The human gastric carcinoma cell line NCI-N87, human prostate cancer cell line PC3, human cervical carcinoma cell line HeLa, and human lung adenocarcinoma cell line A549 were purchased from ATCC (Manassas, VA, USA). All cells were grown in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), supplemented with 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.), 2 mM glutamine, and 1% penicillin/streptomycin/neomycin in a humidified incubator (5% CO₂ in air at 37°C). Avemar was donated by Biropharma USA Inc. (New York, NY, USA). The Avemar was stored as dried powder at 4°C in a bottle until use. Prior to use, it was freshly prepared in sterile water to a final concentration of 400 µg/ml. The solution was centrifuged to remove indissoluble materials and then filtered with a 0.22-µm filter.

A549, PC3 and NCI-N87 cells release VEGF protein constitutively. The augmented release of VEGF protein after 48 h was determined in response to serum starvation in PC3 and NCI-N87 cells, and in response to 1,000 U/ml tumor necrosis factor α (TNF-α) in A549 cells (15). In our previous study, the basal VEGF protein levels were determined at 24, 48 and 72 h following seeding of HeLa cells (5×10³ cells/well) without any stimuli (16).

A human VEGF ELISA kit (cat. no., ENZ-KIT156; Enzo Life Sciences, Inc., Farmingdale, NY, USA) was used according to the manufacturer's protocols in order to determine the possible effects of Avemar on VEGF levels in tumor cells. Briefly, 5×10³ cells were plated in each well of a 96-well plate and were treated with various concentrations (400, 800, 1,600 or 3,200 µg/ml) of Avemar for 24 or 48 h. Samples (100 µl) were then added to the microplates containing VEGF-specific monoclonal antibodies, and the mixtures were incubated for 2 h at room temperature. The plates were then washed three times to remove any unbound substances. Enzyme-linked polyclonal antibodies specific for VEGF were then added to the wells, and the mixtures were incubated for 2 h at room temperature, prior to a further wash to remove any unbound antibody or enzyme reagent. The substrate solution was subsequently added to the wells, and the reaction resulted in the development of a blue color, the intensity of which was proportionate to the amount of VEGF bound in the initial step. Following quenching to cease color development, the intensity of the color was measured at 450 nm with a Multiskan GO Microplate Spectrophotometer (Thermo Fisher Scientific, Inc.) and compared to a standard curve.

The Cox-2 concentration was measured using a human Cox-2 ELISA kit (cat. no., ADI-900-094) provided by Enzo Life Sciences, Inc. Samples were prepared by extracting Cox-2 from the cells and stock solutions were prepared according the manufacturer's protocol. Briefly, the cells were harvested and medium was removed. The cells were re-suspended in radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris, pH 7.4, 0.15 M KCl, 1% NP-40, 5 mM EDTA, 0.5% Sodium deoxycholate, 0.1% SDS). Samples were prepared by sonicating cells in RIPA buffer for 5 cycles of 30 sec, in 1 min intervals on ice. Samples were then added the microplate. The plate was incubated at 37°C for 1 h, then washed prior to the addition of a labeled antibody. The plate was incubated at 4°C for 30 min, washed, and a substrate solution was added. The reaction was stopped and absorbance was measured at 450 nm using a Multiskan GO Microplate Spectrophotometer. Protein concentrations were calculated with reference to the standard curve.

All tumor cell lines were plated in 6-well plates (3×10⁶ cells/well) and allowed to attach for 24 h. Following the incubation period, cells were treated with 400 or 3,200 µg/ml Avemar and the cells were incubated for a further 48 h. Subsequently, the medium in each well was aspirated, and the cells were washed with ice-cold PBS and immediately lysed in 2-mercaptoethanol in RLT buffer from the RNeasy kit (Qiagen GmbH, Hilden, Germany). Further RNA isolation was performed with the RNeasy kit as described by the manufacturer. Total isolated RNA was then quantified, and 100-ng samples of total RNA were reverse-transcribed into cDNA using a QuantiTect Reverse Transcription kit (Qiagen GmbH), according to the manufacturer's protocol, along with random hexamers and oligo-(dT) ₁₆ primers included in the kit. RT was performed at 42°C for 30 min. The total volume for each reverse transcription reaction was 20 µl. The generated cDNA was then used as the template for qPCR in a StepOnePlus™ Real-Time PCR System (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The qPCR was performed under the following conditions: 5 min initial denaturation at 94°C, then 35 cycles of denaturation (30 sec at 94°C), annealing (45 sec at 58°C), extension (45 sec at 72°C), and a final extension at 72°C for 5 min.

The reaction mixtures consisted of SYBR Green I (QuantiTect SYBR Green PCR kit; Qiagen GmbH), 300 µM forward and reverse primers (final concentrations optimized during the assay setup), 100 nM human UPL probe, and 2.5 µl cDNA template. The sequences of the primer pairs are listed in Table I. The expression levels of the tested genes were normalized to that of GAPDH and presented as the fold change compared with the control group (17). All experiments were repeated three times.

All values are presented as the mean ± standard error of mean. Data were analyzed using a one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test. Analyses were performed with GraphPad InStat v.10.0 software (GraphPad Software, Inc., La Jolla, CA, USA). P<0.05 was considered statistically significant.

First, the maximum level of VEGF expression was determined in A549 cells stimulated by TNF-α and in PC3 and NCI-N87 cells stimulated by serum starvation. Basal VEGF protein levels were measured at 12, 24 and 48 h in HeLa cells, and the maximum induction of released VEGF protein was observed after 48 h without any stimuli (Fig. 1).

To determine the possible effects of Avemar on VEGF levels induced by the different stimuli for each cell line, all tumor cells were treated with 400, 800, 1,600 and 3,200 µg/ml Avemar for 24 and 48 h. In all cell lines, the inhibition of induced VEGF levels increased in a dose-dependent manner (Fig. 2). The increase in inhibition, as compared with the control group, was statistically significant in the HeLa and A549 cells that were treated with 3,200 µg/ml Avemar for 48 h (P<0.001). No significant changes in VEGF inhibition were observed in the NCI-N87 and PC3 cells at either time point (P>0.05). In A549 and HeLa cells, RT-qPCR was performed to investigate whether the inhibition of VEGF levels occurred due to alterations at the transcript level. The results revealed consistent alterations in the mRNA (Fig. 3) and protein levels of VEGF (P<0.001). As shown in Fig. 3, Avemar treatment (3,200 µg/ml) yielded 3.2- and 3.4-fold decreases in VEGF mRNA levels in A549 and HeLa cells, respectively (P<0.001).

It was next assessed whether Cox-2 was responsible for the VEGF inhibition following Avemar treatment in HeLa and A549 cells. Unstimulated HeLa cells and TNF-α-stimulated A549 cells were treated with 400, 800, 1,600 and 3,200 µg/ml Avemar for 24 and 48 h, and Cox-2 protein levels were measured by ELISA. The inhibition of Cox-2 levels following Avemar treatment in HeLa and A549 cells increased in a time- and dose-dependent manner (Fig. 4). Compared with the control group, the amount of Cox-2 protein decreased by ~60% in HeLa and A549 cells (P<0.001). RT-qPCR was also performed to examine whether Avemar treatment was also associated with the inhibition of Cox-2 at the transcript level. RT-qPCR verified the ELISA results for Cox-2 levels, with Avemar treatment (3,200 µg/ml) yielding 4.3- and 4.6-fold decreases in Cox-2 mRNA levels in A549 and HeLa cells, respectively (Fig. 5).