The Molecular Mechanism Of Albendazole Inhibits EMT In Malignant Melanoma Cells By Regulating The Phosphorylation Status Of GSK-3β

Albendazole(ABZ)is a broad-spectrum anthelmintic drug of imidazole derivatives,which has been widely used in humans and various animals.It has wide-range and strongest insecticidal effect but less side effects.ABZ has now been included in the WHO essential health standard drug list and is one of the essential drugs.At the same time,the pharmacokinetics and safety of ABZ have been proved,and it is of great significance to study its repositioning in the antitumor effect.Previous articles have reported that ABZ has anti-tumor effects on some different types of cancer,and its mechanism may be through promoting tumor cell apoptosis or inhibiting tumor cell proliferation to produce anti-tumor effects.However,whether ABZ can inhibits the metastasis of tumor cells has not been reported in the literature.It is known that the main reason for the poor prognosis of melanoma patients is also due to the disease progresses rapidly after metastasis.Therefore,in this study,we aimed to explore the inhibitory effect of albendazole on migration and invasion of melanoma cells and its possible mechanism.Our research is divided into three parts:Part 1 Low-dose ABZ can effectively inhibits the metastasis of melanoma cells in vitro and in vivo1.Low-dose ABZ(<0.4μM)could significantly inhibit the migration and invasion of melanoma cells in vitro,but did not affect the proliferation of melanoma cells.(1)The proliferation rate of melanoma cells treated with different concentrations of ABZ for 24 h was detected by CCK-8 assay.The results showed that the proliferation rate of A375 and B16-F10 cells decreased with the increase of ABZ concentration,but when the concentration of ABZ was less than 0.4 μ M,the proliferation rate of melanoma cells was not significantly affected.(2)The wound healing experiment verified the effect of low concentration of ABZ on the migration ability of A375 and B16-F10 cells.The results showed that the percentage of melanoma cell healing in ABZ group was remarkably lower than that in the control group(P< 0.001).(3)Transwell invasion assay confirmed the effect of low-dose ABZ on the invasion ability of A375 and B16-F10 cells.The results showed that the number of cells passing through transwell chamber in ABZ group was significantly lower than that in control group(P< 0.001).2.Low-dose ABZ can significantly inhibits pulmonary metastasis of melanoma cells in vivo.(1)The pulmonary metastasis model of melanoma in nude mice was used to study the effect of ABZ treatment on the metastasis of melanoma cells in vivo.The results of in vivo imaging showed that the fluorescence intensity of lung melanoma cells in the ABZ treatment group was significantly lower than that in the control group,and the fluorescence intensity in the treatment group showed ABZ dose-dependent(P < 0.001).(2)After the nude mice were sacrificed,the lungs were removed and perfused with PBS.The dissection results showed that the pulmonary melanoma nodules of the nude mice in the ABZ treatment group were smaller and the nodules formed in the lungs were less.(3)The tumor nodules on the pulmonary surface were scored according to the number and size,and it was found that the tumor nodule score in the ABZ treatment groups were significantly lower than that in the control group,and the tumor nodule score in the experimental groups showed a negative correlation with the dose of ABZ(P < 0.001).(4)HE staining was performed after pulmonary melanoma tissue sections.The results showed that the number of lung metastatic tumor nodules in the ABZ treatment group was significantly less than that in the control group.Part 2 Low-dose ABZ can significantly inhibits the EMT process of A375 and B16-F10 cells1.Low-dose ABZ inhibits the migration and invasion of melanoma cells by inhibiting the EMT process in vitro.(1)Real time RT-qPCR was used to detect the mRNA expression levels of EMT-related genes in A375 and B16-F10 cells before and after ABZ treatment.The experimental results showed that,compared with the control group,the mRNA relative expression level of E-cadherin in the ABZ treatment group was significantly up-regulated,while the mRNA relative expression levels of N-cadherin,Vimentin,FN1 and Occludin were down-regulated to varying degrees.(2)Western blot assay was used to detect the expression of EMT-related proteins in A375 and B16-F10 cells before and after treatment with ABZ.The results revealed that compared with the control group,the expression of E-cadherin protein was significantly up-regulated,while the relative expression levels of N-cadherin,Vimentin,FN1 and Occludin proteins were down-regulated in ABZ-treated group.2.Low-dose ABZ inhibits the migration and invasion of melanoma cells by inhibiting EMT process in vivo.(1)IHC staining was used to detect the localization and expression of N-cadherin and E-cadherin in pulmonary metastatic melanoma.The results revealed that compared with the control group,the expression levels of E-cadherin and N-cadherin were significantly up-regulated and down-regulated in pulmonary metastatic melanoma tissues in ABZ treatment group,respectively(P < 0.001).Part 3 ABZ mediates Snail degradation by inhibiting pAKT and pGSK-3β/Ser9 and upregulating pGSK-3β/Tyr216 to inhibits EMT progression in melanoma1.ABZ could down-regulate Snail through post-translational modification rather than transcriptional level.(1)Real time RT-qPCR was used to detect the relative expression levels of EMT key transcriptional gene Snail in A375 and B16-F10 cells before and after ABZ treatment.The experimental results showed that compared with the control group,the relative expression of Snail in the ABZ group did not change significantly.