion. This occurs through the repression of EGFR which mediates not only MAPK (Erk1/2) but also Akt signals.

Abbreviations

WB: Western blot analysis

qPCR: quantitative real-time PCR

HEK293/EGFR cell: a wild type recombined HEK293 cell of stable EGFR overexpression

Introduction

Tumor cells promote vessel formation through the expression of angiogenic molecules or their induction in the microenvironment [1]. Among the pro-angiogenic molecules, vascular endothelial growth factor (VEGF), and epidermal growth factor (EGF) have been identified to drive tumor-related angiogenesis [2], [3], [4]. Taspine, isolated for the first time from HMQ Radix et Rhizoma Leonticis (Hong Mao Qi in Chinese, HMQ), has many pleiotropic effects such as anti-inflammatory, cell toxin, antivirus, topoisomerase I and II inhibition, etc. [5], [6], [7]. In previous studies, we have found that taspine has an antitumor effect and inhibits HUVEC proliferation, angiogenesis of chicken chorioallantoic membrane (CAM), and VEGFR-2 mRNA expression [8], [9]. So, future improvements in tumor treatments probably arise from taspine targeting molecular pathways that promote tumor cell survival and growth, the most frequently investigated target being EGFR.

Epidermal growth factor receptor (EGFR or erbB-1) are members of the ErbB family of receptor tyrosine kinases (RTKs). Upon ligand binding, EGFR forms homo- or heterodimers with all other EGFR receptors, leading to activation of various downstream signaling pathways [phosphatidylinositol 3-kinase (PI3K)/AKT and Ras/Raf/mitogen-activated protein kinase (MAPK)] that mediate cell proliferation, migration, invasion, angiogenesis, and metastasis [10], [11]. The expression of EGFR is common in a number of normal epithelial tissues and many human cancers, such as head and neck tumors, colorectal, ovarian, breast, lung, and bladder cancers [12], [13]. Overexpression of EGFR and dysregulation of EGFR-mediated signaling pathways play important roles in tumorigenesis, leading to a poor prognosis [14]. So, EGFR inhibitors are outstanding examples of drug design for tumors [15]. Recent data have highlighted the fact that EGFR has become an important target of cancer drug design, and several selective EGFR inhibitors have now been approved for clinical use, such as the therapeutic antibodies cetuximab, small molecule antagonists targeting EGFR, and gefitinib [16].

The present study aimed to extend the previous observation on the antitumor activity of taspine and to evaluate its antitumor mechanism by modulating the EGFR signaling pathway. Herein, a docking study was used to investigate the interaction between taspine and EGFR, A431 and HEK293/EGFR cells so as to understand the effect of taspine on EGFR and its downstream signaling pathways, and human tumor models xenografted in athymic mice to study the antitumor activity in vivo. A431 and HEK293/EGFR cells were suitable for the study in that the former overexpress EGFR while the latter are wild type recombined HEK293 cells of stable EGFR overexpression and were constructed by gene recombination and named by ourselves [17].

Experimental data presented here showed that taspine displayed antitumor activity in vivo. The antitumor activity of taspine by the EGFR pathway lied in the ability to inhibit A431 cells proliferation and migration, EGF secretion, and to downregulate the activations of EGFR, Erk, and Akt by EGF as well as EGFR, Erk1, and Akt1 expressions at the mRNA level. All these results indicate that taspine with potent antitumor activity in vitro and in vivo might be helpful for tumor treatment.

