Study On The Effect Of Vitamin D On Ferroptosis In Colorectal Cancer Stem Cells And Underlying Mechanisms

BackgroundColorectal cancer(CRC)is a common malignant tumor with high incidence and mortality rate in the world,it ranks third in the incidence spectrum and second in the death spectrum of malignant tumors in the world currently.Studies have shown that the metastasis,recurrence and drug resistance of CRC are the main causes of poor prognosis or even death.Recent studies showed that CCSCs lead to multi-step and multi-stage characteristics in the pathological process of CRC,including chemotherapy failure and tumor recurrence.Thus targeting CCSCs could improve the therapeutic effectiveness in CRC.CCSCs,only a very small portion in colorectal cancer,but with self-renewal capacity and multi-directional differentiation potential,are considered to be the root cause of the occurrence and development of colorectal cancer.CCSCs have a series of malignant biological behavior,including extremely strong ability of self-renewal,proliferation and differentiation,strong tumorigenesis,transfer potential,treatment tolerance,etc.Ferroptosis is an iron-dependent cell death characterized by lipid peroxidation.Previous studies have found that both mesenchymal stem cells and dedifferentiated cancer cells,which are resistant to apoptosis and conventional cancer therapies,are still sensitive to ferroptosis.It has also been found that cancer stem cells,although no longer sensitive to traditional chemotherapy,can still be induced ferroptosis.These suggest that the induction of ferroptosis in CCSCs has great potential value for the treatment of colorectal cancer.There are several regulatory mechanisms of ferroptosis,the most classic of which is system Xc--GSH-GPX4 pathway.System Xc-transports cystine into cells,and then promotes the production of GSH,who is a powerful reductant and a cofactor of GPX4,finally reduces the lipid peroxidation burden of cells.SLC7A11 is the main functional unit of system Xc-,who is a multi-channel transmembrane protein.Studies have proved that SLC7A11 is highly expressed in many malignant tumors including CRC.However,there are also ferroptosis-independent ways for SLC7A11 to promote tumors.Vitamin D is a fat-soluble steroid hormone.Epidemiological and preclinical studies and more and more large-scale clinical trials have proved that vitamin D can reduce the risk of CRC,improve the survival rate and sensitivity to chemotherapy drugs.Some studies have also suggested that vitamin D can inhibit CCSCs.In a study of human mammary epithelial cells,it was found vitamin D could reduce the intracellular GSH content,subsequently mechanism analysis showed this benefit from reduced expression SLC7A11.The project our team are now working on,bioinformation analysis of ferroptosis in CRC and correlative HCT116 cell line experiment,also suggest that vitamin D can inhibit the expression of SLC7A11.Therefore,we hypothesize that vitamin D might promote the ferroptosis of CCSCs by regulating the expression of SLC7A11.Purpose and significanceThere were few studies on vitamin D and CCSCs in the past,here in our study,vitamin D was firstly associated with CCSCs and ferroptosis,correlative mechanism was also further explored.We study on the biological function and corresponding mechanism of vitamin D on CCSCs in vitro and vivo.It is of great significance to find new molecular targets for the treatment of CRC,leading to improve the therapeutic level.Part I:Effect of vitamin D on ferroptosis,self-renewal and drug resistance of CCSCsPurposeThe effects of vitamin D on ferroptosis,self-renewal and drug resistance of CCSCs were investigated in vitro.Methods1.Separate and enrich CCSCs from primary cells of human colon cancer and identify them through qRT-PCR and flow cytometry analysis.2.Different concentrations(0,50,100nM)1,25-(OH)2D3 interfere