(2)Western blot assay was used to detect the relative expression levels of EMT key transcription protein Snail in A375 and B16-F10 cells before and after ABZ treatment.The results showed that ABZ treatment significantly reduced the level of Snail protein in the cytoplasm of melanoma cells(P < 0.001).2.ABZ could promotes the nuclear export of p Snail/Ser246(1)Western blot was used to detect the relative expression levels of phosphorylated Snail protein in the nucleus of A375 and B16-F10 cells before and after ABZ treatment.The results showed that compared with the control group,ABZ treatment significantly reduced the level of p Snail/Ser246 in the nucleus of melanoma cells(P < 0.01).3.ABZ mediates Snail degradation by inhibiting the expression levels of pAKT and pGSK-3β/Ser9(1)Western blot was used to detect the phosphorylation levels of Snail-regulating GSK-3β and its upstream activator AKT in the cytoplasm of A375 and B16-F10 before and after ABZ treatment.Experimental results showed that ABZ treatment significantly reduced the levels of pGSK-3β/Ser9(inactive form)and pAKT/Ser473.(2)The proteasome inhibitor MG132 was added to inhibits the degradation of Snail.Western blot analysis showed that MG132 did not affect the expression levels of AKT and pGSK-3β/Tyr216,but the degradation of Snail was significantly reduced after ABZ treatment,which subsequently led to the up-regulation of its downstream target N-cadherin and the down-regulation of E-cadherin.(3)A375 and B16-F10 cells were transiently transfected with the constitutively activated-AKT(CA-AKT)plasmid.Western blot analysis showed that compared with the ABZ group,the protein levels of pAKT/Ser473 and pGSK-3β/Ser9 were increased after transfection with CA-AKT,and the expression of Snail was also up-regulated.(4)The effect of transient transfection of CA-AKT on the migration of ABZ in inhibiting melanoma cells was detected by wound healing experiment.The experimental results showed that the transfection of CA-AKT plasmid significantly reversed the inhibitory effect of ABZ treatment on the migration of melanoma cells.(5)Transwell invasion assay was used to detect the effect of transient transfection of CA-AKT on ABZ in inhibiting the invasion of melanoma cells.The experimental results showed that the melanoma cells transfected with CA-AKT plasmid significantly reversed the inhibitory effect of ABZ treatment on their invasiveness(P < 0.05).(6)IHC staining analysis of the localization and expression levels of Snail upstream transcription factors in nude mice lung metastatic melanoma nodules.The experimental results showed that the levels of pAKT/Ser473 and pGSK-3β/Ser9 were significantly decreased,and the expression level of pGSK-3β/Tyr216 was significantly increased in the melanoma of the ABZ treatment group(P < 0.001).4.ABZ can inhibits the EMT process of melanoma cells by increasing the accumulation of pGSK-3β/Tyr216(1)Western blot were used to detect the expression of pGSK-3β/Tyr216(active form)in the cytoplasm of A375 and B16-F10 before and after ABZ treatment.The experimental results showed that after ABZ treatment,the expression of pGSK-3β/Tyr216 increased significantly,and its downstream Snail was significantly down-regulated.(2)Stable A375 cell lines expressing wild-type GSK-3β(GSK-3β/WT)and GSK-3β/Y216 phenylalanine(F)mutation(GSK-3β/Y216F)were constructed by lentiviral transduction.After ABZ treatment for 24 h,the protein levels of GSK-3β/Tyr216 phosphorylation levels in the cytoplasm of the three cells were significantly increased compared with the DMSO groups.Compared with ABZ-treated control cells,ABZ promoted more phosphorylation at Tyr216 site of GSK-3β protein in overexpressing GSK-3β/WT cells,but not in cells which overexpressing GSK-3β/Y216 F.(3)Western blot further detected the expression of Snail,N-cadherin and E-cadherin proteins in these three cells after ABZ treatment.Compared with ABZ-treated control cells,overexpression of GSK-3β/Y216 F did not change the protein expression levels of Snail, N-cadherin and E-cadherin.However,in the GSK-3β/WT group treated with ABZ,Snail and N-cadherin were significantly down-regulated,while E-cadherin protein was significantly up-regulated.(4)The effect of ABZ on the migration of A375 cells of overexpressing GSK-3β/WT and GSK-3β/Y216 F was detected by wound healing experiment.The experimental results showed that ABZ inhibited the migration of GSK-3β/WT cells more significantly than that in GSK-3β/Y216 F cells(P < 0.001).(5)Transwell invasion assay was used to detect the effect of ABZ on the invasion of GSK-3β/WT and GSK-3β/Y216 F A375 cells.The experimental results showed that ABZ inhibited the invasion of GSK-3β/WT cells more significantly than that in GSK-3β/Y216 F cells(P < 0.01).In summary,this study found that:1.Low-dose ABZ treatment can significantly inhibits the migration and invasion of melanoma cells in vitro.2.Low-dose ABZ treatment can significantly inhibits the lung metastasis of melanoma cells in mice.3.ABZ can inhibits the metastasis of melanoma cells by reversing the EMT process.4.ABZ can mediates the down-regulation and up-regulation of the downstream proteins N-cadherin and E-cadherin respectively by down-regulating the expression of Snail in melanoma cells,and finally play a role in inhibiting melanoma EMT.5.Transfection of CA-AKT plasmid significantly reversed the inhibitory effect of ABZ treatment on melanoma cell migration and invasion,further proving that ABZ can mediate Snail degradation by inhibiting the expression levels of pAKT and pGSK-3β/Ser9.6.The stable GSK-3β/WT and GSK-3β/Y216 F A375 cell lines were constructed and screened.After treated with ABZ for 24 h,Snail in GSK-3β/Y216 F A375 cell line was dramatically down-regulated.However,there was no significant change in Snail in g SK-3β/Y216 F overexpression group compared with ABZ-treated control group,which further indicated that ABZ could promote Snail degradation by increasing the accumulation of PGSK-3β/Tyr216,thereby inhibiting EMT process of melanoma cells.