Antitumor Effects Of Dioscin In A431 Cells Via Adjusting ATM/p53 Mediated Cell Apoptosis,DNA Damage And Migration

Objective:To study the pharmacological effects of diosgenin on skin cancer and its possible molecular mechanism.1.In vivo,the 4-6 weeks'nude mice were fed for a week,then inoculated with A431 cancer cells in vivo.The cancer cell lines of luciferase labeled Luc-A431 with 5×10~7/m L were suspended in 0.5 m L PBS,and subcutaneous injected into mice underarm area,pay close attention to tumor.Then the animals were randomly divided into three groups(n=5)including control group,low-dose group(20 mg/kg),and high-dose group(80 mg/kg).The mice were given dioscin by gavage for 26 days and the volume was calculated.After the last administration,live animal imaging experiments were performed.In the experiment,the control group and the drug administration group(80mg/kg)were injected with luciferin potassium salt solution,Isoflurane inhalation anesthesia was immediately performed and placed on the black box platform.The imaging background was adjusted and waited for 5minutes until the fluorescence intensity in the nude mice reached its peak,then took photos,and completed the imaging operation,and finally calculated the luminous area of the imaging images.The tumor tissue was embedded in paraffin and cut into 5?m sections by a microtome.Dewaxing,hematoxylin and eosin staining,sealing and microscopic examination were performed to observe the damage of the tumor tissue.In immunohistochemical analysis experiments,using immunohistochemical ldpe-g-nvp kits for testing,through the dewaxing and hydration,antigen repair,to block endogenous peroxidase,closed with normal goat serum work drops,fostering a resistance,marked biotin drops of polymers,goat anti rabbit Ig G marked horseradish enzymes work drops and DAB chromogenic,redyeing,dehydration step,and finally took photos.In the immunofluorescence assay,formalin-fixed tumor tissue was embedded in paraffin and cut into 5?m sections.Tissue sections coated with p53 antibodies were placed in a light-proof wet box at 4°C overnight,followed by fluorescent labeled secondary antibodies and incubated at 37°C for 1 hour.The nuclei were stained with DAPI(5.0 g/m L).The immunofluorescence samples were randomly selected and photographed at 5locations by immunofluorescence microscope,and the image results were analyzed.Finally,western blot were used to investigate the effects of diosgenin on downstream regulation of cell migration,apoptosis,and DNA damage signaling pathway related proteins RHO,CDC42,MMP2,MMP9,Cleaved caspase-3/9,Bax,and Bcl-2 expression.Further cell p53 si RNA transfection experiments were conducted to investigate the effects of diosgenin on apoptosis,migration and invasion of cells and related downstream proteins after the down-regulated expression of target protein p53.2.In vitro,MTT assay was used to detect the cytotoxic effect of dioscin on skin cancer cell A431.After treated with dioscin at different concentrations(0.7,1.4,2.9,5.8 and 11.6 M)for different time(12,24 and 36h),the A431 cells were mixed with MTT(10mg/ml)10?l,and incubated at 37°C for 4h,the supernatant was abandoned and 150 ml DMSO was added to dissolve formazan,then OD value was determined by a microplate reader.Meantime,the morphological changes of cells were observed by the white light experiment under the condition of open field.In the colony formation experiment,A431 cells at logarithmic growth stage were inoculated in 6-well plates at a certain density.The treatment group was treated with dioscin of different concentrations at 2.9,5.8 and 11.6?M separately every 3 days,and the blank group was replaced with fresh medium every 3 days.After 2 to 3 weeks,the supernatant was discarded,fixed with 4%paraformaldehyde and stained with 1%crystal violet solution at room temperature.At last,each group was photographed and the colony formation rate was calculated.In TUENL experiment,different concentrations of dioscin(1.4,2.9,5.8?M)were given,and after overnight incubation,20?l of 1.0?g/m L AO and 1.0?g/m L EB dissolved in PBS were added,and the apoptosis rates of the samples were determined using microscope.In comet assay,logarithmically grown A431 cells were collected and given different concentrations of dioscin(2.9,5.8,and 11.6?M)for 24 h,following the steps Comet electrophoresis detection kits for production,spread glue,cell lysis and electrophoresis steps was used.At last,the fluorescence analysis software was conducted after vision Comet assay software project(CASP)analyzed.In molecular mechanism investigation of dioscin on apoptosis and DNA damage,the total cellular protein was extracted with RIPA lysis buffer containing 1%PMSF by Western blot.Instructions of the protein extraction kit for protein extraction.The BCA protein analysis kit was used to measure the protein concentration.Protein was separated by 10-12%SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane.PVDF membrane was sealed,incubated with specific primary antibody,washed with TBST for three times,and incubated with horseradish peroxidase(HRP)-labeled secondary antibody.After incubation,ECL color development was carried out with enhanced chemilescence substrate detection kit.Image J 5.0software was used for quantitative analysis of protein expression levels.Finally,the effects of dioscin on the expression of ATM,P53,PARP Cleaved caspase-3/9,Bax,and Bcl-2 in downstream regulation of cell migration,apoptosis,and DNA damage signaling pathways were investigated.In immunofluorescence assay,A431 cells at logarithmic growth stage were treated with dioscin(1.4,2.9,5.8?M)for 24h.After washing with PBS,the membranes were fixed at room temperature with 4%formaldehyde,and permeated with 0.2%Triton-X100 at room temperature.The cells were then sealed in 2%skimmed milk solution,added with primary antibody and incubated at 4?overnight.The next day,after washing with PBS twice,the secondary anti-fitc-Conjugated Goat Anti-Rabbit Ig G(1:100)was