Study On The Mechanism Of The Inhibitory Effect Of Costunolide And Dehydrocostus Lactone On Glioma

Objective: Costunolide(CT)and dehydrocostus lactone(DHE),two natural sesquiterpene lactones,have been used for treatment of various diseases with the anti-inflammatory activity.Recently,they have caused extensive interest in researchers due to the anti-cancer abilities in some types of carcinomas.However,the anti-cancer effect and mechanism of CT and DHE in glioma remains poorly understood.The present study conducted to determine the biological effects of CT and DHE on the glioblastoma cells,as well as the possible mechanisms underlying these effects.Methods: To evaluate the anti-glioblastoma effects of CT and DHE,three glioblastoma cell lines(U118,U251 and U87)were treated with different concentrations(0,1,10,25,50 and 100 ?M)of CT and DHE for 12 h,24 h,36 h or 48 h,respectively.And then a MTT assay was performed to determine cell viability.Following this,we employed colony formation assay to evaluate the effect of CT and DHE on the clonogenic capacity of U87 and U251 cells.To identify the effect of CT and DHE on the migration ability of glioblastoma cell lines,wound healing assay was performed under serum-free conditions.The confocal immunofluorescence assay was performed to detect whether DHE resulted in cytochrome c release from mitochondria to the cytosol in U87 cells.Afterward,western blot was used to determine the extent of cytochrome c release fromthe mitochondria into cytosol during treatment.To gain better insight into DHE-induced apoptosis in U87 cells,we measured the expression of major mitochondrial apoptosis signaling associated protein,using western blot.Afterwards,the expression of COX-2 was studied in three human glioblastoma cell lines(U118,U251 and U87)by western blot.After that,we employed U87 and U251 cell lines to determine whether the tumor growth inhibitory effect of DHE was correlated with COX-2 expression.And the effect of DHE treatment on COX-2 protein and m RNA levels were evaluated by western blot analysis and RT-PCR,respectively.To determine whether p300 and p65/p50 NF-?B are involved in the transcriptional regulation of DHE on COX-2 by binding directly to the promoter in U87 glioblastoma cells,nuclear extracts were analyzed by Ch IP using specific antibodies directed against p300,p50 and p65.To further evaluate the effect of DHE on the binding activities of p300 and p65/p50 NF-?B on COX-2 promoter-transactivator complex,we employed the streptavidin-agarose pull-down assay.In brief,nuclear extracts from U87 cells treated with or without DHE were incubated with the 5'-biotinylated COX-2 promoter probe and streptavidin-agarose beads.After centrifugation,transcription activators p65/p50 NF-?B and coactivators p300 present in the complex were analyzed by western blot.Following this,we detected quantitative information of p300 and p65/p50 NF-?B in total nuclear extracts by western blot.To further confirm that DHE inhibited nuclear translocation and interaction between p300 and p65/p50 NF-?B protein,we performed immunoflurescence staining to investigate the localization and distribution of p300 and p65/p50 NF-?B in U87 cells.To fully understand the related molecular mechanism of DHE treatment on glioblastoma cells,we next employed U87 and U251 cells to investigate the effects of DHE on IKK?/ I?B?/NF-?B signaling pathway.And the levels of p-IKK?/?,IKK?,IKK?,p-I?B?,I?B?,p-p65 and p65 protein were analyzed by western blot.Thereafter,we hypothesized that IKK? may be served as a target of DHE in the treatment of glioma.To test this hypothesis,computer molecular modeling assay was performed to simulate the interactions between DHE and IKK?.In vivo study,to determine the transport of DHE across the blood–brain barrier(BBB),Sprague Dawley(SD)rats were intravenous injected with DHE at the dose of 100 mg/kg.After 1 h,CSF was stereotaxically collected from the cisterna cerebellomedullaris of rats.The DHE in the CSF samples was extracted with acetonitrile,and then determined by the mass spectrometry.Then,we established a xenograft nude mice model to further determine the anti-cancer effect of DHE on glioblastoma in vivo.To monitor the toxicity of DHE,the body weight of each mice was recorded every 2 days.The volume of the implanted tumor was measured every 2 days in two dimensions(length and width).All mice were euthanized on day 30.The implanted tumors were excised and weighed,and then fixed in 10% formalin before paraffin embedding.Furthermore,to determine the potential mechanism,we detected the expression of COX-2,p-p65 and p-IKK? in transplanted tumors using immunohistochemistry analysis.Results: When three glioblastoma cell lines(U118,U251 and U87)were treated with different concentrations of CT and DHE for different time,cell viability was measured by MTT assay.We found that CT and DHE could inhibit these cells growth in a dose-dependent and time-dependent manner.At 48 h after treatment,the IC50 values of CT against U118,U251 and U87 cells were 25.24±1.15,34.45±2.8 and 40.18±3.21 ?M,respectively.And the IC50 values of DHE against U118,U251 and U87 cells were 17.16±2.11,22.33±1.93 and 26.42±2.84 ?M,respectively.The clonogenic ability was measured