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Journal of Zhejiang University. Science. B logoLink to Journal of Zhejiang University. Science. B
. 2006 Sep 14;7(10):837–843. doi: 10.1631/jzus.2006.B0837

Inhibition of expression of vascular endothelial growth factor and its receptors in pulmonary adenocarcinoma cell by TNP-470 in combination with gemcitabine

Xue-fen Wang 1,†, Ling-fang Tu 1, Li-hong Wang 2, Jian-ying Zhou 1
PMCID: PMC1599809  PMID: 16972327

Abstract

Angiogenesis is required for solid tumor growth and facilitates tumor progression and metastasis. The inhibition effects of O-(chloroacetyl-carbamoyl) fumagillol (TNP-470), an angiogenesis inhibitor, and gemcitabine, a chemotherapeutic agent, on expression of growth factors were investigated using human pulmonary adenocarcinoma cell line, A549. The A549 cells were divided into four groups: control group, 10−6 mg/ml gemcitabine treated group, 10−4 mg/ml TNP-470 treated group and gemcitabine+TNP-470 treated group. The mRNA and protein expression of vascular endothelial growth factor (VEGF) and its receptors, FMS-like tyrosine kinase-1 (FLT-1) and kinase insert domain-containing receptor (KDR), in different groups were measured. The growth of A549 cell cultured with gemcitabine or TNP-470 was inhibited in an almost dose-dependent manner. Although gemcitabine (10−6 mg/ml) alone and TNP-470 (10−4 mg/ml) alone had no effect on the mRNA and protein expression of VEGF and its receptors (FLT-1, KDR) in A549 cells compared to the control (P>0.05), 10−6 mg/ml gemcitabine in combination with 10−4 mg/ml TNP-470 had significant effect (P<0.01). Moreover, combination of the two drugs significantly inhibited the mRNA expression of VEGF, FLT-1 and KDR compared to either drug alone (P<0.05). This study suggests that combined treatment with TNP-470 plus gemcitabine may augment the antiangiogenic and antineoplastic effects in lung cancer cells in vitro.

Keywords: TNP-470, Gemcitabine, VEGF, FLT-1, KDR

INTRODUCTION

Angiogenesis is required for solid tumor growth and facilitates tumor progression and metastasis (Folkman, 1990; Weidner et al., 1991; Weinstat-Saslow and Steeg, 1994). Moreover, angiogenesis decreased overall survival in patients with various tumors, such as lung cancer (Sauter et al., 1999; Johnson et al., 2005). Angiogenesis is thought to be regulated by a number of growth factors, including basic fibroblast growth factor (bFGF), transforming growth factor (TGF), and vascular endothelial growth factor (VEGF) (Rifkin et al., 1993). VEGF is a disulfide-linked dimeric glycoprotein that increases blood vessel permeability, endothelial cell growth, proliferation, migration, and differentiation (Senger et al., 1993). It is seemingly produced by human tumors solely for the stimulation of tumor vascularization. VEGF mediates angiogenic signals to the vascular endothelium via the receptors of FMS-like tyrosine kinase-1 (FLT-1) and kinase insert domain-containing receptor (KDR) (Miura et al., 2004). O-(chloroacetyl-carbamoyl) fumagillol (TNP-470), an analog of fumagillin derived from Aspergillus fumigatus, inhibits angiogenesis both in vivo and in vitro, regardless of the presence of angiogenesis factors. It is also less toxic than fumagillin (Kusaka et al., 1994). In addition, it was reported that TNP-470 has an inhibition effect on the growth and metastasis of human cell lines and rodent tumors (Yanase et al., 1993; Yamamoto et al., 1994; Shishido et al., 1996). In this study, we investigated the effects of TNP-470 and gemcitabine, a chemotherapeutic agent, against human pulmonary adenocarcinoma cell line A549, which has high metastatic potential among the lung cancers.

