Fifty male Kunming mice aged from 6 to 8 weeks and weighing from 20 to 24 g were obtained from the Animal Experiment Center of Shandong University, China. The mouse model was provided by the Institute of Medicine, Shandong Academy of Medical Science, China. Seven days following S180 cell injection, ascites was extracted from S180 ascites sarcoma mice under an septic condition. Normal saline was then added to adjust the tumor cell concentration to 1×10⁷/ml. Tumors were generated in 40 male Kunming mice by injection of a 0.2-ml tumor cell suspension subcutaneously into the right flank of each mouse. Tap water and food were provided ad libitum.

Aspirin was obtained from Bayer S.p.A. (Milano, Italy). DMSO was obtained from Sigma (St. Louis, MO, USA) to dissolve the aspirin. 5-FU was obtained from Tianjin Jinyao Amino Acids Co., Ltd., Tianjin, China. Western blotting related reagents were purchased from the Shanghai Beyotime Institute of Biotechnology, China.

All tumor-bearing mice were randomly divided into 5 groups: control group, 5-FU group, high-dose aspirin group, low-dose aspirin group and combination group with 10 mice in each group. Treatment was initiated when the diameter of tumors was 5 mm (referred to as day 0) at day 5 following tumor cell injection. The high-dose and low-dose aspirin groups respectively received 250 and 50 mg/kg aspirin by oral gavage once a day for 14 days. Control mice received an equal volume of normal saline. 5-FU (20 mg/kg) was injected i.p. once every 3 days (day 1, 4, 7, 11 and 14). Combination group mice received 50 mg/kg aspirin by oral gavage once every day and 20 mg/kg 5-FU was injected i.p. once every 3 days (day 1, 4, 7, 11 and 14). The volume was measured to be 0.1 ml/10 g. This experiment was repeated three times. The two maximal perpendicular diameters of the tumors were measured every 2 days to document tumor growth (day 0, 2, 4, 6, 8, 10, 12 and 14). Tumor measurements were converted to tumor volume (V) using the formula (V = W² × L/2); where W and L are the perpendicular smaller and large tumor diameters, respectively, and plotted against time. Body weight of the mice was measured twice weekly. Mice were euthanized 24 h after the last treatment. The tumor masses were excised from the mice and were then weighed. Calculation of the tumor inhibitory rate was calculated using the formula: % Inhibitory rate (IR) = [average tumor weight of the control group (g) - average tumor weight of the treatment group (g)]/average tumor weight of the control group (g) × 100.

Part of the fresh tumor specimens was fixed in 10% neutral-buffered formalin, processed and embedded into paraffin blocks. Paraffin-embedded tumor samples were processed into tissue array blocks, which were cut into 4-μm sections for hematoxylin and eosin (H&E) and immunohistochemical staining. Paraffin sections were dewaxed in five changes of xylene and rehydrated through descending concentrations of alcohol. Endogenous peroxidase activities were blocked using 3% hydrogen peroxide. Sections were treated with heating in 10 mmol/l citrate buffer at pH 6.0 inside a water bath. They were incubated overnight in a moist chamber with the primary antibody. The secondary antibodies, biotinylated goat anti-rabbit and anti-mouse IgG (Beijing Biosynthesis Biotechnology Co., Ltd., Beijing, China), were applied at 1:200 in PBS for 1 h at room temperature, stained with DAB and then counterstained with hematoxylin. Finally, sections were dehydrated through graded alcohols, cleared in xylene and mounted in permount. The following antibodies were used for immunohistochemical staining: D2-40 (1:75 dilution; CWBIO, Beijing, China); CD34, VEGF-A, VEGF-C (1:100 dilution; Beijing Biosynthesis Biotechnology Co., Ltd.). Microvessel density (MVD) was analyzed in CD34-stained vascular endothelial cells. CD34 is expressed in the endothelial cells of microvessels. The microvessel count was carried out in accordance with the method of Weidner et al(25). Initially, we selected 3 dense microvessel fields separately at the original magnification ×40 and ×100, and the numbers of CD34-stained cells were then counted at the original magnification ×400 and averaged for statistical analysis. The method of lymphatic vessel count was identical to that of the microvessels.

Sections of fresh tumor specimens were stored at −70°C. They were homogenized with ice-cold lysis buffer, and were then centrifuged at 10,000 rpm for 10 min at 4°C. The protein concentration was determined with the BCA kit. The total protein sample was denaturated at 100°C for 10 min and separated by 10% SDS-PAGE gels and later transferred to Protran nitrocellulose membranes. The membranes were blocked by blocking fluid for 2 h and incubated overnight with an anti-VEGF-A or VEGF-C antibody (1:100 dilution), after extensive washing, and a biotinylated goat anti-rabbit IgG antibody (1:200 dilution) for 1.5 h at room temperature, and then stained with DAB. The band intensities were analyzed by ImageJ software (Wayne Rasband National Institutes of Health, Bethesda, MD, USA).

