Inhibitory Effects Of Vitamin K2 On HSD17B4-caused Proliferation Of Human Hepatocellular Carcinoma Cells And Its Mechanism

Objective: Human hepatocellular carcinoma(HCC),which is the third leading cause of cancer deaths worldwide with high incidence and poor prognosis,there is still lack of effective therapeutic effect.Therefore,looking for effective treatment or auxiliary treatment of hepatocellular carcinoma has important significance.Vitamin K(VK)is a fat-soluble Vitamin.In addition to have a blood coagulation function,VK not only plays a key role in bone metabolism,but has the role of also in resistance to a wide variety of tumor,including hepatocellular carcinoma.Although the molecular mechanisms of VK2 inhibitory effcts on HCC proliferation have been well down,there are still problems remaining to be solved.17?-Hydroxysteroid dehydrogenase(HSD17B4)is a kind of important hormone metabolism enzymes.HSD17B4 can catalyze the conversion of E2,which is the most potent and active form of estrogen,to estrone(E1),which is the less active form of estrogen.Our recent study data showed that HSD17B4 highly expressed in human HCC tissues,and over-expressed HSD17B4 in HepG2 cells decreased E2 level,the later leaded to the activation of STAT3 via the promotion of the protein kinase Akt and MEK/ERK.Then,the activated STAT3 upregulated cyclin D1 and PCNA gene expression,and finally promoted proliferation of HCC.Furthermore,a research recently reported that VK2 can combine with HSD17B4 in HepG2 cell.Base on the reasons described above we consider whether VK2 inhibition on proliferation of HCC cells is due to activity reduction of HSD17B4 by its interaction with HSD17B4,leading to increase of E2 levels?In order to verify the above hypothesis,(1)we observed the size of the subcutaneous tumors in nude mice,which were generated from VK2 stimulated HepG2 cells with HSD17B4-overexpressed plasmid;(2)After VK2 treatment,we observed proliferation,hormone level changes,the interaction of VK2 and HSD17B4,as well as activity change of related key signaling molecular in HepG2 cell to explore the inhibitory mechanism of VK2 on proliferative of hepatocellular carcinoma cells caused by HSD17B4-overexpression,and to provide experimental data for use of VK2 for prevention and treatment of HCC.Methods:1 Cell cultureHuman hepatocellular carcinoma cells(HepG2)were routinely cultured.2 cell treatmentThe HepG2 cells transfection and give VK2 stimulation.3 HepG2 subcutaneous tumor in Nude miceAfter transfection and stimulating HepG2 cells injected into subcutaneous of nude mice,Inducing into the tumor.4 The MTS test cell proliferationDetection inhibition effects of VK2 for proliferative effects of HSD17B4-overexpression on HCC cell.5 The detection of mRNA expression of HSD17B4 by real-time quantitative RT-PCRPromega total RNA extraction kit was used to extract total RNA from HepG2 cells.2 ?g of total RNA was taken to perform reverse transcription.The relative expression of HSD17B4 mRNA was carried out by quantitative RT-PCR with 18 s rRNA as internal control.6 The detection of protein expression of target genes by western blotTotal protein extracted from HepG2 cells after VK2 stimulation was used to determine the protein expression of STAT3,Akt,ERK,MEK and its phosphorylation level,and HSD17B4,cyclin D1,PCNA.7 Detection of E2 by radioimmunoassay.8 Detection the interaction of VK2 and HSD17B4 by ELISA.9 Statistical analysisAll statistical analyses were conducted using SPSS 17.0 software.The data are expressed as means SD.Data were evaluated using One-way ANOVA for multiple group comparison.Statistical significance was set at p < 0.05.Results:1 Vitamin K2 can inhibit the growth of HCC with HSD17B4-overexpression in nude mice.1)VK2 can inhibit the growth of HCC with HSD17B4-overexpression HepG2 cells injected into subcutaneous of nude mice.Nude mice subcutaneously vaccinated with HepG2 cells of transfected empty plasmid(the control group,Empty)and HSD17B4-overexpression plasmid(HSD17B4),and treated HepG2 cells with VK2,to observe the situation of the tumor.Compared with control group,HSD17B4 tumors are bigger;After the two groups were given VK2 treated(Empty + VK2 / HSD17B4 + VK2),the tumors are more smaller than VK2 untreated.The tumors growth inhibition rate of VK2 treated for the control group and HSD17B4 group are the same,about 65%.The results showed that Vitamin K2 can inhibit the growth of HCC with HSD17B4-overexpression in nude mice.2)VK2 can inhibit proliferation of HSD17B4-overexpression on hepatocellular carcinoma cells.Compared with control group,the group of HSD17B4-overexpression raised HepG2 cells proliferation,when giving VK2 processed respectively,the inhibition of HepG2 cells in time and the