CCSCs,the effect of vitamin D on the self-renewal ability of CCSCs was evaluated by sphere formation assay.3.CCK8 and Ki67 staining were used to evaluate the proliferative effect of vitamin D.The contents of ROS,Cys and GSH were detected by kit,and the expression levels of ferroptosis marker genes GPX4,COX2 and SLC7A11 were detected by qRT-PCR and Western blot.The morphology changes of CCSCs were observed by transmission electron microscope.4.CCSCs were divided into control group,1,25-(OH)2D3 intervention group,oxaliplatin group and 1,25-(OH)2D3+oxaliplatin group.Flow cytometry was used to identify the dead and alive cells.Results1.We successfully enriched colonosphere from the primary cells of human colon cancer by serum-free suspension culture system.It was found that CD 133 and CD44,stem-related genes Nanog,Oct4 and Sox2 were all highly expressed in colonosphere cells,which indicated that we successfully enriched CCSCs for follow-up experiments.2.In the group without 1,25-(OH)2D3 intervention,the sphere formation was good and dense;however,the number of sphere in 50nM and 100nM groups decreased.As the concentration of vitamin D increased,the sphere formation gradually decreased.3.Compared with the 0nM group,after 50nM and 100nM 1,25-(OH)2D3 intervention to CCSCs,the cell proliferative ability decreased obviously,and the higher the concentration,the greater inhibition effect.With the increase of vitamin D concentration,intracellular ROS obviously accumulated,and the contents of Cys and GSH obviously decreased.1,25-(OH)2D3 intervention can significantly reduce the level of SLC7A11 and GPX4 in CCSCs,and significantly increase the level of COX2,with statistical significance.On the other hand,transmission electron microscopy showed that CCSCs obtain characteristic manifestations of ferroptosis after 1,25-(OH)2D3 intervention.These results indicate that vitamin D can promote ferroptosis in CCSCs.4.Dead cell ratio of 1,25-(OH)2D3+oxaliplatin group was higher than that in oxaliplatin alone group,with statistical difference,indicating that vitamin D can enhance the sensitivity of CCSCs to oxaliplatin.Conclusions and significanceVitamin D can promote ferroptosis in CCSCs,inhibit self-renewal,and improve the sensitivity to oxaliplatin.Part Ⅱ:Expression of SLC7A11 in colon cancer,and its effect on ferroptosis in CCSCsPurposeTo determine the expression of SLC7A11 in colon cancer tissue and whether it can inhibit ferroptosis in CCSCs.Methods1.qRT-PCR was used to detect the expression level of SLC7A11 in cancer tissues of 20 clinically diagnosed colon adenocarcinoma patients and corresponding normal colon mucosa tissues adjacent to cancer.2.The pLV-puro-SLC7A11 overexpression,pLV-shRNA-puro-SLC7A11,and respective control vector were constructed,then transfected to CCSCs respectively.After stable transfection,the transfection efficiency was detected by qRT-PCR.3.CCK8 and Ki67 staining were used to evaluate the proliferative level of CCSCs in five groups:control,overexpression control,SLC7A11 overexpression,sh-NC and sh-SLC7A11.ROS,Cys and GSH were detected by kit,and the levels of GPX4 and COX2 were detected by qRT-PCR and Western blot.The cell morphology was observed by transmission electron microscope.Results1.qRT-PCR results showed that compared with the adjacent tissues,SLC7A11 expressed higher in colon cancer tissues.2.CCK8 and Ki67 showed over-expression of SLC7A11 could enhance the proliferative activity in CCSCs,while sh-SLC7A11 group decreased.The levels of ROS and COX2 decreased in SLC7A11 overexpression group,but increased in shSLC7A11 group.On the contrary,the levels of Cys,GSH and GPX4 increased significantly in SLC7A11 overexpression group,but decreased significantly in shSLC7A11 group.Transmission electron microscope observation showed that when the expression of SLC7A11 was inhibited,some characteristic manifestations