Experimental Study Of The Combined Effect Of Amlodipine With 5-Fu On Mouse Hepatoma H22 In Vitro And Vivo

Recently,the study of the combination Ca2+ channel block(CCB) with chemotherapeutics in the anticancer effect is increased increasingly. Many investigates indicated that the combination of anticancer drugs with CCB can encrease the cytotoxic effct to multisystem tumor cells,reverse the multidrug resistance of the tumor cells. Our previous study indicated that amlodipine possess more potency than the other CCBs in anti-proliferation effect. In this study we have observed the effect of the combination of amlodipine with 5-Fu on mouse hepatoma cell H₂₂ in vitro and vivo,to expect that amlodipine may be used as a sensitizer of chemotherapy in the treatment of hepatic cancer.Objective: To study the combinated inhibitory effect of amlodipine with 5-Fu on mouse hepatoma cell H₂₂ in vitro and vivo.Methods: The mouse hepatoma cell-line H₂₂ was treated with amlodipine(3.125～50μmol/L)and 5-fluorouracil(12.5～200μg/mL)alone or in combination for 24 ,48,72 hours. MTT assay was used to investigate its inhibitory effect on the proliferation of cancer cells.The method of Chou and Talalay was utilized to assess the type of drug interaction ,i.e. 50% inhibited concentration (IC50) and combined index (CI). Flow cytometry (FCM ) was employed to observe the apoptosis rate (AR) after H₂₂ cells exposed to amlodipine and 5-Fu for 18 hours and cell cycle after 48 hours. Immunocytochemical staining was used to observe the expression of Bax and Bcl-2.The model of transplanted mouse hepatoma cell H₂₂ was established in the right armpit and peritoneal of mice, then divided into four groups randomly: control group, amlodipine group, 5-Fu group, and amlodipine+5-Fu group,treated with normal saline (0.1ml·d-1 ig.), amlodipine(10mg·10g^-1·d^-1 ig.), 5-Fu(10mg·10g^-1·d^-1 ip.at the 3 d,6th and 9th ), amlodipine+5-Fu(amlodipine 10mg·10g^-1·d^-1 ig. , 5-Fu 10mg·10g^-1·d^-1 ip.at the 3d,6th and 9th ) respectively for 10 days. We observed the tumor weight and survival time of mice with H₂₂ tumor, calculated the tumor inhibitory rate and life extension rate.Results:(1) Alone or in combination Amlodipine and 5-Fu could inhibit the growth of H₂₂ cell in a time and dose dependent fashion significantly.The combination of them had synergistic effect on the H₂₂ cell line when the inhibition rate under 95%.(2) The result of FCM confirmed that alone or in combination Amlodipine and 5-Fu could induce the apoptosis of H₂₂ cell .Compraring with the control group, the apoptosis rate of experimental group had a great increase, comparering with alone group, the apoptosis rate of combinated group had a great increase.(3) The result of FCM confirmed that H₂₂ cell was blocked at G0/G1 cell cycle by Amlodipine and S cell cycle by 5-Fu.The combinated group could block cell cycle at G0/G1 and S by Amlodipine with 5-Fu.(4) Amlodipine and 5-Fu could downregulate the expression levels of Bcl-2, upregulate the expression levels of Bax.(5)The inhibitory rates of three groups (Amlodipine group, 5-Fu group, Amlodipine+5-Fu group) for H₂₂ mice solid hepatoma were 35.98﹪, 67.95﹪and 80.68﹪respectively;The life extension rates of three groups (Amlodipine group, 5-Fu group, Amlodipine+5-Fu group) for H₂₂ mice ascites hepatoma were 19.94﹪,24.08﹪and 43.11﹪respectively.Conclusions:(1) Alone or in combination of amlodipine and 5-Fu could inhibit the growth of H₂₂ cells in vitro effectively .The combination of them showed synergistic or antagonistic effects at different concentration.(2) 5-Fu could block the H₂₂ cells in S phase,while amlodipine could block the H₂₂ cells in G1 phase ; the combination of the two drugs exhibit a synergisitic antiproliferation effect by blocking the cell cycle during different phase.(3) It is differ from 5-Fu that the antiproliferation effect of amlodipine mainly by inducing apoptosis of tumor cells,so,the combination of the two drugs produce a more stronger antiproliferation effect than any of the two drugs,and the toxicity will be decreased simultaneously.(4) Alone or in combination of amlodipine and 5-Fu could inhibit the growth of mice-transplanted tumor H₂₂,the volume of the tumor is decreased ,the life span is prolonged in tumor-bearing mice. Such combination exhibits an excellent therapeutic effect.