MATERIALS AND METHODS

Cell proliferation assay

A549 cells (5×103 well−1, obtained from Shanghai Cell Resources Center, China) were cultured in a 96-well plate. Culture supernatants were replaced the next day (day 1) with fresh medium, containing TNP-470 (Takeda Pharmaceutical Company, Japan) or gemcitabine (Eli Lilly and Company, USA) at concentrations of 0, 10−6, 10−5, 10−4, 10−3, 10−2, and 10−1 mg/ml. On day 3, to count viable cells, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide; Sigma) assay was performed. The MTT assay was previously described. Briefly, 20 μl of 5 mg/ml MTT was added and the cells were incubated for 4 h. This product was solubilized by the addition of dimethyl sulfoxide (DMSO). The absorbance of each well was quantified with a NovaPath microplate reader using a test wavelength of 570 nm. The assay was repeated at least four times.

Cell culture and anti-angiogenic treatment

A549 cells were cultured in 9 cm Petri dish at 2×105 cells/ml for 10 ml in RPMI 1640 medium with 10% FCS. Culture media were changed to RPMI 1640 containing no FCS but concentrations of gemcitabine (0, 10−6 mg/ml) with/without TNP-470 (10−4 mg/ml). After 2 d, the cells were collected and cultured in three groups.

Reverse transcription (RT) and polymerase chain reaction (PCR)

RNA was isolated from cell lines using TRIzol reagent (Life Technologies, Inc.). RT was carried out using a kit (Fermentas) according to the manufacturer’s instructions. RT products were stored at −70 °C. The amount of RNA was determined by measuring the specific absorption at 260 nm. Oligonucleotide primers used were (written in the 5' to 3' direction): (1) VEGF sense primer, TCGGGCCTCCGAAACCATGA, and antisense primer, CCTGGTGAGAGATCTGGTTC; (2) FLT-1 outer sense primer, ATTTGTGATTTTGGCCTTGC, and outer antisense primer, CAGGCTCATGAACTTGAAAGC; FLT-1 inner sense primer, CACCAAGAGCGACGTGTG, and inner antisense primer, TTTTGGGTCTCTGTGCCAG; (3) KDR sense primer, GTCAAGGGAAAGACTACGTTGG, and antisense primer, AGCAGTCCAGCATGGTCTG; (4) β-actin sense primer, CGCTGCGCTGGTCGACA, and antisense primer, GTCACGCACGATTTCCCGCT. The VEGF primers correspond to sequences in the untranslated 5' and 3' region resulting in amplication of three different splice variants of size 516, 648 and 720 bp. Primers specific for KDR, FLT-1 and β-actin recognize coding sequences. PCR product sizes are, for FLT-1 outer primer pair, 555 bp; for FLT-1 inner primer pair, 196 bp; for KDR, 591 bp; and for β-actin, 619 bp, respectively.

VEGF, FLT-1 and KDR expression were determined by semi-quantitative PCR using β-actin as an internal standard. The samples were subjected to 35 cycles of amplification as follows: the samples were initially heated to 94 °C for 3 min to ensure complete denaturation of DNA (30 s for subsequent cycles) followed by 30 s at 60 °C to anneal the primers, and then 1-min at 72 °C for extension of the annealed primer. The PCR reaction was concluded by a 30 s elongation phase, again at 72 °C. The PCR products (5 μl) were visualized on 1.5% agarose gel. Electronic images were captured by using a solid state black-and-white video camera (Cohu Electronic), and the intensity of the bands was determined using Kodak Digital imaging software (EDAS290). Every sample was repeated three times.

Protein isolation and Western blot analysis

Cell cultures grown to 70%~80% confluence were lysed with an SDS-based single lysis buffer. The lysate was centrifuged at 12000 r/min, and the supernatant was collected. Total protein concentration of the soluble cell extract was determined based on absorbance. Total protein (40 μg) of each sample was labelled in the presence of SDS before the addition of 2-mercaptoethanol and applied to a discontinuous 10% polyacrylamide gel for electrophoretic separation. Proteins were then electrophoretically transferred to a polyvinylidene difluoride membrane, which was placed in blocking solution (5% nonfat dry milk), probed with VEGF, or FLT-1, or KDR, or β-actin-specific antibody (1:400, Santa Cruz, Inc.) followed by a phosphatase-conjugated second antibody (1:5000, Santa Cruz, Inc.), and washed. Substrates 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium were added for signal detection. Every sample was repeated three times.

