Mechanism Of Artemisinin And Its Derivatives Against Metastasis Of Non-Small Cell Lung Cancer Mediated By HuR

Background Lung cancer is a common type of malignancy.Non-small cell lung cancer(NSCLC)accounts for 80% of the total number of lung cancer.Most of the clinically diagnosed patients have had local spread or distant metastasis.Only a few early stagepatients can undergo surgey or radiotherapy,the recurrence rate and metastasis rate is still high.Therefore,it's the key point for developing new treat concepts for better survival of NSCLC.Artemisinin(ARS),an effective monomer of anti-malaria isolated from compound inflorescence Artemisia annua,was discovered in 1971 by Chinese scientist Tu Yo-yo.Its derivatives include Artemether(ARM),Arteether,Dihydroartemisinin(DHA)and Artesunate(ART),etc.They all contain peroxy bridge structure,belonging to sesquiterpene lactone compounds.ARS drugs are the anti-malarial drugs following thiazolidine,chloroquine and primaquine,which havelow toxicity and strong anti-malarial effect,and they arethefirst choice for the treatment of cerebral malaria and chloroquine-resistant malaria worldly.The anti-malarial mechanism of ARS is relatively clear,which involves the inhibition of heme internalisation,bivalent iron-dependent free radicals and reactive oxygen species(ROS)productionas well as increasing intracellular calcium levels.However,the mechanism of anti-tumor effect of ARS is not clear at present.Most scholars considered that its mechanism of antimalarial action is similar,which is a large amount of reactive oxygen species is the anti-tumor effect molecule of ARSs.ARS drugs are known as causing toxic effects on tumor cells in thepresence of ferrous iron,and the tumor cells contain a high concentration of ferrous iron.Ferrous iron catalyzes the cross-bridge breaking of ARS-based compounds to generate a large amount of free radicals and ROS,leading to the destruction of tumor cell membrane and the leakage of intracellular substances,eventually resulting in toxicity to tumor cells.However,the antitumor effect of antitumor drugs is not limited to its cytotoxic effect.Studies have found that lower concentrations of ARS drugs can induce cell apoptosis.Study group members found that ARS had a strong biological activity to lung cancer,which can directly affect the lymphatic endothelial cells,inhibit tumor-related lymphangiogenesis,lymph node metastasis and distant metastasis,and significantly inhibited tumor invasion and metastasis,while the specific molecular mechanism of anti-tumor metastasis of ARS has to be futher studied.Therefore,the mechanism of ROS dependence couldn't clealy explain the anti-tumor pharmacological effects of ARS drugs,especially anti-angiogenesis,lymphangiogenesis and tumor metastasis and other non-ROS-dependent mechanisms may be involved.HuR Human antigen R(HuR)is a broad-spectrum mRNA-binding protein belonging to the family of embryonic lethal abnormal vision(ELAV),and widely expressed in a variety of cells and tissues and recognized by RNA Motif(RRM)binding to the AU base-rich sequence(ARE)of the 3'UTR of various mRNAs to increase the stability of the mRNA,which allow the transport of mRNA from the nucleus to the cytoplasm to protecting mRNA from nuclease degradation.In addition to a variety of molecular mRNAs,HuR also has the ability to bind and regulate microRNAs(miRNAs)as well as long-chain non-coding RNAs(lncRNAs).HuR is involved in chronic inflammation and pathology in the cardiovascular,nervous and muscular systems.Under normal circumstances,HuR is mainly expressed in the nucleus,but under different stress conditions,such as drugs,hormones,hypoxia,cytokine stimulation,which can shuttle between the nucleus and the cytoplasm,resulting as an increase in the amountof HuR protein in the cytoplasm.The activation of a variety of signaling pathways could change HuR subcellular localization or directly change the expression level of HuR.HuR then through the RRMs and target mRNA ARE and other trans-acting factors,from the nucleus to the