Leukamenin E Induces K8/18 Phosphorylation In HUVEC Cells And Inhibits The Assembly Of Keratin Networks

As the ones of the most abundant keratins in HUVEC cells,the keratin 8(K8)and keratin 18(K18)with the form of phosphorylation mainly regulate various functions of cells,which is an important way of post-translational modification of keratin.We found that phosphorylation of K8 and K18 can regulate the solubility of keratin and participate in the assembly mechanism of keratin network.Meanwhile,keratin can provide the necessary structural support for cells,regulate cell adhesion and cell growth,and resist all kinds of stress.Keratin is deeply involved in various life activities of cells,including cell signal transduction,cell differentiation,cell apoptosis,cell stress response and other important cell life processes.In addition,keratin is also related to cell apoptosis.Recent research of our laboratory showed that the Ent-Kaurene diterpenoid leukamenin E was a kind of plant secondary metabolic products with significant antitumor activity.It has shown growth inhibition,differentiation and apoptotic induction effects on a variety of cancer cell lines,as well as anti-tumor effects such as angiogenesis inhibition,but the molecular mechanism is still not clear.Therefore,it is of great scientific value to further explore the effects of this compound on the functional units of cells and the action targets to elucidate its mechanism and the discovery of new anticancer drugs.With Ent-Kaurene diterpenoid leukamenin E as the tested drug and human umbilical vein endothelial cells(HUVEC)and pancreatic cancer cells(Panc-1)as tested cell lines,the effects of leukamenin E on keratin fibers were studied by MTT,AO/EB double staining,indirect immunofluorescence,western bloting(WB),transwell assay and scratch test.For the first time,we found that leukamenin E could induce the phosphorylation of keratin fibers through the ERK signaling pathway and further block the assembly process of keratin fiber.The main results were showed as follows:1.MTT assay and AO/EB double staining indicated that after the treatment of 2-4μM leukamenin E on HUVEC cells and Panc-1 cells for 24 h,leukamenin E had significant inhibitory effect on the proliferation of both types of cells with concentration dependence.However,it did not cause significant apoptosis and necrosis.A suitable concentration system was established for subsequent studies to exclude cytoskeletal degradation induced by the cell apoptosis.2.Direct and indirect immunofluorescence suggested that,after the treatment of the microfilament depolymerization drug(cytochalasin B)and the microtubule depolymerization drug(colchicine)on the spread and spreading cells to fully depolymerize microfilaments and microtubules,keratin fiber network of HUVEC remained intact,which showed that the keratin fiber network of HUVEC was suitable for the screening of drugs that target the assembly of keratin fiber.3.Direct and indirect immunofluorescence indicated that 2μM and 4μM of leukamenin E significantly reduced the amount of keratin fibers in HUVEC cells,but hardly reduced the number of microfilaments and microtubules in HUVEC cells.For spreading cells,leukamenin E significantly decreased the spreading area of HUVEC and inhibited the assembly of keratin fiber networks.Further WB detection revealed an increase in soluble keratin fibers in the cytoplasm of HUVEC,rather than insoluble keratin fibers,suggesting that leukamenin E may inhibit the formation of keratin fiber networks by increasing keratin fiber solubility.4.Western bloting demonstrated that in the spread cells,the leukamenin E could activate keratin phosphorylation at sites K8-Ser431/Ser7 and K18-Ser52 in a concentration-dependent manner;in the spreading system,the leukamenin E could increase the keratin phosphorylation at sites K8-Ser431/Ser7 and K18-Ser52 with a concentration and time-dependent manner.Besides,the increase of pK8-Ser431 was earlier than pK8-Ser73 and pK18-Ser52.This suggested that the phosphorylation at the 3 sites may lead to an increase in keratin fiber solubility.Indirect immunofluorescence double staining confirmed that,indeed,leukamenin E reduced the localization of pK8-Ser431,pK8-Ser73 and pK18-Ser52 on the HUVEC keratin network.WB also detected an increase in soluble pK8-Ser431,pK8-Ser73 and pK18-Ser52 keratin fibers in the cytoplasm of HUVEC,while insoluble pK8-Ser431,pK8-Ser73 and pK18-Ser52 decreased.This confirmed that the increased levels of pK8-Ser431,pK8-Ser73 and pK18-Ser52 induced by leukamenin E were associated with increased solubility of keratin fibers.5.WB assay showed that leukamenin E could increase the phosphorylation of ERK.Combined with ERK inhibitors,it was found that phosphorylation at sites K8-Ser431,K8-Ser73 and 18-Ser52 could be inhibited.After the treatment of leukamenin E,the significant increase of keratin in superscript and the significant decrease of keratin in sediment could be inhibited by ERK inhibitors in both the spread and the spreading systems.The results showed that the ERK signaling pathway was associated with increased pK8-Ser431,pK8-Ser73 and pK18-Ser52 induced by leukamenin E.6.The results of the scratch test also showed that the leukamenin E significantly inhibited the migration of HUVEC and Panc-1 cells.In summary,in the proliferation-inhibiting system of HUVEC cells,leukamenin E was able to phosphorylate keratin at K8-Ser73/Ser431 and K18-Ser52 via the ERK pathway,increase the cytoplasmic soluble pK8-Ser73/Ser431,pK18-Ser52 and pan-keratin,and possibly further lead to the blocking effect of keratin fiber assembly in cells.Further studies are needed to determine whether leukamenin E can inhibit cell migration via the ERK pathway.