The Research Of The Antitumor Activity And Mechanism Of Bioactive Compounds Of Eriocaulon Sieboldianum And Two HS-173 Derivatives

Aurora kinase is a family of serine/threonine kinases in mammalian and plays a pivotal role in the regulation of cell mitosis.It is frequently overexpressed in human cancer.Overexpression of aurora kinases has been reported to be strongly associated with tumorigenesis,development,invasion and migration,as well as drug resistant and poor prognosis.Aurora kinases are found to be overexpressed in most of hepatoma carcinoma cells and tissues,including HepG2 cells.Therefore,it is a very important target for the development of novel drugs for hepatocarcinoma therapy.Objective:Our previous study found that active fractions from ethyl acetate extract of E.sieboldianum after purification by silica gel column chromatography and some flavonoids isolated from these fractions effectively inhibited the proliferation of K562 cells.Some flavonoids,such as neptin,hispidulin and quercetin-3-O（6"-O-galloyl）-β-D-galactopyranoside（QGGP）,decreased the levels of phospho-Histone H3（Ser10）,suggesting Aurora kinase inhibition.In this study,HepG2 and HepG2/ADM cells with higher Aurora kinase expression were selected to investigate the anticancer mechanism of E.sieboldianum and its active compounds.Methods:To investigate the anti-proliferative effect of the active fraction of E.sieboldianum extract（E3）and its two active compounds,hispidulin（HPDL）and quercetin-3-O-（6"-O-galloyl）-β-D-galactopyranoside（QGGP）,the cytotoxicity of E3 and compounds 1-11 on HepG2 cells was measured by a standard MTT assay.Then,the effects of E3,HPDL and QGGP on the cell cycle distribution were determined using flow cytometry analysis.Moreover,molecular docking studies were performed to investigate the binding affinities and interaction modes for HPDL and QGGP.Next,we detect the related protein levels of Aurora kinase signaling,MAPK pathway and p53-mitochondrial apoptosis pathway by western blotting analysis.Finally,apoptosis in the cells treated with E3,HPDL and QGGP was measured using Annexin V-propidium iodide staining kits.Beides,our previous study showed nepetin not only could inhibit cell growth of K562 cells and decrease the levels of p-Histone H3 at ser10,but also reverse adriamycin resistance of HepG2/ADM cells.Therefore,we further test the antiproliferation effects on HepG2 and HepG2/ADM cells and investigate the anticancer mechanism of neptin in HepG2/ADM cells.Results:1.The active fraction of E.sieboldianum extracts（E3）and its two active compounds,HPDL and QGGP,effectively inhibited the cell growth of HepG2 cells.2.E3,HPDL and QGGP could inhibit Aurora kinase and then lead to cells accumulation of G2/M phase.3.E3,HPDL and QGGP induced cell apoptosis via MAPK signaling in HepG2 cells.4.E3,HPDL and QGGP induce apoptosis of HepG2 cells through increasing the expression of pro-apoptotic proteins,such as p53 and Bax,and decreasing anti-apoptotic proteins such as Bcl-2.In addition,E3,HPDL and QGGP could also induce cell apoptosis via mitochondria apoptosis pathway.5.Nepetin enhanced the sensitivity of HepG2/ADM cells to adriamycin and showed synergistic effects when combined with adriamycin in inducing cell apoptosis.6.Nepetin inhibited the cell growth and proliferation of HepG2 cells via Aurora kinase/Akt inhibition.7.Nepetin increased the intracellular accumulation of adriamycin and Rh123 in HepG2/ADM cells via p-gp inhibition.8.Nepetin induced apoptosis of HepG2/ADM cells through MAPK pathway snd p53-mitochondria apoptosis pathway.Conclusion:E3,HPDL and QGGP induce cell cycle arrest and cell apoptosis via Aurora kinse inhibition and regulation of MAPK signaling and p53-mitochondria apoptosis pathway.Nepetin enhance the sensitivity