High dose concentration administration of ascorbic acid inhibits tumor growth in BALB/C mice implanted with sarcoma 180 cancer cells via the restriction of angiogenesis

Murine sarcoma S180 cells provided by Korea Cell Line Bank were maintained in RPMI-1640 medium supplemented with 10% fetal bovine serum (Hyclone, Aurora, Canada), 100 U/ml Penicillin-Streptomycin (Hyclone), and Non-Essential Amino Acids (Sigma), at 37°C in a 5% CO₂ atmosphere. Female BALB/c mouse (Charles River, Seongnam, Korea) weighing 18-22 g were kept under standard laboratory conditions (tap water, constant room temperature 22°C). Principles of laboratory animal care (NIH publication 85-23, revised 1986) were followed and all experiment was carried out under AAALAC International (Association for Assessment and Accreditation of Laboratory Animal Care International) approval.

Sarcoma 180 cells were cultivated in a CO₂ incubator for five days, adding 9 ml RPMI 1640 medium, in 8 plates of 100 mm in diameter, and then 5 × 10⁵ cells in 200 ml PBS were injected into the abdominal cavities of experimental mice using a 21 G injector. The high dose ascorbic acid dose of 1.7 × 10^-4 mol (30 mg) corresponds to 100 g for a human of 70 kg. The low dose of ascorbic acid was 3.1 × 10^-5 mol (5.5 mg). After each group was treated with ascorbic acid and cancer cells, they were observed and measured over time, and then livers and kidneys were harvested and stored at -70°C for further analysis. BALB/C mice were divided into 7 groups (A - G) with 10 mice per group (Figure 1). Group A was a control group that was treated with phosphate buffer saline (PBS), Group B was treated with low-level ascorbic acid at two-day intervals, and Group C was treated with high dose concentration ascorbic acid at two-day intervals. Group D group was administered Sarcoma 180 cells for cancer induction. Groups E-G received both cancer cells and ascorbic acid. Group E was treated twice with PBS at two-day intervals, injected with S-180 cells, and then treated with high dose concentration ascorbic acid at two-day intervals for Group F was injected with low dose ascorbic acid before injecting cancer cells, and was then treated with high dose concentration ascorbic acid after cancer challenging for 24 days. Group G group was injected with high dose concentration ascorbic acid for four days before injecting cancer cells, and was then treated with high dose concentration ascorbic acid for 24 days after cancer challenging (Figure 1).

RNA was isolated from livers and kidneys of each group. After evenly grinding the samples from each group, 100 mg of each sample were put in 1.5 ml tubes and 1 ml of Corezol (Corebio System, Seoul, Korea) was added. After adding 200 μl of chloroform to the tubes, we centrifuged them at 12,000 g at 4°C for 15 minutes. The supernatants, which contained the RNA, were placed in new 1.5 ml tubes and then precipitated with 700 μl isopropanol. After centrifuging at 12,000 g at 4°C for 15 minutes, we recovered the RNA pellet in 20 μl DEPCed DDH₂O [13]. The RNA concentrations were measured by spectrophotometer and electrophoresis. To identify gene expression in the harvested livers, cDNA was synthesized from 5 μg of total RNA using oligo (dT) primers and Moloney murine leukemia virus (MMLV) reverse transcriptase (SuperBio Co. Daejon, Korea). Six ng mRNA was used for reverse transcription. Primers used for quantitative PCR were designed using Primer3 http://​www.​justbio.​com/​primer/​index.​php and synthesized by Genotech (Daejon, Korea). Angiogenesis genes detected were bFGF (forward primer: CGG CTG CTG GCT TCT AAG TG; reverse primer: CCC GTT TTG GAT CCG AGT TT), VEGF (forward primer: ACA CGG GAG ACA ATG GGA TG; reverse primer: TCT TGA CTC AGG GCC AGG AA) and MMP2 (forward primer: ATG GGG CTG GAA CAC TCT CA; reverse primer: GGG GCC AGT ACC GTC AG); the housekeeping gene was GAPDH (forward primer: TTG CAG TGG CAA AGT GGA GA; reverse primer: GGC TTC CCG TTG ATG ACA AG). PCR amplification was done in a 20 μl total volume containing 4 or 6 μl of 2 × diluted cDNA (duplicate), 0.25 μM each primer, 1 μl 20000 × diluted SYBR Green I (Molecular Probes, Eugene, OR) and 2.5 units Taq DNA polymerase (SuperBio Co. Daejon, Korea) in a reaction buffer composed of 10 mM Tris/HCl (pH 9), 50 mM KCl, 2 mM MgCl₂, 0.5 mM each deoxyribose trinucleotide, and 0.1% Triton X-100, in a Rotor-Gene 3000 (Corbett Research, Sydney, Australia). PCR cycling parameters were 40 cycles of 10 s at 94°C, 15 s at 60°C, and 20 s at 72°C. The products of real-time quantitative PCR were separated by 1% agarose gel electrophoresis to make sure. Two negative controls, missing either RNA template or reverse transcriptase, were included in each experiment. Each data point represents the average of three experiments and the error bars indicate the standard deviation of individual experiments unless mentioned otherwise.

Specimens were fixed in 10% buffered formalin, serially sectioned, and embedded in paraffin. The prepared paraffin blocks were cut at 3 μm thickness and then stained with hematoxylin-eosin [14].

