| BackgroundBreast cancer,which seriously threatens the health of women,is the most common malignant tumor.The current clinical treatments are still based on surgery,radiotherapy,and chemotherapy.The aggressive and metastatic nature of the disease,especially in triple-negative breast cancer,which is insensitive to therapeutic drugs,rapid drug resistance,and easy to relapse,all of which force us to develop new drugs and find new therapeutic methods urgently.The growth of tumors is inseparable from the tumor hypoxic microenvironment,which promotes tumor invasion and metastasis.On one hand,tumor cells in the hypoxic can form tubular structures,communicate with endothelial cells,and form Vasculogenic mimicry(VM)to provide effective nutrition for tumors and promote tumor growth,metastasis,and drug resistance.On the other hand,tumor hypoxic microenvironment can promote epithelial-mesenchymal transition(EMT),which is an early step for tumors to acquire an invasive phenotype.EMT is induced by various transcription factors such as Zinc finger E-box binding homeobox 1(ZEB1).Hypoxia acts as a "start switch" in the above processes,which inhibits the activity of Prolyl hydroxylases 2(PHD2)and the degradation of hypoxia-inducible factor-1α(HIF-1α).The degradation of HIF-1α is reduced,resulting in increasing HIF-1α entry nucleus,forming a heterodimer with HIF-1β and active the hypoxia signaling pathway,thereby increasing the invasion and migration of tumors.PHD2 is an α-KG-dependent dioxygenase,and α-KG plays an important role in maintaining the activity of PHD2.αKG is synthesized in vivo by isocitrate dehydrogenase(IDH).IDH has three subtypes:IDH1,IDH2 and IDH3.At present,IDH1 and IDH2 gene mutations or changes in expression levels have been found in a variety of tumor tissues.However,there are few studies on the role of IDH1 in breast cancer.Especially in the hypoxic microenvironment,the study on IDH1 affects the invasion,metastasis and VM formation of breast cancer through PHD2 has not been reported yet.Icaritin is an active monomeric icariin,which extracted,isolated,and purified from the plant Epimedium.As an original class I new drug independently developed by our country,it has been approved for marketing in the treatment of advanced hepatocellular carcinoma.As a small molecule immunomodulator derived from traditional Chinese medicine,Icaritin can exert anti-tumor effects by anti-inflammation,regulating immune,and improving tumor microenvironment.In addition to hepatocellular carcinoma,Icaritin also has a certain inhibitory effect on melanoma and renal cell carcinoma.However,there are few studies on the treatment of breast cancer with Icaritin.There is no report on the invasion,metastasis and VM formation of breast cancer with Icaritin in hypoxic microenvironment and whether it is related to the IDH1/PHD2 pathway.Above all,this research will explore the following studies in vitro and in vivo:1.The effects and regulatory mechanisms of IDH1/PHD2 on breast cancer invasion,metastasis and VM formation in hypoxic microenvironment.2.The effects of Icaritin on the invasion,metastasis and VM formation of breast cancer in hypoxic microenvironment and whether the mechanism of action is related to IDH1/PHD2 pathways.Methods and Results1.Regulation of IDH1/PHD2 on breast cancer invasion,migration and vasculogenic mimicry in hypoxic microenvironment1.1 Bioinformatics AnalysisThe TCGA public database was used to analyze the relationship between the expression of IDH1 protein and the survival of breast cancer patients.The GEO database was used to analyze the expression of IDH1 protein between normal breast cells and breast cancer cells.The results found that the expression of IDH1 protein in different stages of breast cancer patients was different,and the expression of IDH1 protein in breast cancer tissues and normal breast tissues were different.1.2 Hypoxia modelThe hypoxia model in vitro was constructed by adding Na2S2O4,and the concentration with less effect on cell proliferation was screened by MTT assay.Western blotting was used to detect the expression of HIF-1α protein,and 1 mmol Na2S2O4 was selected to treat the cells for 24 h to construct hypoxia models of MDA-MB-231 cells and MCF-7 cells.1.3 Expression of IDH1 in different breast cancer cellsWestern blotting was used to detect the difference of IDH1 protein expression