of HepG2/ADM cells to adriamycin via Aurora/Akt/p-gp pathway and induce cell apoptosis via MAPK pathway snd p53-mitochondria apoptosis pathway.Receptor tyrosine kinase（RTKs）and its downstream PI3K/Akt/mTOR signaling,which associated with tumorigenesis,development and drug resistance,are very important targets of chemotherapy drugs in non-small cell lung cancer（NSCLC）.Currently,cancer cells have developed drug resistance to EGFR inhibitors successively.C-MET amplification is one of the most important reason resulting in this drug resistance.MET amplification leads to EGFR inhibitors resistances in NSCLC via activating ErbB3/PI3K/Akt pathway.EGFR mutation is another important cause led to drug resistant.However,these drug resistances could be overcomed by the combination of PI3K/Akt/mTOR inhibitor or MET inhibitor and EGFR inhibitors.Thus,PI3K/Akt/mTOR inhibitors,such as PI3K inhibitor,MET inhibitors,mTOR inhibitor,and dual inhibitors of PI3K and mTOR become to the research hotspot in targeted anticancer drugs.Objective:HS-173 is a selective and efficient PI3Ka inhibitor with IC₅₀ of 0.8nM.However,its water solubility is not very good and even somewhat unstable in phosphate solutions of pH=6.7.Therefore,in this study,we performed structure modification taking HS-173 as the lead compound and yield DFX117,a new PI3K inhibitor,which is more effective,stable and easily dissolved in water.In chapter 2 of Part 2,we will investigate the anticancer mechanisms of DFX117 in vitro and in vivo.To explore whether imidazopyridine moiety is essential for its PI3Kα inhibitory activity or not,we replace the imidazopyridine moiety with other group including indole ring and quinazolin.Finally,DFX24 with the best antiproliferative activity was obtained.We will investigate the anticancer mechanisms of DFX24 in vitro and in vivo in chapter 3 of Part 2.Methods:Firstly,we test the antiproliferative activity of DFX24 and DFX117 against different human cancer cell lines,and two most sensitive cell lines,A549 and NCI-H1975,were selected for the further study.Cell cycle distribution was evaluated using flow cytometric analysis.The treated cells were fixed with 70%EtOH and stained with cell cycle analysis kit before subjected to flow cytometric analysis.Then we determined the effects of DFX24 and DFX117 on RTK-PI3K signaling pathway.We also explore the effects of DFX24 and DFX117 on cell autophagy and apoptosis.In addition,RT-PCR was used to analyze the effect of DFX24 and DFX117 on PIK3CA.Annexin V-FITC/PI kit and flow cytometric analysis were used to analyze the effects of DFX24 and DFX117 on cell apoptosis.Finally,A549 nude mouse xenograft models were used to evaluate the tumor growth inhibition in vivo.Results:1.DFX24 and DFX117 effectively inhibited the cell growth of several cancer cells with IC₅₀ of 10 nM to 100 nM.2.DFX24 and DFX117 suppressed PI3k activity significantly and inhibited PI3Ka more selectively with IC₅₀ of 6.5 nM and 0.5 nM respectivetly.3.DFX24 and DFX117 inhibit the PI3K/Akt pathway in a dose-dependent manner.4.DFX24 and DFX117 could induce autophagy of A549 and NCI-H1975.5.The mRNA levels of PIK 3CA were significantly decreased by 0.4 μM DFX117.6.DFX24 and DFX117 induced cell apoptosis via mitochondria apoptosis pathway.7.DFX117 suppressed RTKs,including EGFR,MET and IDF-1R.DFX24 could decrease the protein levels of p-ErbB3.8.DFX24 and DFX117 induce G1 phase arrest via PI3K/Akt pathway and regulation of proteins related to G1 phase.9.DFX24 and DFX117 significantly suppressed the tumor growth in A549 xenograft models.10.DFX24 and DFX117 suppressed PI3K/Akt pathway in vivo.11.DFX24 and DFX117 led to an increase in cleaved PARP,cleaved caspase-3 and cleaved caspase 9 in the tumors and finally induced tumor apoptosis in vivo.