Vitamin D3 Inhibits The Proliferation Of Bladder Cancer Cells By Inducing Autophagy

Background and ObjectiveWorldwide,bladder cancer is one of the common malignant tumors of human,which is the eleventh of malignant tumor.It is the seventh that happened in male patients,while belowthe tenth in female patient s.Similarly,in our country,the incidence of male bladder cancer is higher,ranked seventh,while women in the tenth.There are many kinds of pathological types in bladder cancer,among which the transitional cell carcinoma is more common,accounting for about 90%.With the progress of diagnostic techniques,more and more early clinical diagnoses of bladder cancer come true.The traditional treatments of bladder cancer are surgery,chemotherapy,radiotherapy and immunotherapy,which effects are satisfactory.But all these treatments are invasive and invasive.Although with the atraumatic and prominent curative effect,BCG,one of the treatments,has no significant effect on low-risk bladder cancer.Thus,in order to cure all kinds of bladder cancers satisfactoryly,we must get a non-invasive and widely adapted treatment method for patients.in 1922.McCollum confirmed cod liver oil contains a vitamin and named it"vitamin D",which can cure rickets.Vitamin D3,a fat soluble vitamin,is the main component of vitamin D.Calcitriol(1,25-(OH)2D3),as the main active form of vitamin D3 in body,regulates calcium and phosphorus metabolism in the body and maintain the physiological balance.However,in recent years,with the in-depth study of Calcitriol,biological functions of regulate cell proliferation,differentiation and apoptosis in cancer cells were foun.In addition,it has been reported that calcitriol can also inhibit the proliferation of bladder cancer cells and promote their apoptosis.As the precursor of 1,25-(OH)2D3,does vitamin D3(C27H440)have similar biological functions for bladder cancer cells?And by which biological pathways does vitamin D3 play these biological function?Autophagy is a device/package in eukaryotic cell cytoplasm and cell membrane of the protein degradation of domestic autophagosome formation,and the formation of inclusions and inclusions called autophagy,and finally fuse with lysosomes to form autolysosome,the contents of the package degradation,in order to achieve a steady state cell and organelle update.Autophagy is highly conserved in evolution,pathological and physiological processes occur widely in eukaryotic cells,is one of the programmed cell death,but also self protective mechanism of cells.The research shows that autophagy plays an important role in the nervous system disease,immunity,tumor and apoptosis and so on.There are many kinds of research methods of autophagy,while there are many methods to confirm it.Method of directly observing the autophagosome structure by electronic microscope is the gold standard.But method of detecting the autophagy marker protein LC3 II is relatively simple.This paper is to research the effect of vitamin D3(C27H44O)on bladder cancer cells and its relationship with autophagy.Method1.The concentration of vitamin D3(IC50)assayEJ cells were cultured in 10%fetal bovine serum RPMI 1640 complete medium,5%CO2,37?.In the logarithmic growth phase,adjust the cell concentration of EJ was good for 4×104 per hole were seeded in 96 well plates.Dissoluted by DMSO(Dimethyl sulfoxide),vitamin D3 was diluted as the concentration of 1,1.5,2,2.5,3 mmol/ml respectively and incubated with EJ for 24 hours and 48 hours in complete culture.Meanwhile,we must set up the control group with DMSO only.Microplate determinate the absorption value of each hole that incubated with 10%CCK-8 liquid in serum-free RPMI 1640 culture for 2 hours at 450 nm wavelength.The following formula is used to calculate the cell inhibition rate:Inhibition/%=(ACON-ATRE)/(ACON)*100%.The calculation(SPSS)shows the IC50 of EJ cells.Then,the final concentration of vitamin D3 in the following treament group was estimated by the value of IC50,while the control group was cultured with the same volume of DMSO.2.Cell migration assayCell migration was assayed using transwell chambers(BD Biosciences,San Jose,CA,USA).In both transwell assays,cells,after 24 hours' corresponding treatment,were trypsinized and seeded in chambers at the density of 5 x 104 cells per well,and 500 ?l of 10%FBS RPMI-1640 medium was added to the lower chamber.Migrated cells were fixed with 4%paraformaldehyde methanol for 30 min,and non-migrated Cells were removed by cotton swabs.Finally,cells on the bottom surface of the membrane were stained with hematoxylin for 30 min.Optical microscope(200X,Olympus,Tokyo,Japan)was used to count the number of cells in five random fields of view;mean cell number was calculated for each group.3.Cell cycle distribution analysisCells' DNA was stained with the Cell Cycle Detection Kit(KeyGen,Nanjing,China)according to the manufacturer's protocols.Briefly,70%ethanol was used to fix 1 ×106 cells,which were incubated with VD3 for 24h,overnight with at 4?.After the cells were washed twice with phosphate buffered saline(PBS),cells were incubated with 100?g/ml RNase A 30min at 37?.Then stained the cells using 50?g/ml propidium iodide(PI)under the conditions of protection from light.Results were analyzed with Modfit LT software(Verity Software House,Topsham,ME,USA).4.In vitro cell growth assay and colony-formation assayCells were seeded with 100 ?l medium in a 96-well plate(800/well).Cell proliferation was evaluated by the Cell Counting Kit-8(CCK-8,DOJINDO,USA)according to manufacturer's protocols.Briefly,each well was incubated