Materials and Methods

Materials

Radix et Rhizoma Leonticis were collected in Shaanxi, China. Identification of the plant was done at the Pharmacognosy Laboratory in the Department of Pharmacy, School of Medicine, Xi'an Jiaotong University where a voucher specimen is deposited; the number of the voucher specimen is 2010002. Gefitinib was provided by Astra Zeneca, and its purity was over 99 %. MTT, trypsin, and fibrinogen were from Sigma. Human EGF was from Peprotech Asia. Human EGF ELISA kits were purchased from R & D Systems. Protease inhibitor cocktail and phosphatase inhibitor cocktail were from Roche. EGFR mAb, Akt and phospho-Akt rabbit mAb, p44/42 MAPK (Erk1/2) and phospho-p44/42 MAPK (Erk1/2) rabbit mAb were purchased from Cell Signaling. Rabbit anti-β-actin was purchased from Santa Cruz Biotech. Rabbit anti-mouse IgG, goat anti-rabbit IgG, BCA protein assay reagent kit, and enhanced chemiluminescent (ECL) plus reagent kit were obtained from Pierce. Total RNA extracted kit was from Fastagen. Revert AID™ first strand cDNA synthesis kit was from Fermentas. Other reagents used were analytical grade.

Cell lines

A431 cells were from Shanghai Institute of Cell Biology in the Chinese Academy of Sciences. A431 cells grew in F12 medium containing 10 % bovine serum and antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin), at 37 °C in a 5 % CO₂ atmosphere. HEK293/EGFR cells (overexpressing total cDNA sequences of EGFR stably) were constructed by gene recombination and named by the Research and Engineering Center for Natural medicine, Xi'an Jiaotong University. The HEK293/EGFR cells grew in DMEM medium containing 10 % bovine serum, 200 ng/mL G418, and antibiotics (100 IU/mL penicillin and 100 µg/mL streptomycin), at 37 °C in a 5 % CO₂ atmosphere. Before experiments, cells were incubated overnight in serum-free medium. The positive control was gefitinib, and its concentration was 5.40 µM for A431 cells and 1.60 µM for HEK293/EGFR cells in all experiments.

Animals

All experiments were carried out with mice, which were approved by the School of Medicine, Xi'an Jiaotong University, Animal Ethics Committee 507/CPCSEA (Sanction No. SYXK 2007-003, dated 07.04.26) and were performed in accordance with the International Animal Ethics Committee Guidelines. Female BALB/C nude mice (18–22 g) were supplied by the Experimental Animal Center of Xi'an Jiaotong University. Mice were housed and cared for under standard conditions, with a 12–12 h day/night cycle. All experimental procedures utilizing mice were in accordance with the National Institute of Health guidelines.

Extraction and isolation of taspine

Air-dried and powdered Radix et Rhizoma Leonticis (1 kg) were extracted with 25 L of pH 5–6 hydrochloric acid solution at 90 °C for 2 h. The extract was concentrated and was taken to pH 9–10 with aqueous ammonia, and then the concentrate was cyclically extracted with CHCl₃ until the CHCl₃ phase was colorless. The CHCl₃ fractions were combined to retrieve the CHCl₃, and the precipitate was collected. The precipitate components were separated systematically by column chromatography. Firstly, the sample was subjected to a normal-phase column chromatography (CC, Silica Gel, 400 mm × 30 mm) using a mixture of CHCl₃/CH₃OH/TEA (3 : 1 : 0.1 by vol.) as a mobile phase, to give 4 fractions after recombination by color (1–4). Fraction 1 was subjected to the normal-phase column chromatography (CC, Silica Gel, 200 mm × 15 mm) using a mixture of CHCl₃/CH₃OH/TEA (15 : 1 : 0.1 by vol.) as the mobile phase, to give four 300 mL subfractions after recombination. Taspine was isolated from fraction 1–1 and further purified by crystallization. The structure of taspine was identified by IR, UV, NMR, and MS. The IR, UV, ¹H-NMR, ¹³C-NMR, and MS data of taspine were proved by previous reports. The structure of taspine was shown in [Fig. 1]. The taspine m. p. was 372–374 °C and its purity was over 99 %.

Cell viability assay

The effects of taspine on A431 and HEK293/EGFR cell viability were evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay [18]. Briefly, the cells within the exponentially growing period were harvested and plated in 96-well plates at a concentration of 1 × 10⁴ cells/well and incubated at 37 °C for 24 h. The cells in the wells were respectively treated with various taspine concentrations for 48 h. Then, 20 µL of MTT (5 mg/mL) was added to each well and incubated at 37 °C for 4 h. After the supernatant was discarded, 150 µL DMSO was added to each well, and the absorbance values were determined by a microplate reader (BioRad) at 490 nm.

