| The incidence of esophageal cancer ranked eighth in the world,and it was particularly high in some Asian countries.China is one of the regions with a high incidence,where average annual 150,000 deaths occurred.As the early symptoms are not typical or obvious,many patients are diagnosed as advanced esophageal cancer in the first time,and half of them have distant metastasis.It is very difficult to completely resect,and drug-resistance and radio-resistance are also usual.Although the 5-year overall survival rate of esophageal cancer is about 20%,but when distant metastasis occurs,it can reduce to 4%.Therefore,it is important to develop new,effective and non-toxic chemotherapeutic drugs.Flavonoids which are rich in citrus,lemon and other citrus plants of Rutaceae,have attracted much attention.They have much medicinal value with no obvious toxicity.Flavonoids can scavenge free radicals,exert antioxidation,antimicrobial and anti-cancer activity,reduce vascular fragility,improve vascular permeability,lower blood lipid and cholesterol,and enhance immune ability.They play important roles in the prevention and treatment of cardiovascular disease,liver diseases and many kinds of cancer.Hesperetin,one of the natural flavonoids,has been shown to be involved in regulating proliferation,apoptosis,and invasion of many cancer cells,including liver cancer,breast cancer,cervical cancer,prostate cancer,colon cancer and so on.There are no obvious side effects on normal cells.Much evidence indicated hesperetin induced tumor cells apoptosis by activating mitochondrial-mediated endogenous apoptotic pathway.However,the effects of hesperetin on the biological behavior of esophageal cancer cells are still unclear.5-fluorouracil 5-FU(5-FU),the most commonly used uracil antimetabolic drug,is a basic drug for the treatment of esophageal cancer.It interferes with the synthesis of DNA and RNA,and induces cancer cell apoptosis.Side effects and drug resistance limit its clinical use for esophageal cancer.In order to improve the efficiency of 5-FU and overcome the shortcomings of 5-FU itself,people often use 5-FU-based combination therapy,change the form of 5-FU or employ auxiliary drugs.Therefore,we hypothesized that:(1)Hesperetin can inhibit the proliferation and invasion of esophageal cancer cells and induce the cell apoptosis.(2)Hesperetin can induce cell apoptosis by activating mitochondrial-mediated endogenous apoptotic pathway.(3)Because the apoptosis mechanism induced by hesperetin and 5-FU is different,hesperetin may have synergistic anti-esophageal cancer effect with 5-FU.Materials and methodsl.The effect of hesperetin on the proliferation of esophageal cacer Eca 109 cells was detected by CCK-8 assay and EdU staining.The inhibition rate and IC50 were calculated by GraphPad Prism software.Cell apoptosis was detected by Hoechst 33258 staining and flow cytometry.The cell cycle was detected by PI staining and flow cytometry.The influence of hesperetin on the invasion of Eca 109 cells was detected by transwell chamber precoated with matrix gel.The cell concentration was adjusted to 5×107/mL,and 100 ?L cell suspension was inplanted subcutaneously on the right back of each nude mouse.One week after successful construction of subcutaneous tumor,each group of nude mice were given different concentrations of hesperetin.The general condition of nude mice was observed every day.During the treatment,the size of transplanted tumor was measured by vernier caliper every 3 days.After 21 days of administration,the nude mice were killed by vertebrae dissection.Subcutaneous tumor was isolated and tumor volume and weight were measured.TUNEL staining was used to detect apoptosis rate of tumor cells and HE staining was used to detect distant metastasis.2.The Eca 109 cells were loaded with DCFH-DA,JC-1 probes to detect the changes of intracellular ROS and MMP.After NAC and BSO were used to interfere with the synthesis of GSH in the Eca 109 cells,the effect of hesperetin on cell proliferation,ROS and MMP were detected again.We separated the mitochondria and detected the expression levels of cytochrome C(Cyt C)and apoptosis inducing factor(AIF)by western blot in mitochondria and cytoplasm respectively.Finally,the expression levels of caspase-3,caspase-9,Apaf-1,cysclin D1,BAX,Bcl-2,MMP2 and MMP9 were further detected by western blot.3.The cell viability was detected by CCK-8 assay after single 5-FU or hesperetin or combined treatment.Calcusyn 2.0 software was used to analyze whether combined use of hesperetin and 5-FU exerted synergistic effect on the cell proliferation of Eca 109 cells.Hoechst 33258 staining and flow cytometry were used to detect the effect of apoptosis.The cell cycle was detected by PI staining and flow cytometry.The expression levels of apoptosis-related protein,including caspase-3,caspase-9,BAX and Bcl-2,were detected by western blot.After successful construction of subcutaneous xenograft,the nude mice were randomly divided into 4 groups.The mice were intraperitoneally injected with single drug or combined drug,in order to determine whether hesperetin and 5-FU can synergistically inhibit the growth of tumor cells in vivo.Results1.In the same treatment time,the inhibitory effect enhanced with the increase of hesperetin concentration,and under the same treating concentratioin,the inhibitory effect enhanced with the increase of treatment time.The IC50 was 200 ?M at 72 h.In the follow-up experiment,we treated Eca 109 cells with hesperetin in low,medium,high concontratioin(100,200,300 ?M).The results showed that 100 ?M heperetin inhibited the infiltration of EdU,but there was no significant difference in comparison with control(P>0.05).The inhibition rate increased