Statistical analysis

Data are presented as mean±SD. Datasets containing the four different groups were analyzed by ANOVA. Mean values between two different groups were compared by a least-significant difference (LSD) test, after an F test for homogeneity of variances had been performed. If data failed to meet the requirements for equal variance, a Tamhane’s T2 test was used. Differences were considered statistically significant at a value of P<0.05.

RESULTS

Effects of gemcitabine and TNP-470 on cell proliferation

As shown in Fig.1a, the growth of A549 cell cultured with gemcitabine was inhibited in an almost dose-dependent manner. Half-maximum inhibition of gemcitabine was 10−4 mg/ml approximately. The dose of gemcitabine that we chose to study was 10−6 mg/ml in order to reduce the possible severe side effects induced by high dose drug.

Fig. 1.

Fig. 1

Fig. 1

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Effects of gemcitabine (a) and TNP-470 (b) on in vitro proliferation of A549 cells

The growth of A549 cell cultured with TNP-470 was also inhibited in an almost dose-dependent manner (Fig.1b). Half-maximum inhibition of TNP-470 occurred at approximately 10−3 mg/ml. The dose of TNP-470 that we chose to study was 10−4 mg/ml.

Effects of gemcitabine or/and TNP-470 on mRNA expression of VEGF, FLT-1, KDR

As shown in Fig.2, gemcitabine (10−6 mg/ml) alone and TNP-470 (10−4 mg/ml) alone had no effect on the mRNA expression of VEGF and its receptors (FLT-1, KDR) in A549 cells compared to the control (P>0.05). However, 10−6 mg/ml gemcitabine in combination with 10−4 mg/ml TNP-470 significantly inhibited the mRNA expression of VEGF, FLT-1 and KDR compared to the control (P<0.01). Moreover, the combination of two drugs had significant effect on the mRNA expression of VEGF, FLT-1 and KDR compared to either drug alone (P<0.05).

Fig. 2.

Fig. 2

Fig. 2

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Effects of gemcitabine or/and TNP-470 on mRNA expression of VEGF, FLT-1 and KDR in A549 cells. (a) The representative RT-PCR products from normal control, 10−6 mg/ml gemcitabine treatment, 10−6 mg/ml gemcitabine+10−4 mg/ml TNP-470 treatment, and 10−4 mg/ml TNP-470 treatment; (b) The data expressed by mean±SD for VEGF, FLT-1 and KDR mRNA levels. All the data were showed by the ratio of the expression of aim mRNA and that of β-actin

* P<0.05 vs VEGF expression in gemcitabine+TNP-470 treatment group; † P<0.05 vs FLT-1 expression in gemcitabine+TNP-470 treatment group; ‡ P<0.05 vs KDR expression in gemcitabine+TNP-470 treatment group

Effects of gemcitabine or/and TNP-470 on protein expression of VEGF, FLT-1, KDR

The protein expression of VEGF, FLT-1 and KDR in A549 cells was also examined, which provided a very sensitive approach for the results of relative expression of mRNA (Fig.3). Gemcitabine (10−6 mg/ml) alone and TNP-470 (10−4 mg/ml) alone also had no effect on the protein expression of VEGF, FLT-1 and KDR in A549 cells compared to the control (P>0.05). However, there was significant difference in inhibition of protein expression of VEGF and its receptors (FLT-1, KDR) between the group cultivated using 10−6 mg/ml gemcitabine combined with 10−4 mg/ml TNP-470 and control group (P<0.01). Furthermore, the combination of gemcitabine and TNP-470 enhanced the inhibition of VEGF and its receptors compared to gemcitabine/TNP-470 alone (P<0.05).

