Male Balb/c nude mice were provided by the animal center of Shandong University. BEL-7402 cells were obtained from Shandong Academy of Medical Sciences, China. Immunochemical kit and in situ cell apoptosis detection kit were purchased from Wuhan Boster Technology Company, China. Nitric oxide synthase and NO detection kits were purchased from Nanjing Jiancheng Bioengineering Institute, China. 5-FU was produced by Shanghai Xudong Haipu Pharmacentical Company. L-Arg was produced by Shanghai Xinyi Jinzhu Pharmacentical Company.

Human primary hepatocellular cancer cell line BEL-7402 was maintained in a humidified, 5% CO₂ atmosphere and cultured in DMEM, supplemented with 10% fetal calf serum and penicillin-streptomycin mixture.

BEL-7402 cells were cultured in vitro to the log growth phase, centrifuged and washed with PBS to a concentration of 1 × 10⁷/mL, and 0.2 mL cell solution was subcutaneously xenografted in the neck or the flank of 4-6 wk old male Balb/c nude mice. The mice were housed and maintained in lamina flow cabinets under specific pathogen-free conditions. Ten day after establishment of the model when tumors reached a mean diameter of 0.5 cm, mice were randomly divided into three groups (10 mice per group) and treated as follows: group A was injected intraperitoneally with 0.2 mL normal saline, group B was injected with 5-FU at a dose of 20 mg/kg, and group C was injected with 5-FU at 20 mg/kg and L-Arg at 100 mg/kg. The drugs were injected every other day from d 11 to d 15. The mice were killed at 17d after the experiment finished.

The largest diameter (a) and its vertical diameter (b) of the tumors were measured with caplipers every two days after the tumor appeared. The volume of tumor was equal to 1/2ab². The tumor inhibition rate (TIR) was calculated with the following formula:

TIR = control group (v₁ - v₀) - treatment group (v₁ - v₀)/control group (v₁ - v₀)

(v₀: the volume before injection of drugs, v₁:the volume after injection of drugs).

The sections were stained with hematoxylin-eosin to evaluate the degree and range of the necrosis. It was divided into 4 degrees according to the percentage of the necrotic area: ≤ 25% (+), 25%-50% (++), 50%-75% (+++), > 75% (++++)[5].

TUNEL method was used to detect the apoptosis of tumor cells according to the instructions of the manufacturer. Cells with brown or yellow nuclei were assumed as apoptotic cells. The number of apoptotic cells and total cancer cells was counted under light microscope at 400 × magnification in 5 fields of version and the average values were used for the calculation of apoptosis index (AI) according to the formula: AI = (apoptotic cells/total cancer cells) × 100%.

Tumor specimens were dissected from mice and fixed in 10% buffered formalin solution overnight. They were then embedded in paraffin and sectioned in 4 μm thicknesses. SABC immunohistochemistry was performed according to the manufacturer’s instructions to detect the gene expression of iNOS, P16 and BAX. Briefly, the tissue sections were deparaffinized in xylene at 37°C for 20 min. Endogenous peroxide was blocked by incubating the slides with 30 mL/L H₂O₂ for 10 min at 37°C. Sections were incubated with primary antibodies of iNOS, P16 and BAX at 4°C overnight respectively. Staining was visualized with DAB for 10 min at room temperature. Finally, the sections were counterstained for nuclei by hematoxylin solution. The results were analyzed with a BI2000 pathological image analyzer and expressed as optical density.

NOS catalyzed the formation of NO and L-citrulline from L-arginine and molecular oxygen, and NO reacted with a nucleophile to generate color compounds. The absorbance of NOS at 530 nm was calculated and expressed as U/mg protein. One unit of NOS activity was defined as the production of 1 nmol nitric oxide per second per mg tissue protein. Total NOS activity was measured as follows: 10% tissue homogenate (100 μL) was incubated with 200 μL substrate buffer, 10 μL reaction accelerator and 100 μL color development reagent at 37°C for 15 min after mixing. Then 100 μL clearing reagent and 2 mL stop solution were added, mixed and absorbances were read at 530 nm. For measuring iNOS activity, an inhibitor was added before incubation according to the manufacturer’s instructions. Total protein concentration was determined using the Coomassie blue method with bovine serum albumin as standard.

Tumor samples were thawed, weighed and homogenized 1:9 w:v in 0.9% saline. The homogenates were then centrifuged at 1000 r/min for 5 min at 4°C, the supernatant was taken for NO assay and total protein determination.

NO was assayed spectrophotometrically by measuring total nitrate plus nitrite (NO₃^- plus NO₂^-) and the stable end products of NO metabolism. The procedure was performed by the manufacturer’s instructions. Results were expressed as μmol/g protein.

All data were analyzed with SPSS11.0 statistical software. One-way ANOVA, Bonferroni, Kruskal-Wallis H test and correlated analysis were performed.

5-FU could inhibit the growth of the liver carcinoma in nude mice. When the substrate L-Arg was supplemented, tumor inhibition rate increased significantly (Table 1 ).

There was different necrosis in the tumor tissues. The necrotic range enlarged greatly after treatment with 5-FU and L-Arg (Table 2).

There were a lot of apoptosis cells in 5-FU and 5-FU+L-Arg groups, while only scattered apoptosis cells in NS group. The apoptosis cells are shown in Figure 1. The apoptosis indexes were as follows: NS group 17.4% ± 6.19%; 5-FU group 31.3% ± 12.3%; and 5-FU+L-Arg group 46% ± 15.24%. ^aP < 0.05 group B vs group C; ^cP < 0.05 A vs C; ^eP < 0.05 A vs B. There was significantdifference between the two groups.

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Figure 1 The apoptosis cells in tumor tissues (× 400).

The optical density of iNOS, iNOS activity and NO concentration were increased after treatment with 5-FU. When the substrate L-arg was supplemented, they were increased more significantly (Table 3). The expression of iNOS is shown in Figure 2.

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Figure 2 The expression of iNOS in tumor tissues (× 200).

The expression of P16 and BAX was increased after treatment with 5-FU and L-Arg. P < 0.05 (Table 4). The result of Pearson analysis showed that the concentration of NO was correlated with the expression of P16 and BAX. The correlation coefficient was 0.764 and 0.554.

Hepatocellar carcinoma is one of the most common malignant tumors in China. 5-FU is used extensively for the treatment of liver cancer. The effects of 5-FU alone is not satisfied. It is necessary to find the effective adjuvant for 5-FU.

The expression of iNOS in the human liver carcinoma and the effects of endogenous nitric oxide on the BEL-7402 cells have been examined.

In the present study, the effects of endogenous nitric oxide on the liver carcinoma in nude mice have been studied.

The L-Arg can be used as the adjuvant for 5-FU in the treatment of liver cancer.

L-Arginine (L-Arg) is a semi-essential amino acid that possesses numerous useful physiologic properties. NOS can catalyzethe L-Arg to form the nitric oxide. When the L-Arg is sufficient, the production of nitric oxide can be increased.

This manuscript is very interesting. The title accurately reflects the major contents of the article. The results provide sufficient experimental evidences from which conclusions are drawn. The conclusions are scientifically reliable and valuable.