significantly(P<0.05).when hesperetin dose increased to 200 ?M(P<0.05).Flow cytometry showed that the apoptosis rate increased from 0.9 ±0.2%to 26.4 ±1.2%after exposure to 200 ?M hesperetin for 24 h.When compared to the normal control group,the cell cycle distribution results showed that the percentage of G0/G1 phase cells increased significantly in the group treated with hesperetin((P<0.05).In the tranwell invasion experiment,the number of penetrated cells(100 ?M:30 ±2.5/HP;200 ?M:18±1.6/HP;300 ?M;9±1.1/HP)in the treated group was significantly lower than that in the normal control group(50 ±3.5/HP)(P<0.05).The results showed that hesperetin could significantly inhibit the invasion of Eca 109 cells.In vivo,24 nude mice were successfully constructed subcutaneously xenograft,and were randomly divided into 4 groups.After treated with different concentration of hesperetin for 21 days,it was found that in comparison with control group,the volume and weight of tumor decreased in three hesperetin-treated groups(P<0.05).Tunel staining also showed that the apoptotic rate was higher in the treated group(P<0.05).The histopathological examination showed that no distant metastasis of tumor cells was found in the four groups.2.Human esophageal carcinoma Eca 109 cells were treated with 100,200,or 300?M hesperetin for 24 h,DCFH-DA was used as a probe to detect the intracellular ROS.Compared with the control group,the fluorescence in the treated cells increased significantly(P<0.05).The fluorescence intensity was proportional to the hesperetin concentration.These results indicated that hesperetin could promote the production of ROS in Eca 109 cells.Compared with the single 200 ?M hesperetin-treated group,pretreatment with antioxidant NAC could reduce the increase level of ROS,while combined treatment with BSO could further promote the production of ROS.Different from ROS increase,the content of GSH in the cells decreased significantly after hesperetin stimulation(P<0.05).The GSH level in the 300 ?M hesperetin-treated group decreased to 1/5 of that in the control group.NAC pretreatment can alleviate the decrease of GSH,while BSO can accelerate the reduction of GSH.After using different concentrations of NAC/BSO interfering with the synthesis of GSH,CCK-8 assay was used to detect the effect of 200 ?M hesperetin on the cell viability.The results showed that NAC pretreatment had a protective effect for Eca 109 cells,which was proportional to the concentration of NAC.However,co-treatment with BSO enhanced the toxicity of hesperetin to Eca 109 cells.The cell viability was inversely proportional to BSO concentration.We further used JC-1 probe to detect the MMP.The results showed that with the increase of hesperetin concentration,the red fluorescence decreased gradually,while the green fluorescence increased.The ratio of red to green fluorescence decreased significantly(P<0.05),which indicated that hesperetin could induce the loss of MMP.NAC pretreatment can alleviate the loss of mitochondrial membrane potential decrease,while BSO plays the opposite role.After hesperetin treatment,the expression levels of Cyt C and AIF in mitochondria decreased,while they increased in cytoplasm.The expression of activated caspase-9 and caspase-3,BAX and sufu upregulated,and the expression of Bcl-2 and survivin downregulated.In addition,the expression levels of MMP2 and MMP9 decreased.3.The growth of human esophageal Eca 109 cancer cells was inhibited by 5-FU from 2.5 ?M to 80 ?M or by hesperetin form 25 ?M to 800 ?M.Compared with the single drug treatment group,CCK-8 assay showed that the combined use of hesperetion and 5-FU(molar ration:10:1)had synergistic inhibitory effect on Eca 109 cells growth.More bright-blue condensed nuclei could be seen after Hoechst 33258 staining(P<0.05).The apoptotic rate of the combined treatment group(38.25±4.52%)was significantly higher than that of the single drug treatment group(200 ?M hesperetin:21.38±3.35%;20 ?M 5-FU:13.91±2.16%)(P<0.05).Flow cytometry also showed that the proportion of G0/G1 phase cells in the combined treatment group(70.64±5.65%)was significantly higher than that in the single drug treatment group(200?M hesperetin:63.1±4.81%;20?M 5-FU:58.87±1.53%).The difference was significant(P<0.05).Western blot showed that in comparision with the single drug group,the expression levels of activated caspase-9 and caspase-3,BAX was up-regulated,while the expression of Bcl-2,cyclin D1 was down-regulated in the combined treatment group.Subcutaneous xenografts were successfully constructed in 24 nude mice in vivo.They were randomly divided into 4 groups and were given vehicle,5-Fu 10 mg/kg,hesperetion 60 mg/kg or 5-FU+hesperetin 10 mg/kg+60 mg/kg,respectively.After 21 days' treatment,there was no significant difference in body weight,general status and serological results among four groups,but xenograft weight(control group:0.915 ±0.144 g;5-FU group:0.553 ±0.021 g;hesperetion group:0.443 ±0.033 g;combined treatment group:0.273±0.045 g)was significantly different(P<0.05).The tumor volume in the combined group was smallest,which was significantly different from that in the single drug group or the control group(P<0.05).Tunel staining also showed that compared with the single drug treatment group,the apoptosis rate in the combined group increased significantly(P<0.05).ConclusionsHesperetin can inhibit the proliferation and invasion of human esophageal cancer Eca 109 cells,and activate the mitochondrial-mediated endogenous apoptosis pathway by inducing the increase of ROS and the depletion of GSH.In addition,hesperetin and 5-FU acted synergistically in inducing Eca 109 cells apoptosis. |
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