Migration assay

A cell migration assay was performed using a transwell system, which allows cells to migrate throughout an 8-µm pore size polycarbonate membrane of millicell [19]. Briefly, A431 cells were first serum starved for 24 hours and then plated (100 000 cells/well) in serum-free medium containing taspine at 1.60, 3.20, 6.40 µM concentrations in the upper chamber of a 12-well plate. The lower chamber was filled with 1.5 mL medium containing 10 % FBS. After 12 h, 24 h, 48 h, cells remaining on the upper surface of the membrane were removed with a cell scraper. Membranes were then washed with phosphate-buffered saline, and the cells beneath the membrane were fixed with cold methanol for 15 min and stained with 0.2 % crystal violet. Cell counts were performed by counting six high-power unrelated fields under a light microscope (magnification 40 ×). Experiments were performed in triplicate.

EGF secretion in vitro

A431 (1 × 10⁴ cells per well) and HEK293/EGFR (2 × 10⁴ cells per well) cells were cultured in 24-well culture plates for 24 h. Then, the cells were incubated for another 24 h after the medium was changed to serum free. The same volume with different taspine concentrations (0, 1.60, 3.20, and 6.40 µM for A431 cells and 0.90, 1.80, and 3.60 µM for HEK293/EGFR cells) was added to the wells, respectively. The 48-h cultured medium was collected. EGF protein concentrations were quantitatively measured by a commercially available EGF-ELISA kit at 450 nm [20].

Western blot analysis

The A431 and HEK293/EGFR cells treated by taspine for 48 h were prepared by extracting proteins with RIPA lysis buffer containing protease inhibitor cocktail and phosphatase inhibitor cocktail (Roche) on ice. The EGF (0.1 ng/mL) was added in the cell culture medium for 15 min before extracting proteins. Protein concentration was determined by BCA protein quantitation kit according to the manufacturer's instructions. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (PAGE) loading 30 µL of cell lysates per lane. After electrophoresis, separated proteins were transferred to nitrocellulose membrane and blocked with 5 % nonfat milk in TBST buffer for 2 h. After then, the membranes were incubated with primary antibodies [anti-EGFR, anti-Akt, anti-phospho(Ser473)-Akt, anti-p44/42 MAPK(Erk1/2), anti-phospho(Thr202/Tyr204)-p44/42 MAPK(Erk1/2), and anti-β-actin were used as primary antibodies] at 1 : 1000 dilutions in 5 % nonfat milk overnight at 4 °C with continuous agitation, and then with secondary antibodies conjugated with horseradish peroxidase at 1 : 5000 dilution at room temperature for 2 h according to the manufacturer's recommended protocol. Finally, the blots were detected by ECL reagent (Amersham Pharmacia Biotechnology) and analyzed using Quantity one®, 1-D analysis software (Version 4.4; BioRad) [21].

