Atractylodes Lactone I Targeted MD2Inhibiting TLR4/MYD88/AKT Pathway To Increase The Ovarian Cancer Cell Apoptosis Induced By Paclitaxel

1. Background and ObjectiveThe important cause of the high mortality rate and the poor prognosis ofpatients with ovarian cancer is Drug resistance. Now has confirmed that TLR4/MyD88signaling pathways in ovarian cancer is closely associated with tumourprogression and drug resistance. Lipopolysaccharide (LPS) and paclitaxel couldcombine with TLR4, activate MyD88, leading to the activation of protein kinaseC (PKC/AKT), and further cause the activation of NF-B, ultimately produceproinflammatory factor and influence the apoptosis of ovarian epithelialcarcinoma. Our group preliminary study show that MyD88high expression canbe used as ovarian cancer independent prognostic factors for the short survivalrate, but the MyD88only expression in SKOV3.In the process of MyD88cativation,myeloid differentiation protein2(MD-2) mediated TLR4/MD2complex formation is necessary.Atractylenolide I (Atractylenolide I, AO-1) is extracted from Atractylodes with the effect of anti-inflammatory and anti-tumor. Like LPS, AO-1canrecognize and bind to TLR-4so that antagonist pro-inflammatory effect whichmediate by TLR-4. In our previous study, AO-1as a antagonist ligand to TLR-4which significantly reduced TLR-4signaling pathway mediated MyD88+ovarian cancer cells produce VEGF and Surivin and paclitaxel resistance. It issuggest that Atractylenolide I can increase sensitivity to paclitaxel in ovariancancer cells, and this effect is dependent on TLR-4/MyD88pathway. But Thespecific molecular mechanisms and targets are still unclear.We also performed apreliminary docking of AO-1and paclitaxel to human MD-2by computationalsimulation. We demonstrated,AO-I was shown to fit into the hydrophobic pocketof human MD-2and to partially overlap with the binding site of paclitaxel bydocking simulations, suggesting that AO-I may block the MD-2-mediatedTLR4/MyD88-dependent paclitaxel signaling in MyD881EOCcells. Therefore,AO-I could significantly sensitize the response of MyD88+EOC cells topaclitaxel by blocking MD-2-mediated TLR4/MyD88signaling.To further investigate atractylenolide I antagonize TLR-4/MyD88+/PI3Kpathway down the anti-apoptotic effects of paclitaxel in ovarian cancer. Thispaper use flow cytometry, Western blot techniques to explore its mechanism, tolay a solid foundation for the treatment of chemotherapy-resistant ovariancancer.2Methods2.1Flow cytometry was used to detect different concentrations of paclitaxel, LPS, AO-1and AO-1or LPS combined with different concentrations ofpaclitaxel on human ovarian cancer SKOV-3cells, effect on TLR4-MD2dimer.2.2Western Blot detect the anti-apoptotic protein Akt, PAkt on human ovariancancer SKOV-3, A2780cell which deal with paclitaxel, LPS, AO-1and AO-1orLPS plus paclitaxel.2.3Using flow cytometry detect apoptosis rate on human ovarian cancerSKOV-3and A2780cells deal with paclitaxel, LPS, affecting AO-1and AO-1orLPS plus paclitaxel.3Results3.1With LPS and different concentrations of paclitaxel in MyD88-positiveSKOV-3ovarian cancer cells, the cell surface TLR4-MD2dimer levelsincreased significantly, and with the increasing concentration of paclitaxel,significantly increased dimmer. TLR4-MD2dimer showed no significant changewith Only used Iatractylenolide I in in the SKOV-3cell lines. However, inSKOV-3cell atractylenolide I combined with LPS and different concentrationsof paclitaxel compared with single-use LPS and paclitaxel, the TLR4-MD2dimer levels decreased significantly.3.2After used Taxol, atractylenolide I, LPS, atractylenolide I combined withpaclitaxel and LPS in the SKOV-3cells within24h, the express of total Akt haveno significant changes.After used Taxol, LPS in SKOV-3cells within24h,thephosphorylated Akt (pAkt) expression was significantly upregulated whichlevels increase with time.After used Atractylenolide I, atractylenolide I combined with paclitaxel, atractylenolide I combined with LPS in SKOV-3cells,the phosphorylated Akt (pAkt) expression was significantly decreased.But in A2780cells, After used paclitaxel, atractylenolide I, LPS, atractylenolideI combined with paclitaxel and LPS within24h, the total Akt and thephosphorylated Akt (pAkt) expression with no significant changes.3.3In A2780cells, After used Atractylenolide I, LPS, atractylenolide Icombined with LPS within24h and48h, the apoptosis rate did not changesignificantly. After used Taxol, atractylenolide I combined with paclitaxel within24h and48h, the apoptosis rate increased, but the apoptosis rate was notstatistically significant between monotherapy and combination therapy. InSKOV-3cells, After used Atractylenolide I and LPS within24h and48h theapoptosis rate did not change significantly. After used Atractylenolide Icombined with LPS, the apoptosis rate increased slightly. After used Taxol,atractylenolide I combined with paclitaxel within24h and48h, the apoptosis rateincreased, and when compared with only used paclitaxel,the apoptosis rate withcombination therapy have significantly increased.4ConclusionAfter used paclitaxel in SKOV-3cells,the TLR4-MD2dimer levels in cellsurface increased significantly, the anti-apoptotic proteins phosphorylated Akt(pAkt) expression was significantly upregulated. When paclitaxel combinationwith AO-1, the TLR4-MD2dimer levels in cell surface decreased significantly,the anti-apoptotic proteins pAkt expression was significantly downregulated,while the rate of apoptosis was significantly higher whencompared with paclitaxel alone.But in the A2780cells, TLR4-MD2dimer, Akt,pAkt expression, and the rate of apoptosis hane no significant difference.Suggestthat AO-1is most likely to antagonize TLR-4/MyD88+/PI3K signalingpathway to promote apoptosis of ovarian cancer cells, so that inhibit the growthof ovarian cancer cells.