Representative 3 μm-thick tissue sections for immunohistochemical analysis were mounted on silane coated slides. The sections were deparaffinized in xylene and dehydrated with distilled water through a graded series of ethanol solutions. The slides were pretreated in a microwave oven (20 min) with citrate acid solution for antigen retrieval. After rinsing with APK Wash Solution (Ventana Medical Systems, Tucson, AZ, USA), immunochemistry was performed in a Ventana NexES IHC automated immunostainer (Ventana Medical Systems, Tucson, AZ, USA). The primary antibodies used in this study included MMP-2 and VEGF (ABcam, Cambridge, UK), and bFGF (BD Transduction Laboratories™, San Jose, CA, USA). The prediluted (1:50) primary antibodies were applied for 32 min at 37°C. The sections were then treated for color development with diaminobenzidine (4 min), and counterstaining was done with hematoxylin (4 min) using the iVIEW™ DAB Detection Kit (Ventana Medical Systems).

We used a wound healing assay [15] to identify the degree of migration of cancer cells and normal cells caused by the treatment with ascorbic acid. Wounds were created in confluent H-ras NIH3T3 cells (Biochemistry laboratory, Department of Genetic Engineering, Sungkyunkwan University) using a pipette tip. The cells were then rinsed with medium to remove any free-floating cells and debris. Serum-free medium was then added, and culture plates were incubated at 37°C. Wound healing was observed at 0, 12, 24, and 36 hours within the scrape line, and representative scrape lines for each cell line were photographed. Duplicate wells of each condition were examined for each experiment, and each experiment was repeated 3 times.

We compared angiogenesis gene expression (bFGF, VEGF, MMP-2), survival rate, and ascites genesis rate between experiment groups. All analyses were carried out using the statistic software Sigmaplot (Systat software Inc. Chicago, USA). Data are presented as mean ± SE.

Sizes of ascites and intraperitoneal tumors were measured at 16 days after ascorbate or PBS treatment (Figure 2). Mice developed ascites containing tumor cells between 6 and 12 days after cancer injection. Group D (no ascorbate treatment) developed intraperitoneal tumors rapidly. Groups E, F and G developed tumors both more slowly and later than Group D. The amounts of ascites were quantified by recording weights of each mouse. The weights of Groups A-C were maintained at about 20 g but Groups D-G increased beginning 6 days after cancer injection (Figure 3A).

Significant tumor induction was observed in Group D compared to the other groups. White masses were formed, indicated by red arrows, in each organ (Figure 2); these were tumors formed by cancer cells. In addition, more ascites were generated in Group D than in the other groups into which cancer cell had been injected.

Of the 10 mice in each group, 5 were dissected to measure angiogenesis gene expression and observe abdominal cavities, and 5 mice were observed up to 28 days after injecting ascorbic acid to measure survival rate. Celiectomy was performed around 14 days after the injection of cancer cells, and mice were weighed at that time. The greatest average weight, 27.8 g, was found in Group D at the 14^th day after injecting cancer cells, an increase of 1.39 times from the start of the experiment. Groups G, E, and F groups followed in weight order (Figure 3A). The average body weight at the 18^th day was 25.2 g of Group E, 24.8 g of Group F and 26 g of Group G respectively. These data showed that the treatments of ascorbic acid by challenging with low dose before cancer infection and then treated with high dose of ascorbic acid was more effective (Group E). At the 18^th day, the body weight of Group F was 0.89 times of Group D. Survival rate was measured up to 28 days from the beginning of the experiment. Group D showed a survival rate of 0% after 25 days, and Group E showed a survival rate of 0% after 28 days from the beginning of the treatment. In contrast, F and G groups, which had been treated with ascorbic acid prior to injecting cancer cells, showed a survival rate of 20% at the 28^th day (Figure 3B).

Angiogenesis is an important mechanism in cancer genesis and the growth process. We measured gene expression of genes involved in angiogenesis by staining and real-time PCR. Cancer genesis in each group was identified by H&E staining as shown in Figure 4A. We observed blue staining of giant nuclei followed by cancer cell genesis, and identified cancer genesis in tissue from Group E (Figure 4B, e2). No staining was found in the other groups; they did not differ from the negative control groups to which cancer cells had not been injected. Thus there was a remarkable reduction of cancer genesis in the groups which received prior treatment with ascorbic acid. We also measured gene expression involved in angiogenesis by immunohistochemistry (Figure 4B). Additional histochemical staining was made for A, D, E, and F groups. As a result of staining with antibody of other 3 angiogenesis related protein, applied to this test, in each tissue, no histochemical staining was made in other groups except D group (Figure 4B). We also analyzed expression of genes involved in angiogenesis by Quantitative real-time RT-PCR (Figure 5). In Group D, expression of bFGF was increased by about 18 times over the groups that did not receive injected cancer cells. This increase was 2.5 times, 1.8 times, and about 1.3 times greater than the increase seen in Groups E, F, and G, respectively, groups which had been treated with ascorbic acid after injecting cancer cells. In Group D, expression of VEGF was increased by 4.5-7; for MMP2, the increase was about 5 times. The expression of angiogenesis related genes was thus remarkably reduced in the groups with ascorbic acid treatment compared to the group with cancer cell treatment only. These results suggest that ascorbic acid treatment in high concentration inhibits angiogenesis by inhibiting the expression of angiogenesis related genes.

We used a wound healing assay to compare the inhibition of the expression of angiogenesis related genes and protein synthesis by ascorbic acid with the change of cell migration efficiency (Figure 6). We observed wound recovery at 0, 12, 24, 36 hrs after treating with 2.5 mM or 10 mM ascorbic acid. The H-ras NIH3T3 cells did not recover after wounding and high treatment concentration of ascorbic acid, while artificially formed wounding was recovered in NIH3T3 cell at 12, 24, 36 hrs by cell migration even in ascorbic acid in 2.5 mM and ascorbic acid in 10 mM (Figure 6). Therefore, migration was inhibited according to ascorbic acid concentration in cancer cell and the treatment time.