The Role And Mechanism Of Vitamin D In Enhancing Radiosensitivity Of Colorectal Cancer And Cervical Cancer

1.Vitamin D enhances radiosensitivity of colorectal cancer by reversing epithelial-mesenchymal transitionObjective: Colorectal cancer(CRC)ranks fourth in mortality worldwide,and the main cause of death derives from tumor metastasis and therapy resistance.Radiotherapy(RT)is an important adjuvant treatment,screening potential radiosensitizers and studying underlying molecular mechanism seem to be very crucial for improving the radiotherapy efficacy in CRC.Previous studies have found that vitamin D is closely related to cancer risk in CRC,but its role in RT is still unclear.This study aims to confirm whether vitamin D exerts radiosensitization effect on CRC and verify the potential mechanism.Methods:1.Validate radiosensitivity of vitamin D in vitro.We used CCK8,cell viability and colony formation to prove the influence on cell proliferation with vitamin D in combination with radiation.2.Phenotype validation of radiosensitivity induced by vitamin D.Flow cytometry(FCM)was detected to observe the effect on apoptosis and cell cycle distribution induced by vitamin D and radiation,?-galactosidase staining was determined to assay cell senescence;immunofluorescence(IF)and western bot(WB)were detected to visualize the ?H2AX foci formation(DNA damage marker)and LC3 puncta(autophagy marker),accompanied with protein expression,and transmission electron microscopy(TEM)was further used to observe autophagosome formation;we detected wound healing(WH)under different conditions,and transwell migration and invasion were used to observe cell migratory and invasive capability,as well as epithelial-mesenchymal transition(EMT)markers determined by WB.3.Mechanism validation of radiosensitivity induced by vitamin D.To unveil the dominant phenotype,we used label free proteomics to detect the differentially expressed proteins(DEPs)of cell lines with different treatments.Based on the enrichment and cluster of DEPs,we confirmed the dominant phenotype and target genes.Related phenotype and target genes were assessed to verify the proteomics data,and we observed the alteration of colony formation and related phenotype in cells treated with si RNAs.4.Validate radiosensitivity of vitamin D in vivo.A xenograft mouse model of CRC was used to investigate the synergistic antitumor effects of vitamin D and radiation,and immunohistochemistry(IHC)was determined to detect target protein expression and evaluate toxic effects of mice organs.Results:1.Vitamin D promoted CRC proliferation inhibition induced by radiation.2.Phenotype validation.1)Apoptosis rate was enhanced with vitamin D and radiation treatment,as well as upregulation of pro-apoptosis proteins.2)Combination treatment had no significant influence on cell cycle distribution and cell senescence.3)It was radiation that induced obvious ?H2AX foci but not vitamin D,and the result of WB was in accordance with IF.4)Vitamin D induced elevation of autophagy in combination with radiation,including upregulation of LC3-II,downregulation of p62 and these were further evidenced with chloroquine(CQ,autophagy inhibitor).Meanwhile,more autophagosomes were observed with TEM.5)WH rate,migration and invasion were all remarkably inhibited with combination treatment,accompanied with upregulation of epithelial markers like E-cadherin and Claudin-1 and downregulation of mesenchymal marker Snail.3.Mechanism validation.Analysis of GO and KEGG indicated EMT term was significantly enriched by DEPs,cystatin D and plasminogen activator inhibitor-1(PAI-1)were potential candidate DEPs,which were closely linked with EMT.Next,cystatin D and PAI-1 determined by WB were in parallel with proteomics,and interference with si RNAs reversed the inhibition of cell proliferation and EMT induced by combination treatment.4.In accordance with the results in vitro,treatment with vitamin D and RT significantly suppressed tumor growth in the xenograft CRC model,so as to the expression of cystatin D and PAI-1 detected by IHC and WB.HE staining of mice organs showed no significance in morphology among different groups.Conclusion:Vitamin D exerted a synergistic antitumor effect with RT in vitro and in vivo,and cystatin D and PAI-1 were involved in this additive antiproliferative effect,which was attributed to inhibition of EMT.Vitamin D will be a promising radiosensitizer of CRC in future clinical application,which also seemed to make a contribution to the control of tumor migration and invasion.2.The role and mechanism of vitamin D in enhancing radiosensitivity of cervical cancerObjective: Cervical cancer is the fourth most common female malignant tumor in the world.As the major treatment,radiotherapy(RT)is effective in most patients,but there are still some patients with poor prognosis because of intrinsic resistance or acquired resistance after several rounds of RT.Several studies have reported that apoptosis,autophagy and senescence were all involved in radiation resistance.Therefore,based on the molecular mechanism of radioresistance,exploring potential radiosensitizers will aid in enhancing the RT response in cervical cancer.Previous studies have suggested that vitamin D played an antitumor role in a variety of cancers such as colorectal cancer,breast cancer and prostate cancer.Accumulating evidence indicate that vitamin D can interfere with multiple steps in tumorigenesis,from tumor initiation to metastasis,including regulation of cell behavior like proliferation,differentiation,apoptosis,autophagy and EMT,as well as the interaction between tumor cells and surrounding microenvironment,such as inflammation,immune response and cancer cell stemness.However,the role of vitamin D in cervical cancer has not been clarified,so this study is carried out to unveil the role of vitamin D in radiosensitization of cervical cancer and the underlying mechanism.Methods: Firstly,the correlation between cervical cancer and vitamin D receptor(VDR)was obtained and analyzed by GEPIA and Oncomine databases,and the expression of VDR was verified in cervical cancer cell lines.The effect of vitamin D in combination with radiation in vitro was detected by CCK8 and colony formation.Meanwhile,cervical cancer xenograft model was used to verify whether vitamin D can potentiate the antiproliferative effect of RT in vivo.Next,proteomics sequencing of treated cell lines were detected to reveal the underlying radiosensitization mechanism of vitamin D,and the results of proteomics were further verified in vitro to explore the intricate interaction among different molecular mechanisms.Results: The results indicated that there was significant difference in the expression of VDR between tumor and adjacent normal tissues,and VDR was expressed in Siha and Caski.Vitamin D can enhance the radiosensitivity of cervical cancer in vitro and in vivo,with no additional toxicity.In mechanism,the results of proteomics supported the role of apoptosis and autophagy regulated by combination treatment,with promotion of apoptosis and inhibition of autophagy.Among the DEPs,there were significant differences in the expression of caspase 8 and Ambra1,with upregulation of caspase 8 and downregulation of Ambra1.The regulation of apoptosis and autophagy induced by combination treatment was further validated in vitro.In cancer,the mechanism of tumor cell death is much more complicated.As a single treatment or combination treatment,RT can simultaneously induce priming of apoptosis and autophagy.As a result,the phenomenon finally observed can not simply be attributed to a single form of cell death,and this "mixed" form of cell death of this study may derive from the crosstalk between apoptosis and autophagy.Conclusion: In summary,our study suggested that vitamin D acted synergistically with RT in cervical cancer,crosstalk between apoptosis and autophagy were involved in the potential mechanism.