with the mixture of CCK-8 solution and empty culture medium(10?l CCK-8 solution and 90?l culture medium)for 2h in 5%CO2 at 37?.Then,the absorbance at 450 nm was measured.The cell proliferation assay was performed on days 1,2,3,4 and 5.For colony-formation assay,cells were cultured for 12 days in a 6-well plate which was seeded with 500 cells.Cells colonies were washed with PBS twice and fixed with 4%paraformaldehyde and stained with haematoxylin for 30 min.Then counted the number of clonies which containing more than 50 cells under a microscope.All experiments were repeated for at least three times.Cells were seeded in a 6-well plate and taken cell morphology photos under a microscope in 0h,24h,48h and 72h.5.The expression of LC3 was detectedby Western BlotCells were incubated with VD3 at 37? for 3h,6h.l2h and 24 h and then incubated in ice-cold RIPA buffer[1 M Tris(pH 7.4),5 M NaCl,0.5 M EDTA(pH 8.0),10%SDS,10%DOS,and 10%NP40]with fresh protease inhibitor PMSF over ice for 30 min.The cells were scraped and the lysate was collected in Eppendorff tubes and centrifugated at 10,000 rpm at 4? for 30 min,and the liquid supernatants were collected,aliquoted,and stored at-80? for future use.Proteins(30 ?g)were loaded in 12%SDS-PAGE gels and transferred onto a polyvinylidene difluoride(PVDF)membrane.The membranes were soaked in blocking buffer(5%skimmed milk)under room temperature for 1 h.The blots were washed with PBST.To probe for LC3 and GAPDH(all obtained from Proteintech,China),the membranes were incubated overnight under 4? with relevant antibodies,followed by appropriate HRP conjugated secondary antibodies and ECL detection.Gel Documentation set(TANON,Shanghai,China)was used for picture capture.6.The expression of LC3 was observed by confocal fluorescence microscopy immunofluorescenceFor immunofluorescence staining of talin and vinculin,the cells on the samples were fixed in 4%paraformaldehyde in PBS for 5 minutes(pH 7.4)on day 3 of the culture and day 7 of the culture for the other proteins.Then,the samples were washed three times in PBS(5 minutes),pretreated with 1%bovine serum albumin in PBS containing 0.1%Triton X-100(1 hour),and then incubated in 1%Tween 20 for 20 minutes.After a 5-minute wash in PBS,the primary antibodies,mouse monoclonal anti-LC3(dilution 1:200)was applied overnight at 4?.As the secondary antibodies,Alexa Fluor 488-conjugated F(ab')fragment of goat antimouse Gig(H+L)was used for 1hour at room temperature.A solution of 0.05%Tween 20 in PBS was used to wash the samples.The cell nuclei were counterstained with DAPI for 10 minutes,wash them.Then,an epifluorescence microscope(IX51,DP70 digital camera;Olympus)was used for picture capture7.Statistical analysisStatistical analyses were performed using SPSS20.0 software package.Quantitatives values of all experiments were expressed as the mean ±standard deviation(SD).Differences between variables were assessed by independent t test.Different groups of cell proliferation ability by CCK-8 were analyzed by the use of factorial design analysis of variance.Differences are considered statistically significant at P<0.05.Results1.Concentration of vitamin D3 in the treatment groupIn this experiment,result shows a concentration dependent inhibition of EJ proliferation when incubated with vitamin D3.According to the regression curve corresponding of inhibition rate,IC50 of vitamin D3 was calculated as 0.919-1.436 mmol/ml,and its estimated value was 1.226 mmol/ml;experimental group and control group have significant difference,P<0.05.2.Effect of vitamin D3 on the migration of bladder cancer cellsThere were no significant difference between the effects of vitamin D3 on treatment group and control group of bladder cancer cell EJ and UMUC-3,P>0.053.Effect of vitamin D3 on the cell cycle of bladder cancerThe role of vitamin D3 in bladder cancer Cells(EJ,UMUC-3),cells in S phase and G2/M phase(experimental group)were more than those in the control group,while cells in G1 phase(experimental group)were less than that of the control group.The experimental group and the control group was statistically significant P<0.054.Effect of vitamin D3 on the proliferation ability of bladder cancerMore or equal cells can be seen at 0 hours in the experimental group than the control group.At 24 hours,cells in the experimental group began less than that of control group.At 48 and 72 hours,the difference become more outstanding.Clone law can be seen in the experimental group were significant less than that in control group(P<0.05).All the methods confirmed that vitamin D3 can obviously inhibit the proliferation of bladder cancer cells.The experimental group and the control group has significant difference,P<0.05.5.The relationship between vitamin D3 and LC3The results of Western blot experiment showed the expressions of the LC3 II/1 were higher than those in the control group,expression LC3 II was increased.Obvious green fluorescence dot aggregates were observed in the experimental group by confocal fluorescence microscopy,while the control group not.Conclusion1.The experimental concentration(IC50)of vitamin D3 that works on EJ cells is 0.919-1.436 mmol/ml,and its estimated value is 1.226 mmol/ml.2.Vitamin D3 had no significant effect on the migration ability of bladder cancer cells(EJ,UMUC-3)while its concentration is the estimated value of IC503.Vitamin D3 arrest bladder cancer cell cycle at S phase and G2/M phase to inhibit the proliferation of bladder cancer cells;4.Vitamin D3 can negativelyregulate the proliferation of bladder cancer cells by inducing autophagy.