The Study On Anticancer Activity Of 5-FU And Its Derivatives In Combination With APN/CD13 Inhibitors

There are mainly three methods in the treatment of malignant tumors:surgery, radiation therapy and chemo-therapy treatment. Chemo-therapy is accounted for dominant position in all the cancer treatments. However, Because of the appearance of multi-drug resistance and the existence of cancer stem cells, a single chemo-therapy have been unable to play a very good therapeutic effect, therefore, the combination of different anticancer drug has become popular and been paid more and more attention by people.5-FU is a commonly used chemotherapy drugs, however, because of its presence in bone marrow suppression, serious adverse gastrointestinal reactions, its application in clinical gradually restricted. The emergence of combination therapy provides a new way for 5-FU in clinical use, while scientists have paid more attention on the combination of 5-FU with aminopeptidase N inhibitors.Aminopeptidase N (APN/CD13) belongs to the family of metalloproteinases Ml Gluzincins subfamily, it can degrade the extracellular matrix and play importat role on tumor cell invasion and metastasis. Therefore it has always been regared as a significant anticancer target. That’s why our subject to study the anticancer activity and mechanism of aminopeptidase N (APN/CD13) inhibitors in combination with 5-FU. Through our research,we had several conclusions: (1) In vitro activity evaluation:Firstly, enzyme ativity assay indicated that 5-FU did not affect aminopeptidase N inhibitor binding to the activity site of aminopeptidase N; then the expression of aminopeptidase N on a variety of tumor cell surface were analyzed by flow cytometry and found that human ovarian cancer (ES-2) and small cell lung cancer (A549) were highly expressed aminopeptidase N. We choose ES-2 cell to do cell surface enzyme activity evaluation. Its results further confirmed that 5-FU did interacts with aminopeptidase N nor aminopeptidase N inhibitors. Then in the cell proliferation assay, the combined group inhibited cancer cells growth better than 5-FU alone and Bestatin alone group, where 5-FU:Bestatin 5:1 and 5-FU: Bestatin 10:1 shown best results; normal cells cytotoxicity test proved that the toxicity of 5-FU:Bestatin 5:1 was 1/6 toxicity of 5-FU alone group, while 5-FU: Bestatin 10:1 toxicity was half of it. So the final choice molar ratio of combination group is 5:1 for subsequent experiments. In vitro experiments have initially proved that the combination had better anticancer activity with low toxicity.(2) In vivo activity evaluation:H22 tumor-bearing mice experiments results shown that the inhibition rate of low-dose combination group was close to 5-FU alone treatment group,50.4% and 49.2% respectively, while high-dose combination group inhibition rate reached 62% and Bestatin alone administration group was only 30.6%. Moreover, two mice in the 5-FU group died while no mice in the other groups died, indicating the substantial toxicity of 5-FU and its reduced toxicity when used with Bestatin. In addition,5-FU resulted in weight loss after three days of drug delivery while the Bestatin group and the combination groups tolerated the treatment well. All these results indicated that the combination surely had better anticancer activity with low toxicity.(3) Possible mechanism of combination:To study the mechanism of combination, the apoptosis and cell cycle were analyzed on ES-2 and PLC/PRF/5 cells by flow cytometry. The combination of the two drugs significantly induced cell apoptosis at a rate of 20.86% in PLC/PRF/5 cells and 41.58% in ES-2 cells, while 5-FU or Bestatin alone resulted in relatively low rates of apoptosis in both PLC/PRF/5 cells (< 10%) and ES-2 cells (< 30%). These results indicated that the drug combination inhibited cancer cell growth by greatly inducing apoptosis. The cell cycles of PLC/PRF/5 cells revealed that 5-FU exposure induced cells to remain in the S phase while Bestatin increased the number of cells in the G0/G1 phase. The results of the combination indicated that the cell cycle was arrested in the G0/G1and S phase.(4) Aminopeptidase N inhibitors 4Q, M03 combined with 5-FU:This part is to evaluate anticancer activity of aminopeptidase N inhibitors 4Q, M03 which were designed and synthesized by our laboratory combined with 5-FU. First, enzyme ativity assay and ES-2 cell surface enzyme experiments determined that 5-FU did not affect aminopeptidase N inhibitor bingding to aminopeptidase N. MTT assay proved aminopeptidase N inhibitors 4Q, M03 combined with 5-FU could inhibite a variety of tumor cell proliferation. In vitro experiments, aminopeptidase N inhibitors 4Q, M03 combined with 5-FU shown strong anti-tumor activity.(5) 5-FU derivatives in combination with Bestatin:To keep the same administration, we designed 5-FU derivatives (Capecitabine and Tegafur) in combination with Bestatin for they were all administered orally in clinical. MTT experiments shown that the combination could inhibit a variety of tumor cells’proliferation in vitro, and mostly 1:1 ratio was the best one. This proportion was used in animal experiments. The high-dose combination group inhibition rates were higher than single administration group. All these results indicated that 5-FU derivatives (Capecitabine and Tegafur) combined with Bestatin had good anti-tumor activity.Overall, aminopeptidase N inhibitors combined with 5-FU or its derivatives seemed to have a relatively better antitumor efficacy with low toxicity and well tolerated. It may have an impromising future in clinical treatment of cancer.