| Background and objective:Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the worldwide.The global incidence was more than 62.6 million per year, which make it the fifth frequent cancer, and the mortality rate was nearly 60 million per year ranking the third place in the tumor related deaths. In China, the incidence of liver cancer accounts for about 55% of the world, ranked the second place in the tumor-related deaths, which seriously threaten the health and lives of our people. Until now, the comprehensive treatment of HCC involves multiple disciplines of internal medicine, surgery, interventional and radiotherapy in the treatment of liver cancer, including surgical treatment (surgical resection or liver transplantation) and non-surgical treatment (ablation therapy, hepatic artery embolization, radiation therapy, biological therapy and molecular targeting treatment, etc.).But, there are still no standard systemic chemotherapy drugs or regimens. HCC patients with hepatitis B or hepatitis C virus infection and cirrhosis often accompanied severe liver dysfunction. Because of the ubiquity of multi-drug resistance and the intolerance of serious side effects, traditional systemic chemotherapy is also failed. Thus, finding the drugs which can increase the sensitivity to fluorouracil has an important role in HCC research and provides a possibility in HCC therapy.Allicin, organic sulfur compounds extracted from liliaceae onion garlic bulb, whose main chemical ingredient is Diallyl trisulfide (DATS), is the main active ingredient in garlic.Frequently allicin is used not only in anti-microbial treatment but also in cardiovascular diseases therapy. The growing number of studies found that allicin can inhibit cell proliferation and promote apoptosis to play an anti-tumor effect by inhibiting DNA synthesis and regulating the cell cycle. Recently report shows that allicin has chemo sensitization effect on cancer chemotherapy, but its specific mechanism is not yet clear.Nek2 is the member of cell cycle regulatory protein kinase (NIMA-related kinase,Nek) family which concludes 11 members from Nekl to Nek11 and mainly associates with the regulation of cell cycle. Nek2 is also involved in the regulation of cell cycle with its expression depending on cell cycle. The expression of nek2 is relatively low in phase G1 while it increases rapidly to 3-4 times level in G1/S phase and remains unchanged until phase G2, but it remarkably decrease in M phase. Current study found that the primary physiological functions of nek2 are including centrosome assembly, the regulation of centrosome separation, promoting the condensation of chromatins and so on. In addition, nek2 also plays an important role in the accurate separation of centrosome. In conclusion, nek2 acts as an important role in the maintenance of the stability of genome.Aneuploidy and chromosomal instability usually caused by abnormal cell division and abnormal centrosome separation which are the two most common characteristics of tumor. As an important gene in maintaining genome stability, nek2 is up regulated in many kinds of tumors included ovarian cancer, breast cancer, prostate cancer, lymphoma, renal papilloma, HCC and so on. In the studies of HCC we found that 7 genes including nek2 can reflect the progression from normal liver-cirrhosis-HCC in transcription level. Recent study showed that overexpression of nek2 leads to the instability of chromatin, and nek2 correlates with tumor progression, poor prognosis and chemotherapy drug resistance.In summary, nek2 may be an important target for tumor therapy.This study found that allicin has a synergistic effect with fluorouracil in inhibiting HCC cell proliferation, which increases the sensitivity of hepatocellular carcinoma cells to fluorouracil can by MTT, and preliminary study was performed by Annexin V-FITC/PI double staining flow cytometry, Western blot, JC-1 fluorescent probe staining flow cytometry, DCFH-DA fluorescence labeled flow cytometry to found that allicin promoted the ROS generation and induced mitochondrial apoptosis; Nek2 expression was dramatically increased in HCC cell lines by transfected with pEGFP-N1-Nek2 plasmid, which significantly blocked chemotherapy sensitivity. All suggested that Nek2 played an important role in chemo sensitizing process. This study provided a new idea for HCC therapy research and a theoretical basis for the choice of the chemotherapy drugs.Methods1.Allicin increased the sensitivity to fluorouracil in hepatocellular carcinoma cells.The half inhibitory concentration (IC50) of allicin and fluorouracil in HCC cell line SK-Hep1, BEL-7402 were determined by MTT assay. According to the IC50 values, the appropriate concentration was given (allicin:0,3,6, 10μg/ml and