Study On The Effect And Mechanism Of Chrysophanol On Breast Cancer Cell Cycle And Apoptosis

Purpose: Breast cancer cell lines MCF-7 and MDA-MB-231 were selected as our study subjects and the effect of chrysophanol on the two cell lines was investigated by different methods.We observed the cell proliferation by MTT assay,cell circle and apoptosis by flow cytometry after cells were treated by chrysophanol.The mRNA level was determined by real time quantitative PCR and related protein expression was measured by western blotting.We also revealed the effect of chrysophanol on the NF-?B pathway,as well as clarified the possible mechanism of how chrysophanol affects breast cancer.Furthermore,we observed how chrysophanol enhanced the sensitivity of paclitaxel on breast cancer.The results suggested that chrysophanol may have therapeutic potentials for the herb treatment in clinical use and offer new clues on drug-resistance difficulties of cancer chemotherapy.Material and method: Breast cancer cell line MCF-7 and MDA-MB-231 were purchased from the American Type Culture Collection?Manassas,VA,USA?and cultured in DMEM?Invitrogen;Thermo Fisher Scientific,Carlsbad,CA,USA?with 10% FBS?Invitrogen;Thermo Fisher Scientific,Carlsbad,CA,USA?and 0.01 mg/ml human recombinant insulin?Cat No,I3536;sigma–Aldrich,St.Louis,MO,USA?.The cultures were maintained at 37 ? in a humidified incubator containing 5%?v/v?CO₂ in air.Cells were seeded at a density of 1×10⁶ cells/ml in 6-well plates.1.MTT assay MCF-7 cells(5×10³/well)were plated in 96-well plates and cultured overnight.MTT solution?volume,20 ?l;concentration,5 mg/ml;Sigma,St.Louis,MO,USA?was added to each well and then incubated for another 4 hours.The supernatant was removed and DMSO?150?l?was added for test preparation.Absorbance was measured at 490 nm.Data were obtained from triplicate wells per condition.2.Flow cytometry for cell cycle and apoptosis analysis Cells in 6-well plates were collected using 0.25% trypsase 24 hours after chrysophanol treatment.Cells were washed twice with PBS butter and then resuspended in 250 ?l ofbinding buffer.Cells were fixed in 1% paraformaldehyde for 24 hours and then stained with 5 mg/ml propidium iodide for cell cycle analysis.Cells were stained with propidium iodide and Annexin V/FITC for cell apoptosis analysis.Stained cells were analyzed by FACS Calibur flow cytometer?Becton Dickinson,USA?after incubation in the dark for 15 minutes.3.Western blot Total protein of MCF-7 cells was extracted using lysis buffer?Pierce,Rockford,IL?and Bradford method was used to quantify the protein.When SDS-PAGE assay was performed,30 ?g of the protein was separated and then transferred to polyvinylidene fluoride membranes?Millipore,Billerica,MA,USA?.For primary antibody incubation,cleaved caspase 3,caspase 3,cleaved PARP,PARP,p-p65,p65,p-I?B,I?B,Bcl-2?1:1000;Cell Signaling Technology,Danvers,MA,USA?,and GAPDH?1:2000;Cell Signaling Technology,Danvers,MA,USA?were incubated overnight at 4?.For secondary antibody incubation,peroxidase-coupled anti-mouse/rabbit IgG?1:1000,Cell Signaling Technology,USA?was incubated at 37? for 2 hours.Sample protein was visualized using ECL?Pierce?and detected using a DNR BioImaging System?DNR,Jerusalem,Israel?.Relative protein levels were quantified using Image software.4.Realtime quantitative PCR Total RNA was isolated from MCF-7 cells using TRIzol reagent?Life technology,St.Louis,MO,USA?following the manufacturer's instructions.Then,reverse transcription of total RNA into cDNA was performed using the Reverse Transcription System?A3500;Promega,Madison,WI,USA?and PrimerScript RT Master Mix kit?TakaRa,Dalian,China?.Briefly,a total 20?l of reverse-transcription reaction solution was prepared containing 4?l of 5X RT Master Mix and 800 ng RNA and the mixture was reacted at 85? for 2 min and 37? for 