Molecular Mechanism Of Fangchinoline In Inhibiting Metastasis Of Lung Cancer

Research background and purpose:Lung cancer is one of the most diagnosed aggressive cancers with high mortality in China and even in the world,but at present,it is still of paramount difficulty to inhibit metastasis of lung cancer effectively in clinical treatment.Besides,the treatment for lung cancer metastasis is mainly utilizing precaution,chemotherapy and medication,which has no cure for it in the clinic.Therefore,the research emphasized the way of how to inhibit malignant metastasis of lung cancer.Epithelial-mesenchymal transition(EMT)was a significant event that led to metastasis.Parts of the circulating tumour cell(CTC)managed to ooze out of the vascular system and metastasize to distance which adapted to the microenvironment and created an environment in favour of metastasizing after they successfully survived in circulatory system,and thereby migration pattern was supplanted by proliferation pattern,generating a new metastasis.In this process,EMT played an important role which facilitated the transformation of the cell mode.Hence,having a further understanding of invasion and metastasis of lung cancer and the mechanism of EMT in order to find effective drugs for controlling EMT of lung cancer cells has great guiding significant.Fangchinoline is a kind of bis-benzylisoquinoline alkaloids with complex structure and extensive pharmacological activities which was extracted from a Chinese medical herb.Traditional Chinese medicine researches held that tumour is the pathological products of hot and humid condition with phlegm stagnation in the human body.At present,Fangchinoline was found to effectively inhibit some kinds of tumours,such as breast cancer,hepatocellular carcinoma and stomach cancer.Meanwhile,modern studies indicated that Fangchinoline has specific potentials in inhibiting the invasion and proliferation of glioblastoma multiforme cell line.Therefore,we speculated that Fangchinoline was expected to be a promising drug candidate for inhibiting proliferation and metastasis of lung carcinoma cell.In this study,we intended to conduct cell migration and invasion experiments and detect the proteins of EMT in search of the potential targets for lung cancer cells,discussing the mechanism of action in order to provide theoretical basis for the clinical treatment for lung cancer with Fangchinoline.Method:1.Effect of Fangchinoline on Proliferation of Lung Cancer Cells1.1 CCK8 was performed to detect the effect of Fangchinoline(concentration gradient)on the proliferation of lung cancer cells.1.2 The clone forming tests were adopted to observe that Fangchinoline contributed to the colony formation of lung cancer cells.1.2.3 After the treatment of lung cancer cells with Fangchinoline,CFDA-SE trace method as well as flow cytometry were used to detect the fluorescence intensity of cells in order to show the effect of Fangchinoline on cell division.1.2.4 After the treatment of lung cancer cells with Fangchinoline,5Ethynyl-20-deoxyuridine(EdU)was applied to label the dividing cells whose number thereby were observed with laser confocal micROScopy.1.2.5 Lung cancer cells were strained with propidium iodide(PI),and the impact of Fangchinoline on cell cycle was detected by flow cytometry.2.Effect of Fangchinoline on Migration and Invasion of Lung Cancer Cells wound healing assay and Transwell were used to respectively detect the effect of Fangchinoline on migration and invasion of lung cancer cells.3.Effect of Fangchinoline on epithelial-mesenchymal Transition(EMT)in Lung Cancer Cells After the treatment of lung cancer cell with Fangchinoline,we utilized Western blot to detect the effect of Fangchinoline on the protein expression level of EMT in lung cancer cells,and the fluorescence intensity of Vimentin protein in lung cancer cells was detected by immunofluorescence.4.Effect of Fangchinoline on The Upstream Pathways of metastasis in Lung Cancer Cells4.1 Western blot was used to detect the effect of Fangchinoline on the related proteins in the upstream pathways of EMT in Lung Cancer cells.4.2 Western blot was applied to verify whether SC79(Akt agonist)and Compound