Fig. 3.

Fig. 3

Fig. 3

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Effects of gemcitabine or/and TNP-470 on protein expression of VEGF, FLT-1 and KDR in A549 cells. (a) The representative Western blot products from normal control, 10−6 mg/ml gemcitabine treatment, 10−6 mg/ml gemcitabine+10−4 mg/ml TNP-470 treatment, and 10−4 mg/ml TNP-470 treatment; (b) The data expressed by mean±SD for VEGF, FLT-1 and KDR protein levels. All the data was showed by the ratio of the expression of aim protein and that of β-actin

* P<0.05 vs VEGF expression in gemcitabine+TNP-470 treatment group; † P<0.05 vs FLT-1 expression in gemcitabine+TNP-470 treatment group; ‡ P<0.05 vs KDR expression in gemcitabine+TNP-470 treatment group

DISCUSSION

Tumor angiogenesis plays an important role in tumor growth, maintenance and metastatic potential. Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen and a potent inducer of vessel permeability and angiogenesis in vivo (Senger et al., 1993) and is a marker of tumor invasion and metastasis (Sauter et al., 1999). Non-small cell lung cancer (NSCLC) tissue produces numerous growth factors, which are multifunctional and considered predictive of patient survival. The previous studies showed that a high expression of VEGF (≥25% of cells) was observed in squamous cell carcinomas and adenocarcinomas, and in all cases of large cell carcinomas (Iwasaki et al., 2004; Stefanou et al., 2003). The level of VEGF was independent prognostic factor in cases of NSCLC involving patients who had undergone curative resection (Iwasaki et al., 2004). Furthermore, the VEGF levels showed differences with respect to histological type. The levels of VEGF in adenocarcinoma were higher than for squamous cell carcinoma (Iwasaki et al., 2004). Therefore, VEGF may be important in the growth of lung adenocarcinoma. A number of observations have spurred extensive investigation of VEGF inhibitors as possible therapies for cancer. Inhibitors in development include monoclonal antibody to VEGF and inhibitors of VEGFR activation following ligand binding (Fong et al., 1999; Yukita et al., 2000; Yilmaz et al., 2003).

The VEGF mediate angiogenic signals to the vascular endothelium via high-affinity receptor tyrosine kinase (RTK). To date, three receptors for the VEGFs have been identified, FLT-1, KDR/FLK-1 (fetal liver kinase-1), and FLT-4 (Shibuya et al., 1999; Neufeld et al., 1999; Aprelikova et al., 1992). All three are relatively specific for endothelial cells and have seven immunoglobulin homology domains in their extracellular part and an intracellular tyrosine kinase signaling domain split by a kinase insert. In adults, FLT-1 and KDR/FLK-1 are expressed mainly in the blood vascular endothelium, whereas FLT-4 is restricted largely to the lymphatic endothelium (Veikkola et al., 2000). Current reports show that a number of tumor cell lines, including melanoma, ovarian carcinoma, pancreatic carcinoma, and Kaposi sarcoma, produce VEGF and its cognate receptors and show impaired viability in response to VEGF ablation by either VEGF ASODN (antisense oligodeoxynucleotides) or neutralizing VEGFR antibodies (Lacal et al., 2005; Vieira et al., 2005). We further demonstrate that the human lung adenocarcinoma cell line also expresses VEGF and its receptors, FLT-1 and KDR. The presence of autocrine growth pathways in some tumors implies that VEGF antisense therapy acts on 2 levels: antiangiogenic effects on the tumor vasculature and antineoplastic effects on the tumor cell population (Masood et al., 2001).