cytoplasm and change the RNA space structure,finally inhibits deadenylationand mRNA recruitment to exosomes or block endonuclease recognizing ARE activity,which in turn changes the stability of the mRNA and affects the expression of the relatives proteins.Matrix metalloproteinase(MMP)-9(type IV collagenase;gelatinase B),one of the most important enzymes that break down the extracellular matrix,plays a key role in tumor cell invasion and metastasis.Chemokine receptors are G protein-coupled receptors with a transmembrane structure that bind to their specific ligands,which involved in the proliferation and metastasis of malignant tumors.HuR,MMP-9 and CXCR4 are known to be significantly higher expressed in NSCLC than normal lung tissues.Our previous results showed that cytoplasmic HuR positive involvement with tumor progression,lymph node metastasis.We propose that HuR participate in the post-transcriptional regulation of MMP-9 and CXCR4 and participate in angiogenesis and lymphangiogenesis,tumor growth and metastasis.Based on the current research status both home and abroad,combined with the previous research on HuR protein involved in the growth and metastasis of lung cancer and the anti-tumor of ARS,our study hypothesized that the mechanism of ARS and its derivatives anti-NSCLC metastasis involves non-ROS pathway;ARS modifies HuR expression by activating related kinase signaling,inhibiting the nuclear-cytoplasmic transport of HuR or directly affects protein expression,which in turn affects the expression of downstream tumor-associated molecules and ultimately inhibits tumor growth and metastasis.PurposesBased on the previous studies and relevant researches home and abroad,the molecular mechanism of artemisinin-based drug against NSCLC metastasis is further to be explored,which provides evidence for the anti-tumor effect of artemisinin-based drugs.Methods The work is divided into three parts to study.The first part of artemisinin and its derivatives on NSCLC proliferation and invasion.First of all,CCK-8 experiments were conducted.ARS drugs with different concentrations of drugs intervened A549,95 D and H1975 cells and IC50 was calculated to verify the effect of ARS on the proliferation of tumor cells.Transwell chamber experiments were carried out.The cells were treated with different concentrations of ART to observe the changes of cell invasiveness.Finally,a reasonable drug concentration was designed.The effects of ART on mRNA and protein expression of anti-proliferation,invasion and angiogenesis were observed by RT-PCR and Western blot.The second part of HuR involved in NSCLC proliferation and invasion.First,24 hours after transient transfection of A549 cells with siHuR,CCK-8 assay was used to observe cell viability.Transiently transfected A549 cells with siHuR for 24 hours,Transwell chamber experiments were performed to observe the effect of reduced HuR on cell invasiveness.After transient transfection of A549 cells with siHuR for 24 hours,the effects of ART on mRNA and protein expression of anti-proliferation,invasion and angiogenesis in NSCLC cells were observed by RT-PCR and Western blot.The third part of artemisinin drugs through HuR to study NSCLC biological effects of molecular mechanisms.A549 and 95 D cells were treated with different concentrations of ART.HuR subcellular localization was observed by laser confocal microscopy.Theexpression of HuR cytoplasm and nucleus was quantified by Western blot.After treated with actinomycin D(ActD),The effect of ART on mRNA degradation of metastasis-related genes was analyzed.Results The first part of artemisinin and its derivatives on NSCLC lung cancer cell proliferation and invasion.1.ARS and its derivatives inhibited the proliferation of the three NSCLC lines.The IC50 values of ARS,ART and DHA on A549 cells were respectively 2013.91?g / ml,105.77?g / ml and 76.44?g / ml.The IC50 values of ARS,ART and DHA on 95 D cells were 1304.66 ?g / ml,66.89 ?g / ml and 61.81 ?g / ml,respectively.The IC50 values of ARS,ART and DHA on H1975 cells were 1889.68 ?g / ml,94.34 ?g / ml and 90.77 ?g / ml,respectively.Compared with A549 and H1975,95 D cells are more sensitive to ARS and its derivatives.For the same type of lung cancer cell lines,the IC50 value of ART and DHA is smaller than that of ARS.2.40?g/mlART group on A549 cells,the number of cell penetrating cells less than 0?g / mlART group [(12.0000 ± 2.0000)vs(32.6667 ± 2.51661),t =-11.136,P <0.05].The number of cell penetrating cells was less than 40?g/ml ART group [(5.333 ± 0.55535)vs(12.0000 ± 2.0000),t =-5.547,P <0.05] after 90?g / ml ART treatment.There was a negative correlation between the number of cells penetrating the membrane and the dosage of ART.The invasion ability of ART inhibiting A549 cells was positively correlated with the concentration of ART.The expression of CXCR4 protein in A549 cells was significantly lower than that in 0?g / mlART group(0.4763 ± 0.01658 vs 0.7408 ± 0.06384,t =-6.945,P <0.05).ART inhibited CXCR4 mRNA expression levels A549 cell in a certain concentration range(P <0.05).The second part of HuR involved in NSCLC proliferation and invasion mechanism.The HuR mRNA-treated A549 cells had significantly lower HuR mRNA levels than the negative control group [(0.8862 ± 0.09870)vs(2.4225 ± 0.13347),t =-16.029,P <0.01].HuR siRNA-treated A549 cells showed significantly lower HuR protein levels than the negative control group [(0.5227 ± 0.04638)vs(0.9475 ± 0.06127),t =-9.574,P <0.01].The mRNA level of MMP-9 in HuR siRNA-treated A549 cells was significantly lower than that in the negative control group [(0.6131 ± 0.34299)vs(1.0868 ± 0.35277,t =-5.157,P <0.05].The MMP-9 protein level of HuR siRNA-treated A549 cells was significantly lower than that of the negative control group [(0.0682 ± 0.02903)vs(0.4654 ± 0.13072),t =-5.138,P <0.05].The CXCR4 mRNA level of HuR siRNA-treated A549 cells was significantly lower than that of the negative control group [(0.6629 ± 0.29973)vs(1.6367 ± 0.45143),t =-3.113,P <0.05].The CXCR4 protein level of HuR siRNA-treated A549 cells was significantly lower than that of the negative control group [(0.1301 ± 0.00199)vs(0.7147 ± 0.00752),t =-130.132,P <0.01].HuR siRNA transfected cells showed lower cell proliferation ability than the negative control group(t test,P <0.05).HuR siRNA decreased the invasion ability in A549 cells [(10.6670 ± 2.08167)vs(27.6667 ± 2.08167),t =-10.002,P <0.05].The third part of artemisinin drugs through HuR NSCLC biological effects of molecular mechanisms.1.Cells transfected with HuR siRNA showed higher ability of ART to inhibit proliferation than the negative control(P <0.05 for both groups).ART reduced HuR mRNA,there's no difference for protein expression.Laser confocal results showed that ART inhibited HuR cytoplasm distribution in a dose-dependent manner.4.Westernblot results show that ART can reduce the cytoplasmic distribution of HuR in a dose-dependent manner.5.ActD experimental results show that in the presence of ActD,CXCR4,MMP-9 mRNA decreased with time.ART experimental group showed that the ART concentration of 40 ug / ml within four hours,CXCR4,MMP-9 mRNA decreased significantly compared with the control group.The mRNA of MMP-9 showed a relative increase trend after four hours,and ART did not reduce the relative expression level of MMP-9 within twenty-four hours..Conclusions 1.ARS and its derivatives inhibit the proliferation and invasion of NSCLC cells,and inhibit the expression of MMP-9 and CXCR4 at the post-transcriptional level,and decrease the mRNA and protein levels of CXCR4.2.HuR knockdown can reduce the mRNA and protein levels of CXCR4 and MMP-9.3.ART at low concentrations did not reduce HuR mRNA and protein levels,but changed the nuclear-cytoplasmic transport of HuR.Artesunate affects MMP-9 and CXCR4 mRNA levels after transcription.4.Artemisinin-like drugs,represented by artesunate,inhibit the nucleus-cytoplasm transport of HuR,then affect the mRNA stability of downstream CXCR4 and MMP-9 after transcription,reduce the expression of CXCR4 protein,inhibit the proliferation and invasion of NSCLC cells.