between MDA-MB-231 cells and MCF-7 cells.It was found that IDH1 protein expression was lower in highly invasive MDA-MB-231 cells and higher in low invasive MCF-7 cells.Therefore,in subsequent experiments,plasmid with IDH1 overexpression was used to transfect the MDA-MB-231 cells,and small interfering RNA(siRNA)of IDH1 was used to transfect MCF-7 cells.1.4 Effects of IDH1 overexpression on MDA-MB-231 cells in hypoxic microenvironmentMDA-MB-231 cells were transfected with IDH1 overexpression plasmid.Clone formation assay and Transwell assay results showed that IDH1 overexpression had no effect on the proliferation of MDA-MB-231 cells under hypoxia.Significantly,IDH1 overexpression inhibited both the invasion and migration of MDA-MB-231 cells.By examining the effect of IDH1 overexpression on the VM formation of MDA-MB-231 cells under hypoxia,it was found that IDH1 overexpression significantly inhibited the VM formation in MDA-MB-231 cells.Next,Western blotting was used to detect the expression of PI3K,VE-Cadherin,MMP-2,p-AKT,AKT,E-Cadherin,N-Cadherin,ZEB1,PHD2,and HIF-1α under hypoxia.It was found that overexpression of IDH1 inhibited the protein expression of ZEB1,N-Cadherin,PI3K,p-Akt,VE-Cadherin,MMP-2,and HIF-1α,and increased the protein expression of E-Cadherin and PHD2.Next,to further explore the effect of IDH1 overexpression on the PHD2-HIF-1α related pathway,the PHD2 inhibitor IOX4 and the substrate α-KG were used.The results showed that IOX4 could reverse the effect of IDH1 overexpression on the migration of MDA-MB-231 cells,and α-KG could inhibit the migration of MDA-MB-231 cells,which is like the effect of IDH1 overexpression.Western blotting showed that IOX4 could significantly inhibit the expression of PHD2 and increase the expression of HIF1α,while α-KG could increase the expression of PHD2 and reduce the expression of HIF-1α.1.5 Effects of siRNA of IDH1 on MCF-7 cells in hypoxic microenvironmentMCF-7 cells were transfected with siRNA of IDH1.Clone formation assay and Transwell assay showed that IDH1 interference had no significant effect on the proliferation of MCF-7 cells under hypoxia,but promoted the invasion and migration of MCF-7 cells.Next,Western blotting was used to detect the effects of IDH1 interference on related protein expression of PI3K,VE-Cadherin,MMP-2,p-AKT,AKT,E-Cadherin,N-Cadherin,ZEB1,PHD2,and HIF-1α under hypoxia.The results showed that interference with IDH1 could promote the protein expression of ZEB1,NCadherin,PI3K,p-Akt,VE-Cadherin,MMP-2,and HIF-1α,and reduce the protein expression of E-Cadherin and PHD2.Subsequently,the PHD2 inhibitor IOX4 and PHD2 substrate α-KG were used to further investigate the effect of interfering IDH1 on PHD2-HIF-1α-related pathways.Transwell assay showed that α-KG could reverse the promotion of interfering IDH1 on MCF-7 cell migration,and the addition of IOX4 could promote the migration of MCF-7 cells,which is similar to the effect of interference with IDH1.Western blotting showed that IOX4 significantly inhibited the expression of PHD2 and increased the expression of HIF-1α,while α-KG significantly increased the expression of PHD2 and promoted the degradation of HIF-1α induced by hypoxia.2.The effect and mechanism of Icaritin on breast cancer cells in hypoxic microenvironmentBreast cancer MDA-MB-231 cells,MCF-7 cells were treated with Icaritin for 24 and 48 h.The results of MTT assay showed that Icaritin(5,10,15 μM)with less effect on the proliferation of two cells.The concentration was selected for the subsequent experiments.The effects of Icaritin on the invasion and migration of breast cancer cells were detected by scratch assay and Transwell assay,and the VM formation ability of breast cancer cells was detected by VM formation assay.The results showed that Icaritin could significantly inhibit the invasion,migration and VM formation of breast cancer cells.Next,we investigate whether the effect of Icaritin on breast cancer cells is related to the IDH1/PHD2 pathway.MDA-MB-231 cells were transfected with IDH1 overexpression plasmid under hypoxia,and then treated with Icaritin.The effects on invasion and migration of MDA-MB-231 cells were detected by Transwell assay,and the effects on VM formation ability of MDA-MB-231 cells were detected by VM formation assay.The expression of PI3K,VE-Cadherin,MMP-2,p-AKT,AKT,ECadherin,N-Cadherin,ZEB1,PHD2,and