The angiogenesis inhibitor TNP-470 has been reported to inhibit neovascularization by preventing endothelial cell proliferation (Shishido et al., 1996; Kato et al., 1994). One possible mechanism for its effect is that it acts on endothelial cells to inhibit growth factor-induced DNA synthesis. TNP-470 has inhibitory activities against both tumor growth and metastasis (Yanase et al., 1993; Yamaoka et al., 1993a). Antitumor and antimetastatic activities of TNP-470 were evaluated in various human cell lines (Yamaoka et al., 1993b; Tanaka et al., 1995). In this study, we also found the inhibitory effect of TNP-470 on tumor cell proliferation by using a human lung adenocarcinoma cell line, A549. However, TNP-470 is rapidly cleared from the circulation with a short terminal half-life ((0.88±2.5) h) (Figg et al., 1997). Thus, for treatment of lung adenocarcinoma, which, because of its high biological malignancy, requires prolonged maintenance of effective blood concentrations of drugs, we should employ combination chemotherapy.

Gemcitabine (2′,2′-difluorodeoxycytidine) is a novel nucleoside analogue that exerts its antitumor activity via multiple action mechanisms. These include inhibiting DNA replication and cell growth and masked DNA chain termination (Storniolo et al., 1997). It is widely used in lung adenocarcinoma as a first-line chemotherapy and has been shown to produce significant survival benefits (Ferrigno et al., 2004). However, single-agent systemic chemotherapy achieves objective response rates ranging from 10% to 25%. Systemic polychemotherapies achieve variable response rates, but with consistent toxicity (Andre et al., 2004). Therefore, it is important to find a combination chemotherapy that is safe and well tolerated.

To our knowledge, this is the first report describing the antiangiogenic activity of TNP-470 in combination with gemcitabine in a human lung adenocarcinoma cell line. In this study, we investigated the inhibition of expression of VEGF and its receptors, FLT-1 and KDR, in human lung adenocarcinoma cell line using the established model, A594. In order to reduce the toxicity of drugs, we choose the dose of TNP-470 and gemcitabine lower than the half-maximum inhibition occurring at approximately 10−3 mg/ml and 10−4 mg/ml respectively. Although the expression of VEGF and its receptors, FLT-1 and KDR, was not reduced in human lung adenocarcinoma cell line treated with either TNP-470 or gemcitabine. The inhibition effects of the combination of TNP-470 and gemcitabine were stronger than those of TNP-470 alone or gemcitabine alone. This study suggests that combined treatment with TNP-470 plus gemcitabine may augment the antiangiogenic and antineoplastic effects in lung cancer cells in vitro. The present findings may be of importance for further exploration of the potential application of this combined approach in the treatment of lung cancer.

Although the mechanism of this combination therapy is not clear, it appeared to be due to a synergistic effect. In in vitro experiments treatment of KoTCC-1 cells with gemcitabine and TNP-470 decreased the IC 50 of gemcitabine by greater than 50% and markedly enhanced gemcitabine induced apoptosis (Muramaki et al., 2004), then it may reduce the expression of VEGF and its receptor, FLT-1 and KDR. Moreover, gemcitabine enhanced the aggregation of TNP-470 and increased its cell concentration (Jia et al., 2005), which may strengthen the inhibition effects of TNP-470 on VEGF, FLT-1 and KDR.

In summary, by combining antiangiogenic agents with each other and/or with other modalities in the treatment of cancer, the limitations of each therapeutic approach could be overcome, leading to enhanced efficacy with diminished toxicity (O′Reilly, 2002). Antiangiogenic therapy (TNP-470 in combination with gemcitabine) shows significant anti-tumor and anti-metastatic effects and is helpful for reducing the dosage for cytotoxic drugs and the side effects (Lin et al., 2005). Our angiocytotoxic therapy (TNP-470 in combination with low dose gemcitabine) also showed excellent inhibitory effects of VEGF and its receptor on A549 model of human lung adenocarcinoma cell line. These results suggest that angiocytotoxic therapy may provide a new safe and effective strategy for treatment of lung adenocarcinoma. However, before these agents can be used in clinical practice, a better understanding of their action mechanism and regulation is needed.

LIMITATION

Limitations of our lab prevent our carrying out specific experiment in vivo and in vitro to show the anti-angiogenesis and anti-tumor effect of the treatment by gemcitabine and TNP-470. It should be done in our subsequent experiment.

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