RNA isolation and real-time RT-PCR

Quantification of EGFR, Erk1, Erk2, Akt1, Akt2, Akt3, and an internal reference gene (β-actin) was done using a fluorescence-based real-time detection method. Total RNA of the A431 and HEK293/EGFR cells treated by taspine was isolated using a total RNA extracted kit. The total RNA was reversely transcribed in 20 µl reaction solution using the Revert AID™ first strand cDNA synthesis kit. The sequence details of individual pairs of primers of EGFR, Erk1, Erk2, Akt1, Akt2, Akt3, and β-actin were as follows: 5′-CGTGCCCTGATGGATGAAGAAGAC-3′ (sense), 5′-CGGTGGAATTGTTGCTGGTTGC-3′ (antisense) for the EGFR gene; 5′-GGACCTGATGGAGACTGACCTG-3′ (sense), 5′-TGGCGGAGTGGATGTACTTGAG-3′ (antisense) for the ERK1 gene; 5′-GAAGACACAACACCTCAGCAATG-3′ (sense), 5′-GTTGAGCAGCAGGTTGGAAGG-3′ (antisense) for the ERK2 gene; 5′-CTACTTCCTCCTCAAGAATGATGGC-3′ (sense), 5′-CAGCGGATGATGAAGGTGTTGG-3′ (antisense) for the AKT1 gene; 5′-ACAAGCGTGGTGAATACATCAAGAC-3′ (sense), 5′-GCAGGCAGCGTATGACAAAGG-3′ (antisense) for the AKT2 gene; 5′-AAGAAGGTTGGGTTCAGAAGAGG-3′ (sense), 5′-AATGTGTTTGGCTTTGGTCGTTC-3′ (antisense) for the AKT3 gene; 5′-ATCGTGCGTGACATTAAGGAGAAG-3′(sense), 5′-AGGAAGGAAGGCTGGAAGAGTG-3′ (antisense) for the β-actin gene. The real-time PCR reactions were performed with the iCycler iQ Real-Time PCR Detection System (BioRad Laboratories) in 96-well reaction plates. Reaction volumes were 25 µL, containing 2 µL cDNA and 100 µM of each pair of primers and iQi SYBR Green Supermix (BioRad). Thermal cycling conditions included preincubation at 95 °C for 2 min followed by 40 PCR cycles at 95 °C for 20 s and 60 °C for 1 min. All reactions were run in triplicate. iCycler software was used to analyze the calibration curve by plotting the threshold cycle (Ct) vs. the logarithm of the number of copies for each calibrator. The relative amount of mRNA for each gene was normalized based on that of the housekeeping gene β-actin [E^-△△Ct]. Measurements yield Ct values that are inversely proportional to the amount of cDNA in the tube. For example, a higher Ct value means that more PCR cycles are required to reach a certain level of cDNA detection. Gene expression values (relative mRNA levels) are expressed as ratios (differences between the Ct values) between the gene of interest and an internal reference gene (β-actin). This reference gene provides a baseline measurement for the amount of RNA isolated from a specimen [22].

Docking study

In an effort to elucidate the binding modes of taspine with EGFR, it was constructed with the Sybyl/Sketch module and optimized using Powell's method with the Tripos force field with convergence criterion set at 0.05 kcal/(Å mol) and assigned with the Gasteiger-Hückel method [23]. The docking study was performed using the Sybyl/FlexX module, and the residues in a radius of 6.5 Å around the (PDB ID 3B2V) were selected as the active site. Other docking parameters implied in the program were kept as the default.

Antitumor effect of taspine on A431 cell lines xenografted in athymic mice

Solid tumor models were developed from A431 cell lines. The female immunodeficient BALB/C nude mice (18–22 g) were randomized and then implanted with 0.2 mL A431 cells (2 × 10⁷ cell/mL) s. c. into the right axilla. Tumors were measured once every three days and tumor volumes (V_t) [(L × W²)/2] were calculated from caliper measurements. The relative tumor volume (RTV) was expressed as the V_t/V₀ index, where V_t is the tumor volume on the day of measurement and V₀ is the volume of the same tumor at the start of the treatment. The results were expressed as median T/C where T/C (%) equals median RTV of treated animals/median RTV of control animals × 100. Mice were injected with Gefitinib (50 mg/kg in 0.5 % CMC-Na; n = 8) or taspine (2.5 mg/kg and 5.0 mg/kg in 0.5 % CMC-Na; n = 8), or vehicle alone (normal saline; n = 8). Drugs were given once a day for 14 days. Mice weight and tumor volume were recorded when the animals were killed. Animal care was in accordance with institutional guidelines.

Statistic analysis

Data were expressed as mean ± SD. Statistical analysis was performed using the statistical software SPSS10.0. ANOVA was used to analyze statistical differences between groups under different conditions. P < 0.05 was considered significant.