C(AMPK inhibitor)could reverse the result that Fangchinoline inhibited protein expression of EMT in lung cancer cells.4.3 Wound healing assay was used to respectively detected whether SC79 and Compound C could reverse the inhibitory effect of Fangchinoline on the migration of lung cancer cells.4.4 Wound healing assay was used to respectively detected whether SC79 and Compound C could reverse the inhibitory effect of Fangchinoline on the invasion of lung cancer cells.5.Effect of Fangchinoline on Oxidative Sress of Lung Cancer Cells5.1 DCFH-DA staining was used to detect the effect of Fangchinoline(concentration gradient)on ROS in lung cancer cells.5.2 Western blot was used to verify whether H2O2 could reverse the inhibitory effect of Fangchinoline on the expression of related proteins of EMT in lung cancer cells.5.3 Wound healing assay and Transwell assay were respectively used to detect whether H2O2 could reverse the inhibitory effect of Fangchinoline on migration and invasion of lung cancer cells.5.4 Mitochondrial ROS interference was excluded by establishing a lung cancer mode with mitochondrial DNA deletion(ρ0 lung cancer cell)2,and mitochondrial DNA(mtDNA)expression of ρ0 lung cancer cells was detected by qRT-PCR.5.5 DCFH-DA and MitoTracker were co-stained,and thereby laser confocal was used to show the effect of Fangchinoline on ROS in ρ0 lung cancer cells.5.6 ROS in lung cancer cells were stained with DCFH-DA,and flow cytometry was applied to detect the effect of Fangchinoline on ROS in ρ0 lung cancer cells.6.Effect of Fangchinoline on NADPH oxidase Nox family in lung cancer cells6.1 mRNA expression level of Nox family in lung cancer cells was determined by qrt-PCR.6.2 Western blot was used to show the effect of Fangchinoline on Nox4 protein in ρ0 lung cancer cells.6.3 The Nox4 plasmid was transfected into ρ0 lung cancer cells,and the ROS in lung cancer cells were stained with DCFH-DA.Besides,flow cytometry and laser confocal analysis were conducted to observe whether the Nox4 plasmid could reverse the inhibitory effect of Fangchinoline on ROS in lung cancer cell membrane region.6.4 Western blot was applied to detect the effect of Fangchinoline on related proteins in EMT and pathways in transfected ρ0 lung cancer cell of Nox4 plasmid.6.5 The effect of Fangchinoline on the migration and invasion of transfected ρ0 lung cancer cells of Nox4 plasmid were detected by wound healing assay and Transwell assay,respectively.Results:1.Fangchinoline has indicated a certain potential anti-cancer effect on the lung cancer cells through the cell line-relied mechanisms.The CCK8 assay showed that Fangchinoline inhibited the proliferation of lung cancer cells at a dose-dependent fashion.The IC50 of Fangchinoline on NCI-H1299 and NCI-H1975 cells for 24 h were 13.64 μM and 10.49 μM,respectively.With prolonged exposure to Fangchinoline for 48 h,the IC50 were 11.20 μM for NCI-H1299 cells and 7.81 μM for NCI-H1975 cells.The low dosage of Fangchinoline(≤5μM)could suppress the colony formation,and the inhibitory effect showed dose-dependent manner.Fangchinoline dosedependently decreased the CFDA-SE fluorescence intensity.The results were also verified by EdU incorporation assay that indicated the dramatically decreased number of EdU-positive cells following Fangchinoline treatment.Altogether,these results suggested Fangchinoline at the lesser toxic concentration showed the remarkable inhibitory effect on the DNA replication of lung cancer cell.The conclusion was corroborated by the cell cycle analysis of cells with PI staining that Fangchinoline induced G0/G1-phase arrest.2.With the treatment of Fangchinoline,the metastatic phenotype was impaired.The typical assays including the wound healing experiment and the transwell assay indicated that the inhibited invasion and migration of lung cancer cells following the treatment of Fangchinoline.With exposure to Fangchinoline,E-cadherin,Claudin-1,and Z0-1 in lung cancer cells dramatically increased(vs.control group),while N-cadherin,Slug,Snail,and Vimentin decreased in a dose-dependent manner.In addition,the expression level of Vimentin protein was also evaluated by immunofluorescence assay,which was consistent