HIF-1α of MDA-MB-231 cells was further detected by Western blotting.The results showed that compared with the Icaritin group,the MDA-MB-231 cells in the Icaritin+IDH1 overexpression group had significantly reduced ability to invade,metastasize,and VM formation.The expression of ZEB1,NCadherin,PI3K,p-Akt,VE-Cadherin,MMP-2,and HIF-1α decreased,and the expression of E-Cadherin and PHD2 increased.Next,MCF-7 cells were transfected with IDH1 siRNA under hypoxia,and then treated with Icaritin.The effect on the invasion and migration of MCF-7 cells were detected by Transwell assay.The expression of related proteins PI3K,VE-Cadherin,MMP-2,p-AKT,AKT,E-Cadherin,N-Cadherin,ZEB1,PHD2,and HIF-1α was further detected by Western blotting.The results showed that compared with the Icaritin group,the invasion and metastasis ability of the Icaritin+si-IDH1 group was enhanced,the expression of ZEB1,N-Cadherin,PI3K,p-Akt,VE-Cadherin,MMP-2,and HIF-1αincreased,and the expression of E-Cadherin and PHD2 decreased.Above results indicated that the mechanism of Icaritin on breast cancer may be through the up-regulation of IDH1 protein expression,increase the expression of PHD2 protein,reduce the accumulation of HIF-1α induced by hypoxia,thereby reducing the expression of VE-Cadherin,N-Cadherin,MMP-2 proteins.3.In vivo19-2 1g female BALB/c-nu nude mice(8 weeks of old)were selected and randomly divided,namely Control group,Vector group,IDH1 overexpression group,Vector+Icaritin group,IDH1 overexpression+Icaritin group,5 in each group.The tail vein was inoculated with untreated,empty plasmid-transfected or IDH1-overexpressing MDA-MB-231-luc-D3H2LN cells respectively.Nude mice administered by gavage with normal saline or Icaritin(10 mg/kg),every 2 days,for 10 consecutive doses.The body weight of nude mice was recorded,and the lung metastasis of breast cancer was observed in vivo fluorescence imaging system.After the nude mice were sacrificed,the lung tissue was removed,and the nodules were observed under microscope.The tumor metastasis of the lung tissue was detected by HE staining,and the VM formation was detected by CD34/PAS double staining.The results showed that there was no significant difference in the body weight of the mice in each group.The luminescence intensity was detected in the lungs of the mice in control group,and there were obvious lung metastatic nodules under the microscope.HE staining revealed a large number of tumor cells,and VM formation was detected by CD34/PAS double staining.Compared with Control group,the luminescence intensity of the nude mice in the Vector group was slightly reduced,but there was no significant difference in lung metastatic nodules,HE staining,and VM formation.Compared with Vector,IDH1 overexpression group and Vector+Icaritin group had weaker luminescence intensity,less metastatic nodules in lung tissue,and less tumor cells by HE staining,making it difficult to find VM formation.Compared with the Vector+Icaritin group,the IDH1 overexpression+Icaritin group had no luminescence,no metastatic nodules were found in the lung tissue,and no tumor cells and VM formation were found.Conclusions1.IDH1 overexpression could inhibit the invasion,migration and VM formation of breast cancer in hypoxic microenvironment,by inhibiting the PI3K/Akt pathway,the expression of PHD2 was up-regulated,which promoted the degradation of HIF-1αinduced by hypoxia,and inhibited the expression of VE-Cadherin,MMP-2 and ZEB1mediated EMT process.2.Icaritin had good inhibitory effects on breast cancer invasion,metastasis and VM formation.The mechanism may be achieved by up-regulating IDH1 expression,inhibiting the PI3K/Akt pathway to increase the expression of PHD2,promoting the degradation of HIF-1α,inhibiting the expression of VE-Cadherin,MMP-2 and ZEB1mediated EMT process.SignificanceThis study explored the effects of IDH1 on the invasion,metastasis and vasculogenic mimicry of breast cancer in hypoxic microenvironment,as well as the effect and mechanism of the compound Icaritin on breast cancer in vitro and in vivo.By affecting the IDH1/PHD2/HIF-1α signaling pathway,it has a better inhibitory effect on the invasion,migration and VM formation in breast cancer,which provides theoretical basis for breast cancer treatment and drug development in the future. |
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