Results

The effect of taspine on A431and HEK293/EGFR cell viability was examined by MTT. Taspine treatment exhibited significant inhibition on growth in A431cells and HEK293/EGFR cells in a dose-dependent manner. The 50 % viability inhibitions (IC50) of taspine on A431 and HEK293/EGFR cells were 3.2 µM and 1.8 µM, respectively. In addition, the results indicated that HEK293/EGFR cells were more sensitive to the antiproliferative effect of taspine.

In solid tumors, cell growth, migration, and metastasis are critical steps. In order to test the effect of taspine on in vitro A431cell migration, cells were treated with taspine. As shown in [Fig. 2], results indicated that taspine could significantly reduce cell motility in a time- and dose-dependent manner at concentrations of 1.60 µM, 3.20 µM, and 6.40 µM, respectively.

ELISA for EGF showed that taspine could inhibit EGF production in a dose-dependent manner at low concentrations (0.9–6.4 µg/mL) compared with the control group in A431 and HEK293/EGFR cells (p < 0.05). The EGF expressions obviously decreased at different concentrations ([Fig. 3]). There were significant differences between the taspine and the control groups.

To further identify the effect of taspine on EGFR and its downstream signaling pathways that might contribute to growth inhibition, the present study examined the phosphorylation of several key regulators involved by Western blot analysis. [Fig. 4 A] and [4 C] showed that the EGFR expression was decreased in the taspine-treated group. At the same time, Akt and phosphorylation of Akt were significantly inhibited by taspine treatment in A431 and HEK293/EGFR cells. Consistently with the inhibition of Akt activity, Erk1/2 and phosphorylation of Erk1/2 were reduced. [Fig. 4 B] and [4 D] showed the quantitation of protein expressions which exhibited the same trend in A431 and HEK293/EGFR cells. These results suggest that EGFR activated by EGF and its downstream signaling pathway proteins could be downregulated by taspine in a dose-dependent manner.

Quantitative PCR was carried out to understand whether taspine could influence the synthesis of EGFR, Akt, and Erk1/2 transcript. As shown in [Fig. 5], the mRNA levels of EGFR had a statistically significant correlation with those of Akt and Erk1/2. Compared to the negative control (p < 0.05), the EGFR and Akt3 mRNA expressions in the taspine-treated group were significantly downregulated at a high concentration of taspine, and the Erk1 and Akt1 mRNA expressions in a dose-dependent manner. There were identical changes in mRNA in A431 and HEK293/EGFR cells. This indicates that taspine could regulate the mRNA levels of EGFR, Akt, and Erk1/2.

Docking of taspine in the active site of EGFR showed six H-bond interactions between oxygen and nitrogen of taspine and amino acid residues of the receptor ([Fig. 6 A]). We found that one oxygen-1(O-1) formed a hydrogen bond to TRP453 with a distance of 3.08 Å, O-2 formed a hydrogen bond to LYS454 with a distance of 2.72 Å, O-3 and O-4 formed two hydrogen bonds to TRY32 with distances of 3.42 Å and 2.84 Å, O-5 formed a hydrogen bond to SER-30 with a distance of 2.89 Å. The nitrogen of the side chain formed a hydrogen bond to ASN449 with a distance of 3.17 Å. Also, virtual docking of EGFR in complex with taspine showed that taspine was bound to the binding pocket of the receptors domain ([Fig. 6 B] and [C]). As seen from [Fig. 6 B], the glycosyl group extended to one hole of the active site. [Fig. 6 C] indicated the hydrogen bond density on the surface of the receptors. All the above showed that taspine had a good action on EGFR.

The antitumor properties of taspine were evaluated using human tumor models xenografted in athymic mice, with gefitinib employed here as a positive control group. Taspine significantly inhibited tumor growth in A431 xenografted athymic mice in a dose-dependent manner ([Table 1]). Compared with the control group, the group treated with taspine significantly inhibited tumor growth at a rate of 14.68 % and 56.42 %, respectively. Furthermore, there was no substantial change in athymic mice body weight during the experiment, which could be considered as the antitumor activity of taspine taking precedence over the toxicity on athymic mice.