with the western blot results.3.We treated lung cancer cells with Fangchinoline and found the downregulation of p-mTOR,suggesting mTOR might be a key player mediating the actions of Fangchinoline.We examined the typical upstream kinases of mTOR,including AKT and AMPK.The data showed the downregulation of p-AKT and upregulation of p-AMPK,which accorded with the change of mTOR.With addition of SC79,the EMT-related marker proteins were reversed,and correspondingly the metastatic capacity of lung cancer cells was rescued;in contrast,Compound C failed to counteract the effect of Fangchinoline,suggesting the inhibitory effect of Fangchinoline on lung cancer EMT was associated with AKT/mTOR pathway.4.GO analysis of the transcriptome sequencing data indicated the oxidoreductase activity was significantly under-regulated,suggested Fangchinoline might disrupt the intracellular redox balance.Sole H202 treatment downregulated E-cadherin and ZO-1 and up-regulated N-cadherin,Snail and Vimentin,which suggested ROS promoted EMT.In the combination treatment,H2O2 could reverse the inhibition effect of Fangchinoline on EMT.The claim was evidenced by the examination of the EMT-related marker and the analysis of cellular mobility capacity.H202 could upregulate the phosphorylated AKT/mTOR in lung cancer cells either alone or in combination with Fangchinoline.Further,DCFH-DA labelled ROS was analyzed by flow cytometry.Fangchinoline at the low concentration(no more than 5 μM)could reduce the total amount of ROS.However,when increasing the dosage of Fangchinoline as much as 10 μM,the level of ROS did not show significant change.To figure out why the effect of Fangchinoline on ROS level was independent of dosage,we observed the in-situ ROS labelled by DCFDA fluorescence in lung cancer cells by confocal micROScopy,finding that ROS virtually distributed unevenly in the cells and following drug treatment the level of ROS in different localization altered diversely.Around the nuclear,the abundant(bright green fluorescence)ROS augmented with the induction of drug;in contrast,green color diffused in the vicinity of the cell membrane attenuated.We used ethidium bromide(EB)reagents to deprive preferentially to mitochondrial DNA.The constructed ρ 0 cells were validated by the examination of Cox2,the mitochondria marker.The expression of Cox2 in the lung cancer cells was remarkably reduced to less than 1%.In the ρ 0 cells,Fangchinoline treatment resulted in the dosedependent reduction of ROS.5.The results of qRT-PCR showed that only Nox4 dramatically decreased in mRNA level in Fangchinoline-treated cells.The results were further confirmed by western blot.To further confirm the critical roles of Nox4,we constructed the Nox4 overexpression plasmid,which was transiently transfected into lung cancer cells.With the overexpression of Nox4,ROS measured by flow cytometry indeed increased,the quantity of ROS,however,returned to normal with the intervention of Fangchinoline.Consistently,in-situ observation through confocal microscopy showed Fangchinoline decreased cytoplasmic ROS.In addition,the phosphorylation forms of AKT and mTOR were also upregulated in the Nox4-overexpressed lung cancer cells,suggesting Fangchinoline inhibits Nox4 from catalyzing the increase of ROS,which in turn activates AKT/mTOR pathway.Although Fangchinoline,identical with the previous experiments,blocked EMT marked by E-cadherin,N-cadherin etc.,and the overexpression of Nox4 reversed the effect.Similar results were also obtained by the examination of cell mobility i.e.,the wound healing assay and the transwell assay.Conclusion1.Fangchinoline can inhibit the proliferation of lung cancer cells,leading to the block in the stage of G0/G1.2.Fangchinoline can inhibit the migration,invasion,and EMT progress of lung cancer cells.3.Fangchinoline can block metastasis progress in lung cancer cells by inhibiting the Akt/mTOR signaling pathway.4.Fangchinoline inhibited cytoplasmic ROS to restrain the metastasis of lung cancer cells.5.Fangchinoline was able to decrease Nox4 derived ROS to inhibit the Akt/mTOR signaling pathway.Nox4 was expected to an important target that can inhibit the metastasis of lung cancer cells.