fluorouracil:0,100,300μg/ml); the capability of proliferative was detected by using MTT assay. The interaction of the two drugs was verified through the CI formula: CI=(D)1/(DX)1+(D)2/(DX)2.2.Allicin collaborated with fluorouracil induced mitochondrial apoptosis in hepatocellular carcinoma cells.Experimental groups are as follows:control group, the allicin group, the fluorouracil group, allicin combined with fluorouracil group. By Annexin V-FITC/PI double staining, the early apoptosis level was detected between different groups after drug treatments. The apoptosis-related proteins:full-length and cleavage PARP, caspase-3,Bcl-2 were detected thought Western blot.JC-1 fluorescence probe staining was used to measure the mitochondrial membrane potential of single drug or two-drug treatment cells by flow cytometry; DCFH-DA fluorescent labeling by flow cytometry to detect the amount of reactive oxygen species (ROS) generation3.Allicin enhanced the sensitivity of fluorouracil by the regulation the Nek2 of expression.Nek2 protein level of the HCC cell line expression was detected by Western blot after a single drug or two-drug treatment. The pEGFP-N1-Nek2 plasmid was constructed and transfected into BEL-7402 which made Nek2 expression unregulated. The expressions of apoptosis-related proteins were detected by Western blot.Results1. Allicin increased the sensitivity to fluorouracil in hepatocellular carcinoma cells.Through MTT assay the IC50 for allicin in liver cancer cell lines SK-Hepl, BEL7402 was 10.389μg/ml 10.004μg/ml respectively. For fluorouracil IC50 were 438.945μg/ml,410.336μg/ml respectively. Selected the appropriate concentration of allicin as 0,3,6,10μg/ml,and fluorouracil 0,100,300μg/ml. Using formula CI= (D) 1/ (DX) 1+(D) 2/(DX) 2, CI values were calculated, the value of both cell lines less than 1, that means allicin cooperative fluorouracil to inhibit cell proliferation. Select the concentration of allicin for 3μg/ml, the fluorouracil concentration for 100μg/ml to treat HCC cells; the difference in groups was compared by one-way ANOVA statistical methods, LSD Act to pairwise comparisons. The results found that allicin combined with fluorouracil compared with single-agent fluorouracil group can significantly inhibited cell proliferation (SK-Hepl:P= 0.005; BEL-7402:P<0.05).2. Allicin collaborated with fluorouracil induced mitochondrial apoptosis in hepatocellular carcinoma cells.After drug treatment, the cells in each group was stained by Annexin V-FITC/PI, and the level of apoptosis were detected by flow cytometry, results suggest that, the early apoptosis rate of two-drug group (SK-Hepl:8.067%±1.097%; BEL-7402: 13.667%±5.5003%) compared to single-agent fluorouracil group (SK-Hepl:6.700% ±.6928%; BEL-7402:8.100±3.8743), single-agent allicin group (SK-Hepl: 1.133%±0.4041%; BEL-7402:2.867%±1.8475%), control group (SK-Hepl: 0.533%±0.057%; BEL-7402:1.600%±0.2646%) was significantly higher, and the difference was statistically significant (P<0.05). The detection of mitochondrial membrane potential showed the early apoptosis cell percentage in control group were 3.933%±1.7214%(SK-Hepl) and 9.300%±3.3719%(BEL-7402); in allicin group were 6.400%±4.2790%(SK-Hepl) and 18.600%±5.806%(BEL-7402); in fluorouracil group were 16.767%±8.3680%(SK-Hep1) and 28.233%±4.8211% (BEL-7402);in two-agent group were 6.467%±1.9858%(SK-Hep1) and 46.800%±6.2923%(BEL-7402), which suggested the two-drug treatment induced apoptosis through the mitochondrial pathway. Then, by DCFH-DA fluorescent labeling the cellular ROS level was measured by mean fluorescent, SK-Hepl cell line control group:573.67±39.627; allicin group:746±22.113; fluorouracil:794±24.062; two-agent group:3914.33±36.295. BEL-7402 cell line control group:400.33±30.827; allicin group:736.33±13.051; fluorouracil group:627.67±42.712; two-agent group: 2042±61.262. The result showed that the two-drug group was significantly higher (P<0.001).3. Allicin enhanced the sensitivity of fluorouracil by the regulation the Nek2 of expression.Western blot analyzed the expression of Nek2 between different groups. The results showed that Nek2 expression of the two-drug treatment group compared to single-agent treatment group and the control group was decreased. Compared with un-transfected cells, protein levels of cleavage PARP, caspase-3 were significantly attenuate after pEGFP-N1-Nek2 plasmid was transfected.Conclusions:1. Allicin collaborated with fluorouracil can synergistically inhibit HCC cells proliferation.2. Allicin combines with fluorouracil increases the generation of intracellular ROS and induce the mitochondrial apoptosis pathway.3. Allicin increases the sensitivity of fluorouracil by down regulating Nek2 expression. |
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