Discussion

Cancer cells proliferate via several signal transduction pathways. One such mechanism is by the EGFR pathway which is a frequently unregulated site of tumor signaling [24].

Results in the present study provide evidence that taspine inhibits proliferation and migration in A431 and HEK293/EGFR cells via inhibiting the autocrine/paracrine release of EGF. Accordingly, taspine reduces EGFR, Erk, Erk phosphorylation, Akt, and Akt phosphorylation activation induced by EGF in those cells. However, taspine does not affect EGF-induced Erk2 and Akt2 mRNA expressions in A431 and HEK293/EGFR cells. Previous studies have reported that taspine was screened through the HUVEC CMC procedure. Taspine had retention characteristics similar to anti-KDR (anti-VEGFR-2) on the cell membrane stationary phase (CMSP) and could inhibit angiogenesis by inhibiting KDR mRNA expression [9], [10]. There was a good interaction between EGFR and taspine in previous reports [25]. So, the aim of this study was to investigate the action on EGFR by taspine and correlations between the expression levels of EGFR downstream molecules and their response to taspine.

EGFR is closely related to tumorigenesis and development of various cancers. Overexpression of EGFR widely exists in various malignant tumors, especially solid ones. The EGFR family plays an essential role in normal organ development by mediating morphogenesis and differentiation through effects on cell proliferation, differentiation, apoptosis, invasion, and angiogenesis [26], [27]. Unlike normal cells that have tight regulatory mechanisms controlling EGFR pathways, tumor cells often have disregulated EGFR signaling through receptor overexpression and/or mutation. This leads to proliferation under adverse conditions, invasion of surrounding tissues, and increased angiogenesis as well as resistance to radiation and chemotherapy. Therefore, EGFR is a legitimate therapeutic target [28]. This study investigated the effect of taspine on cell viability and migration in A431 and HEK293/EGFR cells overexpressing EGFR. The results demonstrate that taspine could inhibit A431 and HEK293/EGFR cells viability and migration in a dose-dependent manner.

EGF in coordination with VEGF has profound effects on cell growth, migration, and new vessel growth [2], [3]. Akt and MAPKs have been implicated in promoting cell migration and tumorigenesis. Specific inhibitors of PI3K/Akt and MEK/Erk1/2 display a similar inhibitory effect on cell migration and invasion [29]. In this study, the action of taspine on EGF secretion was analyzed; the expression of EGFR, Akt, phospho-Akt, p44/42 MAPK (Erk1/2), and phospho-p44/42 MAPK (Erk1/2) was examined in A431 and HEK293/EGFR cells by Western blot, and the mRNA expression of EGFR, Erk1, Erk2, Akt1, Akt2, and Akt3 was then studied by quantitative PCR. The findings indicate that taspine could inhibit EGF production which inhibited A431cell growth and migration and blocked the two-way paracrine action. Results from the Western blot assay indicated that the EGFR expression was decreased in the taspine-treated group. At the same time, the Akt and phosphorylation of Akt induced by EGF were significantly inhibited by taspine in A431 and HEK293/EGFR cells. Consistently with the inhibition of Akt activity, Erk1/2 and phosphorylation of Erk1/2 induced by EGF were decreased. These results suggest that EGFR activated by EGF and its downstream signaling pathway proteins could be downregulated by taspine in a dose-dependent manner. In addition, the EGFR and Akt3 mRNA expressions in the taspine-treated group were significantly downregulated at a high concentration of taspine (p < 0.05), and the Erk1 and Akt1 mRNA expressions in a dose-dependent manner (p < 0.05), compared to the negative control. The protein and mRNA levels of EGFR had a statistically significant correlation with Akt and Erk1/2. There were identical changes of protein and mRNA expression in A431 and HEK293/EGFR cells. The docking study showed a good binding model between EGFR and taspine. Antitumor activity of taspine in vivo showed that taspine inhibited A431 cell xenografted in athymic mice. All this confirms that taspine has an antitumor action by modulating the EGFR signaling pathways.