added and incubated at room temperature and in dark for 1h.Lastly,DAPI(1.0 g/m L)was dyed for15min,washed with PBS,and observed and photographed with fluorescence microscope.In vitro transfection experiment of p53si RNA,p53si RNA and control si RNA were respectively dissolved and then mixed with Lipofectamin 2000 reagent to form liposome inhibitor complex.After transfection for 24 h,cell viability,apoptosis,DNA damage,and protein expression levels of p53,PARP,Cleaved caspase-3/9,Bax,Bcl-2,were detected.3.In the study of migration and invasion of skin cancer A431 cells by dioscin,the effects of dioscin on cell invasion were determined by scratch and Transwell migration experiments.In scratch assay,the cells were wounded with a sterile pipette tip on the cell monolayers and washed with serum-free medium to remove detached cells.Then giving different concentrations of dioscin(2.9,5.8 and 11.6?l)treatment for a night,4%paraformaldehyde fixed and observed under microscope,the ability of cells migration was determined by changes in scratch width.In Transwell invasion experiment,after maintaining A431 cells under serum starvation conditions for 24h,the cells were digested and loaded onto the top of a24-well migration chamber in 200?l serum-free medium containing different low concentrations of dioscin(2.9,5.8 and 11.6?M),and the 500?l medium containing 10%FBS was added to the lower chamber for 24 h culturing.lastly,the cells that had migrated into the lower surface of the filter were fixed with 10%formaldehyde and stained with hematoxylin for 30 min.In molecular mechanism investigation of dioscin on cell migration and invasion,the total cellular protein was extracted with RIPA lysis buffer containing 1%PMSF by Western blot.Instructions of the protein extraction kit for protein extraction.The BCA protein analysis kit was used to measure the protein concentration.Protein was separated by 10-12%SDS-polyacrylamide gel electrophoresis and transferred to PVDF membrane.PVDF membrane was sealed,incubated with specific primary antibody,washed with TBST for three times,and incubated with horseradish peroxidase(HRP)-labeled secondary antibody.After incubation,ECL color development was carried out with enhanced chemilescence substrate detection kit.Image J 5.0 software was used for quantitative analysis of protein expression levels.Finally,the effects of dioscin on the expression of RHO,CDC42,MMP2,MMP9 in downstream regulation of cell migration signaling pathways were investigated.In vitro transfection experiment of p53si RNA,p53si RNA and control si RNA were respectively dissolved and then mixed with Lipofectamin 2000 reagent to form liposome inhibitor complex.After transfection for 24 h,cell viability,cell proliferation,migration and invasion ability,apoptosis degree,DNA damage degree,and protein expression levels of RHO,and CDC42 were detected.Results:1.In vivo results showed that,dioscin significantly inhibited tumor growth and 80 mg/kg dioscin notably decreased tumor weight by 73.3%and tumor volume by 80.4%.In addition,immunofluorescence results showed that dioscin significantly increased the expression of target protein P-ATM,p53,PARP and cleaved caspase-3,while Western blot showed that dioscin significantly increased the expression levels of cleaved caspase-9 and Bax,and significantly decreased the expression levels of RHO,CDC42,MMP2,MMP9 and Bcl2.2.The results of MTT in vitro showed that compared with the control group,dioscin treatment significantly reduced the activity of skin cancer cell line A431.Colony formation test proved that dioscin could inhibit cell proliferation.Comet assay and TUNEL experiment showed that dioscin could induce apoptosis of A431 cells and DNA damage.The results of molecular mechanism experiments in vitro showed that dioscin regulated the expression of p53,caspase and other apoptosis-related proteins in A431 cells by regulating ATM/p53pathway,and induced the apoptosis of tumor cells.Moreover,p53si RNA could inhibit the apoptosis of A431 cells,and the regulatory effect of p53si RNA on cells could be reversed after dioscin administration.3.The scratch test and Transwell test migration and invasion.The results of molecular mechanism experiments in vitro showed that dioscin regulated the expression of p53,caspase and other apoptosis-related proteins in A431 cells by regulating ATM/p53 pathway,and and inhibit the migration and invasion of tumor cells.Meanwhile,dioscin can regulate the expression of Rho,CDC42 and other migration-related proteins in A431 cells by regulating ATM/P53 pathway.Moreover,p53si RNA could inhibit the apoptosis of A431 cells and the migration and invasion of A431 cells,and the regulatory effect of p53si RNA on cells could be reversed after dioscin administration.Conclusion:1.Dioscin significantly inhibited the tumor growth of A431 cells in tumor-bearing nude mice.Dioscin regulates the expression of p53,caspase and other apoptosis-related proteins in A431cells by regulating ATM/p53 pathway,and induces apoptosis of cells in tumor tissues.By regulating the expression of Rho,CDC42 and other migration-related proteins,the migration and invasion of cells in tumor tissues were inhibited.2.Dioscin could significantly promote apoptosis and DNA damage in skin cancer A431 cells in a dose-dependent manner.Furthermore,this effect was achieved by regulating the expression of apoptosis and DNA damage related proteins ATM,p53,PARP,Cleaved caspase-3/9,Bax,and Bcl-2.3.Dioscin could significantly inhibit the migration and invasion of skin cancer A431 cells in a dose-dependent manner.This effect is achieved by regulating the expression of migration and invasion-related proteins Rho,CDC42,MMP2 and MMP9.In summary,dioscin has a good anti-skin cancer effect,which is exerted by up-regulating the expression of P-ATM and p53,and then regulating the signaling pathways of cell apoptosis,cell migration and DNA damage.In a word,dioscin has a great research and development value in anti-skin cancer.