by colony formation assay.We found that treatment of these cells with CT or DHE at the different concentrations result in a dose-dependently reduced the number of colonies,compared with untreated control group.The migration ability was measured by wound healing assay.We found that the migration rate of cells significantly decreased with increasing concentrations of CT and DHE,compared with the control group.Thereafter,the confocal immunofluorescence assay was performed to detect whether DHE resulted in cytochrome c release from mitochondria to the cytosol in U87 cells.In the control group,cytochrome c was mainly localized in the mitochondria,however,increased concentrations of DHE resulted in elevated levels of cytosolic cytochrome c as evidenced by a diffused green fluorescence.Afterward,we employedwestern blot assay to measure the levels of major mitochondrial apoptosis signaling associated protein.The results indicated that DHE statistically increased the levels of cytosolic cytochrome c and cleaved caspase3/9 proteins in a concentration-dependent manner.However,the levels of Bcl-2 protein and anti-apoptotic Bcl-2/BAX ratio were reduced.Then,the expression of COX-2 protein in U118,U251 and U87 cell lines were studied by western blot.The result revealed that COX-2 protein was strong expressed in U87 and U251 cells,whereas the expression of COX-2 was relatively weak in U118 cells.Thus,we employed U87 and U251 cell lines to determine whether the tumor growth inhibitory effect of DHE was correlated with COX-2 expression.Western blot and RT-PCR results suggested that DHE effectively inhibited COX-2 expression at both protein and m RNA levels in glioblastoma cells by a dose-dependently manner.To further determine whether p300 and p65/p50 NF-?B were involved in the transcriptional regulation of DHE on COX-2 by binding directly to the promoter in U87 glioblastoma cells,nuclear extracts were analyzed by Ch IP and streptavidin-agarose pull-down assay.These results indicated that DHE significantly inhibited p300 recruitment and p65/p50 binding directly to the COX-2 promoter in a dose-dependent manner,compared with the control group.Following this,western blot results suggested that p300 and p65/p50 NF-?B protein levels in nucleus significantly decreased after DHE treatment,compared with the control group.After that,we performed immunoflurescence staining to investigate the localization and distribution of p300 and p65/p50 NF-?B in U87 cells.The result indicated that coactivators p300 and p65/p50 NF-?B obviously decreased in nuclear localization and increased in cytoplasmic localization following DHE treatment,compared with the control group.Hence,to fully understand the related molecular mechanism of DHE treatment on glioblastoma cells,we next employed U87 and U251 cells to investigate the effects of DHE on IKK?/ I?B?/NF-?B signaling pathway.Western blot results revealed that the levels of p-IKK?/?,p-I?B? and p-p65 protein were markedly down-regulated by treated with DHE in a dose-dependent manner,however,the whole levels of IKK?,IKK?,I?B? and NF-?B p65 did not significantly change during this study.This indicated that DHE might suppress NF-?B activationthrough inhibiting IKK? kinase activity.And then,molecular docking studies predicted that DHE could bind to ATP binding site of IKK?.All of these results supported that IKK? was a target site of DHE in the NF-?B signaling pathway to suppressed COX-2 expression.The mass spectrometry analysis of the CSF samples showed a single peak at a retention time of 1.81 mins.The result suggested that DHE can rapidly cross blood–brain barrier in mouse model.Thus,we established a xenograft nude mice model to further determine the anti-cancer effect of DHE on glioblastoma in vivo.As expected,compared with the control group,both the tumor volume and the tumor weights were dramatically reduced in the treated groups.Therefore,these results may indicate that DHE exhibits strong properties in suppressing the growth of glioblastoma in vivo.Furthermore,the immunohistochemistry results indicated that DHE could suppress the activation of the IKK?/NF-?B/COX-2 pathway in U87 cells transplanted tumors,which might be partially responsible for the inhibition of the xenograft growth.Conclusion: The present study conducted to determine the biological effects of CT and DHE on the glioma,as well as the mechanisms underlying these effects.In our study,the glioblastoma cells were significantly inhibited in their viability,proliferation and migration by CT and DHE treatment.At the meantime,DHE also induced mitochondria-mediated apoptosis by promoting the release of cytochrome c into cytosol,which activating caspase signaling pathway.Furthermore,our results fully demonstrated that DHE significantly suppressed COX-2 expression by inhibiting the phosphorylation of IKK? via targeting the ATP-binding site,thereby abrogating NF-?B binding and p300 recruitment to COX-2 promoter.Moreover,the current study firstly demonstrated that DHE can cross blood–brain barrier.In addition,treatment with DHE markedly inhibited neoplastic weight and volume in the xenograft nude mice model,and these effects may be mediated through inhibition of the IKK?/NF-?B/COX-2 signaling pathway.