The Effect And Its Mechanisms Of Hesperetin On The Proliferation And Apoptosis Of Gastric Cancer Cells

BackgroundGastric cancer?GC?is one of the most common and malignant gastrolintestinal cancer.According to the GLOBOCAN 2015 data,almost one million new cases of GC were estimated to have occurred in 2012?951,000 cases,6.8%of the total?,making it the fifth most common malignancy in the world,after cancers of the lung,breast,colorectum and prostate.And GC is the third leading cause of cancer death in both sexes worldwide?723,000 deaths,8.8%of the total?.More than 70%of cases?677,000 cases?occur in developing countries?456,000 in men,221,000 in women?,and half the world total occurs in Eastern Asia?mainly in China?.In addition,according to the Cancer Statistics in China 2015,there were almost 679000 new cases and 498000 deaths of GC in 2012 in China.The high morbidity and mortality,malignancy,and unsatisfactory treatment effects lead to a serious threat on people's health.Currently,main therapeutic modalities involve surgery,radiation,and chemotherapy.However,about 70%gastric cancer patients have a locally advanced or metastatic disease at the time of initial diagnosis which compromises the effects of surgery and radiation greatly.Chemotherapeutic drugs,including 5-fluorouracil,cisplatin,and adriamycin,have become restricted due to drug resistance and cell toxicity.Therefore,it is of great value to discover the natural anticancer compounds that have high efficacy and low toxicity in the treatment of GC.Hesperetin?3',5,7-trihydroxy-4-methoxyflavanone?,also referrde to as hesperitin,is a member of flavanone,subclass of flavonoids,and occurs in fruit sources including citrus.Like most flavonoids,hesperetin?the aglycone form?exists in nature in its glycoside form,hesperedin?glucose is attached to hesperetin?.Dietary hesperidin is deglycosylated into hesperetin by intestinal bacteria before absorption.As a natural plant monomer,hesperetin has previously been shown to have various activities such as anti-oxidation,anti-inflammatory,anti-bacterial,anti-tumor,inhibit liver fibrosis and immune regulation by regulating PI3K/Akt,NF-?B,Notch as well as Keap/Nrf2 signal pathways.And we found that hesperetin has impacts on the proliferation and apoptosis of GC cancer cells,however the mechanism is still unclear.To confirm the effect of hesperetin on GC cells and investigate the underlying mechanism,in the first part,we treated GC cells?SGC-7901?MGC-803 and HGC-27?with hesperetin of various dose to check the effects of hesperetin on the proliferation and apoptosis of GC cells;in the second part,we used H₂O₂ and NAC to change the intracellular ROS level and confirm the role of ROS played on hesperetin-induced apoptosis and make mechanisms clear;in the third part,we constructed nude mice model of subcutaneous xenografts of gastric cancer by intraperitoneally injecting hesperetin into nude mice to explore the effect of hesperetin on the proliferation and apoptosis of gastric cancer cells in nude mice.Methods1.CCK-8 and colony formation assay were used to examine the effect of hesperetin on the proliferation of SGC-7901?MGC-803 and HGC-27;Hoechst 33258 staining and western-blot were used to check the effects of hesperetin on the apoptosis.2.H₂O₂ and NAC were used to interrupt the hesperetin-induced ROS accumulation,and then the intracellular ROS levels in control group,hesperetin group,hesperetin+H₂O₂ group and hesperetin+NAC group were checked and the proliferation rate of SGC-7901 were detected by CCK-8 and colony formation.3.H₂O₂ and NAC were used to interrupt the hesperetin-induced ROS accumulation,and then the intracellular ROS levels in control group,hesperetin group,hesperetin+H₂O₂ group and hesperetin+NAC group were checked and the apoptosis rate of SGC-7901 were detected by Hoechst 33258,annexin-V PI/FITC and western blot;the mitochondrial membrane potential???m?was examined using JC-1 kit.4.Cyclosporin A?CsA?and Ru360 were used to inhibit mitochondrial permeability transition pore?mPTP?and mitochondrial calcium uniporter?MCU?,respectively.Apaf-1,AIF andCyt C in mitochondrial and cytoplasma in control group,hesperetin group,hesperetin+H₂O₂ group,hesperetin+NAC group,CsA group,Ru360 group,hesperetin+ CsA group and hesperetin+ Ru360 group were detected by western blot.Meanwhile,the Ca²⁺ levels in mitochondrial were checked by Fluo-3AM.5.Nude mice model of subcutaneous xenografts of gastric cancer were established and hesperetin were intraperitoneally injected into nude mice to explore the effect of hesperetin on the proliferation and apoptosis of gastric cancer cells in nude mice by HE staining and TUNEL assay.Results1.The results of CCK-8 assay showed that hesperetin could inhibit the proliferation of