All data were processed by SPSS11.5 and presented as means ± SEM. The t-test was performed to compare means and distributions for different treatment groups. Statistical significance was based on two-tail P<0.05.

Tumors grew relatively rapidly in the mice. Treatment with 5-FU (20 mg/kg) alone, either high-dose (250 mg/kg) or low-dose (50 mg/kg) aspirin alone, and a combination of 5-FU (20 mg/kg) and aspirin (50 mg/kg) resulted in inhibition of tumor growth compared to the control group. The inhibitory rate was 64.1, 33.5, 22.2 and 70.1%, respectively (Table I, Fig. 1; P<0.05 for each comparison). The inhibitory effect was stronger in both the combination and 5-FU groups (P<0.01). Although the inhibitory rates of the high- and low-dose aspirin groups were lower, the mice in both groups were in a good condition with an increased body weight after the experiment (Table I). This demonstrated that aspirin not only inhibited the growth of S180 sarcoma, but also reduced the body wasting effect caused by the tumor.

H&E staining showed that S180 sarcoma cells were distributed as sheets and nests. Tumor cells varied in size and shape. High- and low-dose aspirin, and the 5-FU and combination groups exhibited morphological changes characteristic of apoptotic tumor cells such as nuclear pyknosis and karyorrhexis. Microvessel density was significantly lower than that in the control group (Fig. 2). In the high-dose aspirin, 5-FU and combination groups the tumors exhibited large patchy necrosis along with the frequent observation of monocyte infiltration.

Based on immunohistochemical staining, VEGF-A and VEGF-C were expressed in the cytoplasm of tumor cells. Cells positive for VEGF-A and VEGF-C were stained brown (Fig. 3). The expression of VEGF-A and VEGF-C in the control group was markedly higher than that in the other treatment groups. Grey scale intensity variants of VEGF-A and VEGF-C immunoreactivity were evaluated by Leica Qwin V3 software. Sections were evaluated in each of 5 randomly selected positive regions at the original magnification ×200. Fig. 4 indicates an inverse relationship between the grey scale intensity and the protein expression. Higher grey scale intensity indicates weaker protein expression, and lower intensity indicates stronger protein expression. Treatment with 5-FU and aspirin resulted in a reduction in VEGF-A and VEGF-C expression. VEGF-A and VEGF-C expression decreased significantly in both the 5-FU alone and combination groups when compared with the other treatment groups showing a dose-dependency on high- and low-dose aspirin. In each comparison, there was a significant difference (P<0.05).

VEGF-A and VEGF-C expression was normalized to β-actin expression by band intensity. As known in Fig. 5, VEGF-A and VEGF-C expression was reduced in the high- and low-dose aspirin, 5-FU and combination groups. Band intensities were analyzed by ImageJ software. VEGF-A and VEGF-C expression decreased significantly in the S180 sarcoma tumors treated with 5-FU alone or with the combination with aspirin. The decreased expression also showed a dose-dependent trend in the high- and low-dose aspirin groups.

Cells positive for CD34 were stained brown. Microvessel distribution is shown in Fig. 6A. Necrotic tumor cells were frequently present in the 5-FU-treated group and in the high-dose aspirin group. The 5-FU alone, high- and low-dose aspirin and combination groups all demonstrated inhibition of MVD in comparison to the control group (P<0.05 for each comparison), which suggests that aspirin inhibits angiogenesis (Table II).

D2-40-positive tubular structures were stained brown. The vessel walls were thin and irregular in structure (Fig. 6B). The 5-FU alone, aspirin and the combination groups had an inhibitory effect on LVD. The high-dose aspirin (250 mg/kg) group showed a noticeable inhibition of LVD in comparison to the control group (P<0.05) (Table II). The high-dose aspirin group exhibited positive tumor cells generating non-endothelial cell-lined channels (Fig. 6B, arrow fourth panel). The low-dose aspirin group displayed a tumor cell around a lymphatic vessel (Fig. 6B, arrow, middle panel). The result revealed that LVD of the control group was the highest in the 5 groups and the lumen showed obvious expansion. The 5-FU, high- and low-dose aspirin groups had an inhibitory effect on LVD and VEGF-C. High-dose aspirin (250 mg/kg) markedly inhibited lymphatic vessel density when compared with the control group.