Effects And Mechanisms Of 5-FU Combined With L-Arg On The Human Liver Carcinoma In Nude Mice

Background and Objective: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in our country. Surgical resection of the carcinoma is the best choice. But many patients could not be operated for diagnosis too late. After curative resection, the most common problem is recurrence. The complex treatments are adopted to improve the curative effects of the liver carcinoma. Chemotherapy becomes the important mean in liver cancer complex treatments.5-FU is one of the most common chemotherapy drugs for liver cancer. It is necessary to find the new mechanisms of 5-Fu and it is a new direction for liver cancer treatment to look for the adjuvant of 5-FU. NO is a small molecular gas. It has the dual effects to the tumor cells. NO can promote the tumor growth at low concentrations, on the other hand it will inhibit the tumor growth and induce the apoptosis of tumor cells at high concentrations. It will be benefit to prevent and treat the tumors by modulating the concentration of NO. L-Arg is the precursor of NO. It will generate the NO under the action of NOS. It is well known that in vitro L-Arg at low concentrations gently promoted the growth of BEL-7402 cells; on the other hand it could inhibit the growth and proliferation and induce the apoptosis of BEL-7402 cellsat high concentrations.5-FU can induce the expression of iNOS in BEL-7402 cells. The concentration of NO should be increased when the 5-FU and L-Arg are used together. It will increase the anti-tumor effcts of 5-FU.In our study, we established the transplanted tumor model of human liver carcinoma in nude mice with BEL-7402 cell. The mice were treated with 5-FU and L-Arg. The effects and mechanisms of 5-FU and L-Arg on the human liver carcinoma in nude mice were observed.Methods: BEL-7402 cells were cultured at 37 °C , 5%CO2 humid conditions. The culture medium was DMEM with 10%FBS. The cells were digested by 0.25% trypsin. A single cell suspension of BEL-7402 cells with the concentration of 5.OX 106/ml was prepared for injecting. The 0.2ml cells were injected into subcutaneous tissue of nude mice under the aseptic condition. The mice were raised at the aseptic constant temperature, constant humidity enviroment and were observed every day. The tumor size was measured every day after the tumor was appeared. The growth curve of transplanted tumors was draw by the tumor volumes. The morphology features of transplanted tumor tissue were observed by miscrocope. The expression of iNOS in BEL-7402 cells and tumor tissues was detected by immunohistochemistry method.The forty nude mice were injected with the BEL-7402 cells subcuneously and the mice were divided intoA, B, C, D 4 groups at random when the tumor grew to 5mm. The mice were injected intraperitoneally with 0.2ml normal saline solution 0.2ml(A group), 20mg/kg 5-FU(B group), lOOOmg/kg L-Arg(C group), 20mg/kg 5-FU+1000mg/kg L-Arg(D group) respectively, for 5 times on every other day. The changes of tumor volumes were measuredcontinuously. The growth curve of every group was draw and the tumor inhibition rate of each group was calculated. We observed the morphologic changes used HE staining by light microscope and measured the apoptosis rate by TUNEL staining method. The expression of iNOS, PI 6, BAX, VEGF in the tumor tissue was detected by immunohistochemistry method. The activity of iNOS was tested by chemical colorimetry and the concentration of NO in tumor tissue was detected by nitrate reductase method.Results: The tumorgenic rate of BEL-7402 cells in nude mice was 100%. The tumorgenic time of neck was more centralized than other two sites. The growth curve of transplanted human liver carcinoma in nude mice with BEL-7402 cells was exponential curve. The morphological feature and cell arrangement of tumor tissue had some characteristic features of human liver cancer. The reaults of immunohistochemistry showed the positive expression of iNOS in BEL-7402 cells and transplanted tumor tissues.Both 5-FU and L-Arg could inhibit the growth of transplanted tumor in nude mice. The tumor inhibition rate was 31.6% and 23.4% in 5-FU group and L-Arg group. 5-FU combined with L-Arg could inhibit the tumor growth more appearently. The tumor inhibition rate of 5-FU+L-Arg groupe was 52.9%. 5-FU, L-Arg could induce the implanted tumor cells to undergo apoptosis. The apoptosis index of A, B, C, D groupe was 6.2 + 4.16%, 19.2 ±9.59%, 18.5 ±6.45%, and 33.5 + 14.87% respectively.Immunohistochemistry staining showed that both 5-FU and L-Arg could induce the expression of iNOS in the tumor tissue. L-Arg had the agonist effects with 5-FU of inducing the expression of iNOS. The iNOS optical density of A, B, C, D groupe was 0.4621 ±0.0115 , 0.4783 ±0.0107, 0.4850 ±0.0116, 0.5269±0.0034. The optical density of BAX in each group was0.4501+0.0114, 0.4788 + 0.0068, 0.4744 + 0.0164, 0.5045 + 0.0199. The optical density of P16 in each group was 0.4565 ±0.0139, 0.4939±0.041, 0.5009 + 0.0258, 0.5451+0.027 and the optical density of VEGF was 0.4529 + 0.0151, 0.4993 + 0.0235, 0.4895 + 0.012, 0.5366 ± 0.0243. The iNOS activity in each group was 4.86 + 2.5u/mgprot, 10.37 + 2.57u/mgprot, 9.83 + 2.31u/mgprot,15.1+4.91u/mgprot. The concentration of NO in each group was 6.58±3.2umol/L> 17.97 + 6.16umol/L, 20.59 + 6.1 umol/L > 30.41 ± 8.8umol/L. The concentration of NO was correlated with the expression of BAX, PI6, VEGF. The correlation coefficient was 0.697, 0.500, 0.667 respectively.Conclusions: BEL-7402 cells have good tumorgenic ability in nude mice. The human liver carcinoma in nude mice conserves some characters of human liver cancer. The transplanted tumor can express the iNOS protein. In vivo, both 5-FU and L-Arg can inhibit the growth of transplanted tumor in nude mice and induce the tumor cells to undergo the apoptosis and necrosis. 5-FU combined with L-Arg can inhibit the tumor growth more appearently and increase the apoptosis index and necrotic ratio. 5-FU and L-Arg can induce the expression of iNOS and increase the activity of iNOS. The concentration of NO is related with BAX, PI6, VEGF. Nitric oxide can raise the apoptosis-related gene and antioncogene. It can also increase the expression of VEGF and vascular in tumor. Endogenous nitric oxide is an effective factor for 5-FU to treat the human liver carcinoma in nude mice. It will increase the effects of 5-FU by supplyment of L-Arg.