of ferroptosis appeared in CCSCs.Conclusions and significanceSLC7A11 is highly expressed in colon cancer tissue,and it can inhibit ferroptosis in CCSCs.Part Ⅲ:Vitamin D promote ferroptosis in CCSCs by inhibiting SLC7A11PurposeTo explore the mechanism of vitamin D promoting ferroptosis in CCSCs.Methods1.After intervened with different concentrations(0,50,100 nM)1,25-(OH)2D3,the expression of SLC7A11 was detected by qRT-PCR and Western blot in CCSCs,immunofluorescence was also used to detect the expression level and cell localization.2.CCSCs with SLC7A11 overexpression,normal-expression and sh-SLC7A11 were intervened with 1,25-(OH)2D3(0 or 100 nm),finally divided into normal group,VD group,sh-SLC7A11 group,VD+sh-SLC7A11 group,SLC7A11 overexpression group and VD+SLC7A11 overexpression group.The proliferative activity was detected by CCK8 kit.The self-renewal ability was detected by sphere formation assay.Evaluation of ferroptosis:ROS,Cys and GSH were detected by kit,GPX4,COX2 and SLC7A11 were detected by qRT-PCR and Western blot.Results1.The expression of SLC7A11 gradually decreases with the increase of 1,25(OH)2D3,which indicates that vitamin D can inhibit the expression of SLC7A11.2.CCK8 showed that SLC7A11 overexpression could enhance the proliferative ability in CCSCs,but decreased after the joint intervention of VD(P<0.05),which indicated the effect of SLC7A11 overexpression on the cell proliferative ability could be saved by vitamin D.Sphere formation assay showed the same result.3.The levels of Cys,GSH,GPX4 and SLC7A11 increased in SLC7A11 overexpression group,while COX2 and ROS decreased.The levels of Cys,GSH,GPX4 and SLC7A11 descended after the joint intervention of VD,and,the levels of COX2 and ROS rised after the joint intervention of VD,all with statistical differences.The inhibitory effect of SLC7A11 on ferroptosis could be saved by vitamin D.Conclusions and significanceVitamin D promote ferroptosis in CCSCs by inhibiting SLC7A11.Part IV:Study on the effect and mechanism of vitamin D on CCSCs in vivoPurposeExplore the effects of vitamin D and SLC7A11 on tumorigenicity and ferroptosis of CCSCs through subcutaneous tumorigenesis in nude mice.Methods1.The experiment was divided into 6 groups,there are 5 BALB/c nude mice of 46 weeks old in each group,and 106 cells in each group were injected subcutaneously into nude mice.The tumor size was measured every week,and after 5 weeks,tumor tissues were collected,weighed and measured,and then frozen sections or tissues were extracted respectively.2.The levels of ROS,Cys and GSH in tumor tissues were detected by kit in control group,SLC7A11 overexpression group and sh-SLC7A11 group respectively.Results1.Compared with the control group,the tumorigenic volume and weight of nude mice in vitamin D intervention group were significantly reduced.2.Compared with the control group,SLC7A11 overexpression increased the tumorigenic volume and weight,and increased Cys and GSH levels,while decreased ROS.sh-SLC7A11 group showed the opposite trend.3.SLC7A11 can promote the tumorigenesis,but the ability is reduced by the joint intervention of vitamin D.The promotion of SLC7A11 on the tumorigenesis can be saved by vitamin D.Conclusions and significanceVitamin D can inhibit the tumorigenesis of CCSCs,while SLC7A11 can promote tumorigenesis and inhibit ferroptosis in tissues,vitamin D affects tumorigenesis through SLC7A11.Full text summaryIn this study,firstly,the hypothesis that vitamin D promotes ferroptosis in CCSCs by inhibiting SLC7A11 was constructed through previous literature evidence,and then the hypothesis was verified by subsequent experiments in vivo and vitro.Finally,we proved that vitamin D can inhibit the stemness of CCSCs,promote ferroptosis,and reduce the resistance to oxaliplatin.Vitamin D promote ferroptosis of CCSCs and inhibit the stemness of CCSCs by acting on SLC7A11.