Antitumor Activity Of Xanthohumol In Vitro And Its Molecular Mechanisms

INTRODUCTION: Traditionally,Xanthohumol(XN),a natural polyphenol derived from the hop plant,has been used to as additive to add bitterness to beer.However,nowsdays,XN has received much attention for a broad spectrum of biological properties and the role of it can prevent and treat cancers.Several researches have described the antiproliferation activity of XN in different human tumor cell lines including human prostate cancer,fibrosarcoma,leukemia,colon cancer,ovarian cancer and breast carcinoma,but the molecular mechanism remain unclear.In the present study,we have assessed the efficacy of XN on human promyelocytic leukemia cells HL-60 and mouse/rat neuroblastoma/glioma hybrid NG108-15 cells,from apoptosis,autophagy,paraptosis and cell cycle,we tried to explore the anti-malignancy mechanism of XN and its molecular basis,which would provide datas support and establish the theoretical basis for the functional food as well as clinical application.METHODS: Trypan blue exclusion assay was used to measure the proliferation of cells treated with various concentrations of XN;morphological changes of cells treated with different concentration of XN were observed under light microscope;apoptosis was examined by both Annexin V/PI double-staining and immunofluorescence;The expressions of caspase-3,Beclin-1,LC3-?,p62,CHOP,GRP78/BIP,poly-ubquitinated proteins,ERK1/2,p-ERK1/2,p38 and p-p38,JNK and p-JNK,c Jun and p-Jun were examined by Western blot;proteasome activity was tested following the instructions for reagent kits;The source of cytoplasmic vacuoles induced by XN and intracellular location of LC3-? was observed by accurate transmission electron microscopy and laser scanning confocal microscope;Intracellular ROS,mitochondrial membrane potential and cell cycle were estimated with the fluorescent probe DCFH-DA,JC-1and PI by flow cytometry,respectively.RESULTS: Part? The Inhibition of HL-60 Cell Proliferation by Xanthohumol and Its Molecular Mechanisms 1.XN dose-dependently inhibited HL-60 cells proliferation and IC50 was about 11.9 ?M,then microscopic observation revealed that XN treatment induced cytoplasmic vacuoles.2.XN induced caspase-independent apoptosis in a concentration dependent manner.However,the pan-caspase inhibitor z-VAD-fmk can't reverse XN-induced cell death and cytoplasmic vacuoles,meanwhile,XN treatment induced cell cycle arrest at G1 phase and G2/M phase.3.XN treatment caused LC3-II accumulation through blocking of autophagosome maturation,as well as the failed protective effect from 3-MA and Baf A1.4.XN treatment induced paraptosis.Cytoplasmic vacuolization,derived from endoplasmic reticulum(ER),results in irreversible cell injury to HL-60 cells.XN treatment triggered the dilatation of endoplasma reticulum(ER)and induced ER stress by upregulating CHOP and GRP78/Bip.XN-induced cell death and cytoplasmic vesicles were suppressed in the presence of 4-PBA(an ER stress inhibitor),the same results appeared while the exest of de novo protein synthesis inhibitor CHX.p38 MAPK signaling pathway regulated XN-induced paraptosis and p38 inhibitor SB203580 reduced XN-induced cell death and cytoplasmic vesicles.XN-treated cells accumulated poly-ubquitinated proteins and inhibited proteasome activity.Meanwhile,XN treatment enhanced intracellular ROS,decreased mitochondrial membrane potential.Part? The Inhibition of NG108-15 Cell Proliferation by Xanthohumol and Its Molecular Mechanisms 1.XN dose-dependently decreased cell viability of NG108-15 cells,cells exhibited strong inhibition at the 40 ?M Xn.2.XN treatment arrested the cell cycle at G1/S phase but can't induce apoptosis.3.XN treatment triggered the phosphorylation of the estrogen-regulated kinase(ERK1/2)and p38 mitogen activated protein kinase(MAPK),XN treatment inhibits the proliferation of NG108-15 cells and arrests the cell cycle at G1/S phase via activating ERK1/2 signaling pathways.CONCLUSIONS: Part? The Inhibition of HL-60 Cell Proliferation by Xanthohumol and Its Molecular Mechanisms 1.XN dose-dependently decreased cell viability of HL-60 cells,the main mechanism of XN-induced cell death is paraptosis.XN-induced paraptosis depended on ER stress and de novo protein synthesis and p38 MAPK pathway may play a critical role in the XN induced-paraptosis.2.XN induced caspase-independent apoptosis and induced cell cycle arrest at G1 phase and G2/M phase.XN treatment caused the rise of ROS and decreased mitochondrial membrane potential.3.XN inhibit the fusion of autophagosomes and lysosomes in HL-60 cells,thus effectively suppress the maturation of autolysosome,finally,leading to block cell autophagy flux.Part? The Inhibition of NG108-15 Cell Proliferation by Xanthohumol and Its Molecular Mechanisms 1.XN treatment inhibits the proliferation of NG108-15 cells through cell cycle arrest at G1/S via activating ERK1/2 signaling pathway.2.XN treatment activates p38 MAPK pathway but doesn't induce apoptosis in NG108-15 cells.In conclusion,this paper found that XN has strong antitumor activity in vitro,and it showed up cell speciticity for inhibiton of different tumor cell lines.