The Synergistic Effects Of Allicin On 5-Fluorouracilagainst Hepatocellular Carcinoma And Its Mechanism

Objective: Hepatocellular carcinoma(HCC) is one of the most common malignant solid tumors of digestive system. The morbidity of HCC in China is 350,000 every year which is the highest in the world, making it a huge threat to the national physical health and life quality. HCC has a quite insidious onset. The symptoms of HCC in the early stage, usually atypical abdominal pain, are very easy to be ignored and misdiagnosed. Some the patients are even diagnosed with advanced HCC which is barely unresectable at the first treatment. Patients with advanced HCC are forced to choose chemotherapy, interventional treatment, radiotherapy, biotherapy, and Chinese medicinal therapy other than surgical operation. For this part of patients, the therapeutic effects of chemotherapy might determine their final survival rates. 5-Fluorouracil(5-FU)is one of the most widely used and fundamental cell cycle specific chemotherapeutic drugs for HCC. Calcium folinate(CF) is the most classical chemotherapeutic sensitizers of 5-FU, and they two are often used in combination. However, this chemotherapy was found limitations like questionable safety, poor tolerance, bad respond, and drug resistance. Allicin is an organic sulphureous compound extracting from garlic. Recently, allicin has been reported having multiple anti-tumour effects like blocking cell cycle and sensibilizing chemotherapy drugs. Allicin could improve the cytotoxic effects of chemotherapy drugs, reduce the drug dosage, alleviate the side effects, and inhibit drug resistance. Allicin helps achieve higher effects at relatively low toxicity. The aim of this study is to investigate the possibility that allicin replace CF as a sensitizers and its underlying mechanisms. Methods: Human HCC SMMC-7721 cells at the logarithmic phase were randomly divided into the negative control group, 5-FU(100mg/L)group, low concentration allicin(25mg/L) + 5-FU(100mg/L)group, middle concentration allicin(50 mg/L) + 5-FU( 100mg/L) group, high concentration allicin(100 mg/L) + 5-FU(100mg/L)group, and low concentration CF(5mg/L)+ 5-FU(100mg/L)group, middle concentration CF(10mg/L)+ 5-FU(100mg/L)group, and high concentration CF(20mg/L)+ 5-FU(100mg/L)group. CCK-8 was used to invest the cell proliferation ability of each group, and the inhibitory rates were calculated. According to the results of CCK-8, we chose the best concentration to continue the study. Flow cytometry(FCM) was used to detect the cell cycle in each group, and to observe the cell cycle arrest effect. Hoechst 33258 staining was conducted to testify the cell apoptosis, and the apoptosis rates were concluded by cell counting. The expression of proteins which were closely related to the inducement of multidrug resistance like P-glycoprotein(Pgp) and multidrug resistance-associated protein-1(MRP-1) were measured by western blot. All data involved were analyzed by the statistical software of SPSS17.0.T test method was used to compare the variables of the two groups. The difference was statistically significant when P<0.05. Results: The statistics of the high concentration CF+5-FU group were not evaluated because they caused a widespread cell death and could not continue the following experiment. The data of CCK-8 indicated that all groups had inhibitory effects on the proliferation ability of SMMC-7721 cells. In different concentration groups of allicin combined with 5-FU, the higher concentration was, the better synergistic effect behaved(P<0.05). In low and middle concentration groups of CF combined with 5-FU, the middle concentration had a more obvious effect(P<0.05). Compared between high concentration allicin + 5-FUgroupand middle concentration CF+ 5-FUgroup, the inhibiting effect of high concentration allicin + 5-FUgroup was stronger(P<0.05). The treatment effects of combined therapies were better than 5-FU alone(P<0.05). According to the results of CCK-8, we chose high concentration allicin+5-FU group and middle concentration CF+ 5-FU group to continue the subsequent experiments. FCM discovered that 5-FU group, high concentration allicin+5-FU group, and middle concentration CF+5-FU group can all arrest the cell cycle at S phase. The arrest effects of high concentration allicin+5-FU group were better than the rests(P<0.05). High concentration allicin+5-FUgroup can also arrest G2/M phase cells(P<0.05). Hoechst 33258 staining showed the apoptosis rates of high concentration allicin+5-FU group were higher than 5-FU group and middle concentration CF+5-FU group(P<0.05). Western Blot revealed that compared with 5-FU group and middle concentration CF+5-FU group, high concentration allicin+5-FUgroup can down-regulated the expression of Pgp and MRP-1 more efficiently(P<0.05). Conclusion: The synergistic effects of allicin in cell proliferation inhibition, apoptosis, and cell cycle arrest were stronger than CF. And the synergistic effects were time and concentration dependent. The underlying mechanism may be related to that allicin could block the cell cycle and reverse multidrug resistance in SMMC-7721. In a conclusion, allicin in combination with 5-FU is a potential candidate to replace CF in combination with 5-FU in the future, and to treat HCC in a more efficient and less toxic way.