SGC-7901,MGC-803 and HGC-27 gastric cancer cells and was in a dose-and time-dependent manner.The IC50 of hesperetin on SGC-7901,MGC-803 and HGC-27 after incubating for 36h were 300?M,420?M and 450?M.In the colony formation assay,with the increase of Hesperetin dose,the numbers of colony were decreased?P<0.05?.Hoechst 33258 staining showed that percentages of apoptotic cells in the control group,low-dose hesperetin group?100?M?,mid-dose hesperetin group?200?M?,and high-dose hesperetin group?400?M?were 1.3 ± 0.46,8.1 ± 1.01,17.6 ±1.73,and 31.4 ± 1.52%,respectively?P<0.05?.The results of western blot showed that,compared with that in control group,the expressions of Bax,cleaved caspase-3 and cleaved caspase-9 in hesperetin group were up-regulated and the expression of Bcl-2,procaspase-3 and procaspase-9 were down-regulated?P<0.05?.Meanwhile,ROS levels in hesperetin group were improved when compared with that in control group?P<0.05?.2.After H₂O₂ and NAC were used to interrupt the hesperetin-induced ROS accumulation,compared with hesperetin-only group,the ROS levels in hesperetin+H₂O₂ group were increased and that in hesperetin+NAC group were decreased?P<0.05?.The results of CCK-8 assay showed that,compared to hesperetin-only group,the inhibition rate in hesperetin+H₂O₂ group were increased and that in hesperetin+NAC group were decreased?P<0.05?.The results of colony formation assay showed that the numbers of colony in hesperetin+H₂O₂ was less than hesperetin-only group,but hesperetin+NAC was higher than hesperetin-only?P<0.05?what indicated that intracellular ROS levels played a critical role in the hesperetin-induced inhibition on the proliferation of SGC-7901 cells.3.The results of Hoechst 33258 staining showed that the apoptic rate in hesperetin-only group,hesperetin+H₂O₂,and hesperetin+NAC group were 18.1 ±1.01%,28.7 ± 1.33%and 10.6 ± 1.19%,respectively?P<0.05?.The result of FACS showed that the apoptic rate in control group,hesperetin-only group,hesperetin+H₂O₂,and hesperetin+NAC group were 18.1 ± 1.01%,28.7 ± 1.33%and 10.6 ± 1.19%?P<0.05?.The results of western blot showed that,compared with hesperetin-only group,the expressions of Bax,cleaved caspase-3 and cleaved caspase-9 in hesperetin+H₂O₂ group were increased and that of Bcl-2,procaspase-3 and procaspase-9 were decreased,on the contrary,the expressions of Bax,cleaved caspase-3 and cleaved caspase-9 in hesperetin+NAC group were decreased and that of Bcl-2,procaspase-3 and procaspase-9 were increased?P<0.05?.The results of Hoechst 33258,FACS and western blot indicated that that intracellular ROS levels were important for hesperetin-induced apoptosis of SGC-7901 cells via mitochondrial apoptotic pathway.4.The results of mitochondrial Ca²⁺ showed that,compared with control group,the mitochondrial Ca²⁺ level in Hesperetin-only were increased,but in Ruthenium Red-only and Ru360-only group were decreased.However,when compared with Hesperetin-only group,the mitochondrial Ca²⁺ level in Hesperetin+H₂O₂ group is increased and that in Hesperetin+NAC group and Hesperetin+Ru360 group decreased?P<0.05?what indicated that Hesperetin could increase the mitochondrial Ca²⁺ level via inducing the intracellular ROS accumulation.The results of western blot showed that,compared with control group,the expressions of Apaf-1,AIF and Cyt C were decreased in mitochondria and increased in cytoplasma in Hesperetin-only group,but increased in mitochondria in CsA-only and Ru360-only group?P<0.05?what mean that Hesperetin could induce the translocation of Apaf-1,AIF and Cyt C from mitochondria to cytoplasma and CsA and Ru360 inhibited it.When compared with that in Hesperetin-only group,the expressions of Apaf-1 AIF and Cyt C were increased in mitochondria in Hesperetin+NAC,Hesperetin+CsA,Hesperetin+Ru360 group,but decreased in Hesperetin+H₂O₂ group?P<0.05?.These western blot data indicated that Hesperetin could induce the translocation of Apaf-1,AIF and Cyt C from mitochondria to cytoplasma via the open of mPTP via intracellular ROS accumulation.5.In nude mice model,we found that Hesperetin could inhibit the growth of tumor and the tumor volume in control,low-dose Hesperetin,moderate-dose Hesperetin and high-dose Hesperetin groups were ₂O₂3 ± 137.6 mm3,1274.7 ± 123.8 mm3,983.4 ±99.6 mm3 and 726.8 ± 81.3 mm3?P<0.05?and the tumor weight of these groups were 1.89 ± 0.23g,1.13 ± 0.19g,0.78± 0.13g and 0.46 ± O.11g?P<0.05?.The result of TUNEL assay showed that the apoptic rate in these groups were 11.56 ± 2.12%,23.08±3.41%,31.47 ±3.15%and 49.56%± 5.73%?P<0.05?.ConclusionHesperetin could induce the apoptosis of gastric cancer cells by the translocation of Apaf-1,AIF and Cyt C from mitochondria to cytoplasma via the open of mPTP induced by intracellular ROS accumulation.