concentration dependence reduces;After down-regulate HSD17B4 with siRNA,cell proliferation significantly lower,at this point,the processing of VK2 had inhibitive effects on cell proliferation.The results showed that Vitamin K2 can inhibit proliferation of HSD17B4-overexpression on hepatocellular carcinoma cells.2 Vitamin K2 can interact with HSD17B4 and reduce HSD17B4 activity of HCC cells.1)VK2 can inhibit the activity of HSD17B4.HSD17B4 catalytic estradiol(E2)into estrone,so this experiment used the levels of E2 to reflect the activity of HSD17B4 with the same conditions.With different concentrations of VK2(0,20,50,100 ?mol/L)to deal with the HepG2 cells of control group(Empty)and the HSD17B4-overexpression group after 48 h,tested the level of E2 in medium.The results showed that following the increase of VK2 dose,medium E2 level also will increase.The results showed that VK2 inhibits the activity of HSD17B4 in HepG2 cells.2)VK2 plays no role in HSD17B4 expression of mRNA and protein.Dealing HepG2 cell with control group and the HSD17B4-overexpression group after VK2 treated,the expression of HSD17B4 mRNA and protein level were no significant changes in HepG2 cells.Similarly,down-regulate HSD17B4(SiHSD17B4)and the control group(SiNC)were treated by VK2 in Hep G2 cells,also do not affect.The results showed that VK2 do not inhibit the activity of HSD17B4 through its expression of mRNA and protein.3)VK2 can interact with HSD17B4 and then change the activity of HSD17B4.Treated the control group(Empty)and HSD17B4-overexpression group with different concentrations of VK2(0,10,20,50,100 ?mol/L)to deal with 48 h in HepG2 cells,and precipitated HSD17B4 with HSD17B4 antibody,testing content of VK2 in precipitation,the results showed that VK2 combined with HSD17B4.ELISA results showed that with the increase of VK2 dose,VK2 content of precipitation in the control group does not change with the processing of VK2 concentration,but in the HSD17B4-overexpression group,VK2 content increases in the sediment with the concentration of VK2 processing.The results showed that VK2 can interact with HSD17B4.3 Vitamin K2 can inhibit the expression of proliferation related protein in HSD17B4-overexpression on HCC cells.The results of Western blot shows that treated the control group(Empty)and HSD17B4-overexpression group with VK2(50 ?mol/L)after 48 h,cell proliferation related genes cyclin D1 and PCNA protein levels were reduced.However,treated down-regulate HSD17B4(SiHSD17B4)group and the control group(SiNC)with VK2,cyclin D1 and PCNA protein levels almost have no effect.The results showed that VK2 can inhibit the expression of cell proliferation related genes of protein with HSD17B4-overexpression.4 Vitamin K2 can inhibit the activation of signaling pathway of Akt and MEK/ERK and STAT3 activation of HSD17B4-overexpression on HCC cells.1)VK2 can inhibit STAT3 activation of HSD17B4-overexpression.According to the results of Western blot,HepG2 cells was treated up-regulate and down-regulate HSD17B4 with VK2(50 ?mol/L)after 48 h,VK2 significantly reduced the phosphorylation of STAT3 level of HSD17B4-overexpression group on HepG2 cells;but VK2 had little effects on the phosphorylation of STAT3 level of the down-regulate HSD17B4 group on HepG2 cells.The results showed that VK2 can inhibit STAT3 activation of HSD17B4-overexpression.2)VK2 can inhibit the activity of Akt and MEK/ERK signaling pathway of HSD17B4-overexpression.Because the signaling pathways of Akt,ERK and MEK kinase are associated with proliferation,and promoting of STAT3 activity,therefore,we detecte the activation of these kinases levels in HepG2 cells,which were treated up-regulate and down-regulate HSD17B4 with VK2.So that prove whether or not they are related to inhibition of the above STAT3 activation.According to the results of Western blot,VK2 significantly reduced the phosphorylation of Akt,ERK,MEK level of HSD17B4-overexpression group on HepG2 cells,but VK2 has little effects on the phosphorylation of Akt,ERK,MEK level of the down-regulate HSD17B4 group on HepG2 cells.The results showed that VK2 can inhibit the activation of Akt,ERK and MEK of HSD17B4-overexpression,associating with the inhibition of STAT3 activation.Conclusions:Vitamin K2 provents proliferation of HCC cells via inhibiting the activity of HSD17B4.Its mechanism is as follows:1)Vitamin K2 reduces the activity of HSD17B4 by combination with HSD17B4,and then increases levels of E2 in HepG2 cells.2)Increased E2 inhibits the activation of Akt and MEK/ERK signaling pathway leading to decrease in STAT3 activation.3)The lower levels of activated STAT3 decrease the expression of cyclin D1,PCNA and other growth related genes,eventually leading to proliferation suppression of HCC cells.