The study explored a new mechanism whereby taspine was a multitarget inhibitor. In conclusion, the anti-angiogenic action and modulation of the EGFR signaling pathways of taspine lie in the ability to inhibit cell proliferation and migration by reducing EGF secretion. This effect occurs through the repression of EGFR which mediates not only MAPK (Erk1/2) but also Akt signals. Therefore, it is postulated, on the basis of these findings, that the pharmacological inhibition of taspine could potentiate the anti-tumor effect.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (Grants No. 30730110 and 81001447), the Natural Science Foundation of Shaanxi Province (Grant No. 2010JQ4017), the scientific research grant by the Xi'an Jiaotong University (The tumor angiogenesis differences analysis by taspine), and the new teacher scientific research grant by the Xi'an Jiaotong University (Screening of taspine derivates and their mechanism of inhibition to tumor angiogenesis).

All authors declare no conflict of interest.

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• 23 Mou J, Fang H, Jing F, Wang Q, Liu Y, Zhu H, Xu W. Design, synthesis and primary activity evaluation of L-arginine derivatives as amino-peptidase N/CD13 inhibitors.  Bioorg Med Chem. 2009;  17 4666-4673
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• 25 Sun M, Ren J, Du H, Zhang Y M, Zhang J, Wang S C, He L C. A combined A431 cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening compounds from total alkaloid of Radix Caulophylli acting on the human EGFR.  J Chromatogr B. 2010;  878 2712-2718
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Langchong He

Institute of Materia Medica
School of Medicine, Xi'an Jiaotong University

No. 76, Yanta Weststreet #54

Xi'an, Shaanxi Province 710061

P. R. China

Phone: +86 29 82 65 54 51

Fax: +86 29 82 65 54 51

Email: helc@mail.xjtu.edu.cn

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• 23 Mou J, Fang H, Jing F, Wang Q, Liu Y, Zhu H, Xu W. Design, synthesis and primary activity evaluation of L-arginine derivatives as amino-peptidase N/CD13 inhibitors.  Bioorg Med Chem. 2009;  17 4666-4673
  CrossrefPubMedSearch in Google Scholar
• 24 Li J, Li Y, Feng Z Q, Chen X G. Anti-tumor activity of a novel EGFR tyrosine kinase inhibitor against human NSCLC in vitro and in vivo.  Cancer Lett. 2009;  279 213-220
  CrossrefPubMedSearch in Google Scholar
• 25 Sun M, Ren J, Du H, Zhang Y M, Zhang J, Wang S C, He L C. A combined A431 cell membrane chromatography and online high performance liquid chromatography/mass spectrometry method for screening compounds from total alkaloid of Radix Caulophylli acting on the human EGFR.  J Chromatogr B. 2010;  878 2712-2718
  CrossrefPubMedSearch in Google Scholar
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  PubMedSearch in Google Scholar
• 27 Dutta P R, Maity A. Cellular responses to EGFR inhibitors and their relevance to cancer therapy.  Cancer Lett. 2007;  254 165-177
  CrossrefPubMedSearch in Google Scholar
• 28 Partanen A. Epidermal growth factor and transforming growth factor-alpha in the development of epithelial-mesenchymal organs of the mouse.  Curr Top Dev Biol. 1990;  24 31-55
  PubMedSearch in Google Scholar
• 29 Giannelli G, Sgarra C, Porcelli L, Azzariti A, Antonaci S, Paradiso A. EGFR and VEGFR as potential target for biological therapies in HCC cells.  Cancer Lett. 2008;  262 257-264
  CrossrefPubMedSearch in Google Scholar

Langchong He

Institute of Materia Medica
School of Medicine, Xi'an Jiaotong University

No. 76, Yanta Weststreet #54

Xi'an, Shaanxi Province 710061

P. R. China

Phone: +86 29 82 65 54 51

Fax: +86 29 82 65 54 51

Email: helc@mail.xjtu.edu.cn