30 min.PCR was performed using 7500 Realtime PCR System?Applied Biosystems?and SYBR Green master mix kit?Applied Biosystems?.The relative expression of target genes were calculated as ?Ct=Ct gene –Ct reference,and the fold change of target gene expression was calculated by the 2^-??Ct method.GAPDH was used as the reference gene.Experiments were repeated in triplicate.Statistical analyses were performed using SPSS version 19.0 for Windows.The ANOVA was used to compare differences among control and treatment groups.P<0.05 was consideredto indicate statistical significance.Results:1.Chrysophanol inhibits breast cancer cell proliferationMCF-7 and MDA-MB-231 cells were treated with chrysophanol by different concentration in different time.MTT assay results showed proliferation rates of MCF-7 and MDA-MB-231 cells were decreased significantly when treated with chrysophanol in a concentration-dependent manner.When 5?M chrysophanol was used within 12 h,cell proliferation rate showed no significant change.And when chrysophanol concentration increased to 10 and 20?M within 24 h,proliferation rates were decreased significantly.When chrysophanol concentration ?40?M or time ?36h,proliferation rate level was lower than 50%.2.Chrysophanol inhibits breast cancer cell cycle progression.Cell cycle analysis in MCF-7 and MDA-MB-231 showed that G1 percentage increased while S percentage decreased after chrysophanol treatment but G2 percentage had no significant change.Results proved chrysophanol can respress breast cancer cell cycle progression in G1-S phases.3.Chrysophanol regultes protein and mRNA levels of cell cycle related genesWestern blot results showed that chrysophanol exposure dramatically inhibited expression of cyclin D1,cyclin E while upregulated p21 levels in a concentration dependent manner in both MCF-7 and MDA-MB-231 cell lines.In accordance with western blot results,PCR results showed cyclin D1 and cyclin E m RNA levels decreased when treated with chrysophanol.The m RNA expression of p21 was upregulated in both cell lines in a dose dependent manner.4.Chrysophanol regulates cell apoptosis and related proteinsMCF-7 and MDA-MB-231 cells were treated with chrysophanol and stained with Annexin V/PI.Percentage of apoptotic cells was increased significantly when treated with chrysophanol in both cell lines.MCF-7 and MDA-MB-231 cells were treated with paclitaxel?PTX;5nM,12hours?and chrysophanol?20?M,24 hours?,then apoptosis rate was examined.The results showed that chrysophanol significantly increased apoptosis rate in paclitaxel treated breast cancer cells.Western blot results showed that chrysophanol treatmentupregualted cleaved caspase 3 and cleaved PARP levels in both cell lines.In cells treated wit paclitaxel chrysophanol also significantly upregulated caspase 3 and PARP cleavage in bot cell lines.5.Chrysophanol regulates chemosensitivity through NF-?B signaling pathwayWestern blot analysis showed that expression of Bcl-2,p-I?B,p-p65 was significantly decreased after treatment with chrysophanol,demonstrating that NF-?B activity was suppressed.MCF-7 and MDA-MB-231 cells were treated with NF-?B inhibitor?PDTC?.Apoptosis analysis was performed and the results showed that difference of PTX induced apoptosis rate between PTX+ chrysophanol + PDTC and PTX+PDTC groups was not as significant as that between PTX and PTX+ chrysophanol gruops.In addition,in PDTC treated cells,the role of chrysophanol on Bcl-2 reduction was not significant.Conclusion:1.Chrysophanol inhibits malignant growth and cell cycle of breast cancer cells by inhibiting phosphorylation of NF-?B and its downstream NF-?B/cyclin D1 pathways.2.Chrysophanol induces apoptosis of breast cancer cells by inhibiting NF-?B signaling pathway.3.Chrysophanol also inhibits NF-?B/Bcl-2 pathway,which facilitates PTX induced apoptosis.