| BackgroundBreast cancer is among the most frequent tumors worldwide.In recent years,the continuously rising incidence has come to the first place in women,which seriously threatening their lives and health.Breast cancer is a disease with high heterogeneity in morphology and genetics,and tumor progression and metastasis are the main factors affecting the clinical prognosis.For advanced breast cancer patients,radiotherapy is still an important treatment especially for local and regional adjuvant therapies.At present,radiation insensitivity occurs from time to time when patients are required to accept radiotherapy treatment.Therefore,enhancing the radiosensitivity of tumors and improving the therapeutic benefit ratio of tumor radiotherapy are urgent problems to be solved in modern medicineClinically,breast cancer can be divided into different subtypes according to the expression of molecular protein indicators in tumor samples,among which hormone receptor positive breast cancer patients could express positive estrogen receptor(ER+),show progesterone receptor(PR+),or owe even both of them.Palbociclib is a CDK4/6 molecule-targeted inhibitor that,in combination with letrozole,has received FDA conditional accelerated approval as a first-line endocrine-based therapy for ER+patients with metastatic breast cancer.In terms of cellular and molecular studies,it has been reported that palbociclib can coordinate the effect of endocrine drugs and chemotherapy drugs on inhibiting the proliferation and progression of estrogen receptor-positive breast cancer.However,the specific effect on radiotherapy and underlying mechanisms are still unclear.In this study,we hope to determine the effect of palbociclib on radiotherapy in breast cancer.We tried to determine the sensitization effect on radiotherapy of breast cancer and explore its molecular mechanism preliminarily.In addition,we further selected the sensitizing target genes through bioinformatics analysis and verified the possible linear co-expression relationship through experimental validation.We hope to provide thinking for the clinical intervention of radiotherapyMaterials and methodsEstrogen-receptor-positive breast cancer cell lines(McF-7 and T47D)were treated with graded concentration of palbociclib,and the lethal concentration(IC50)of the drug against both cell lines were determined by cytotoxicity assay.The experimental group pretreated by 4 ?M palbociclib and the control group treated with DMSO were exposed to under gradient doses of x-rays after irradiation(0,2,4,6,8 Gy),respectively.Then the cells were spread into the cell culture plates under standard condition.At fixed time points,cells were intercepted and observed via cloning experiment,comets(single cell gel electrophoresis experiment)and immunofluorescence test,which could detect the phenotypic states and changes in proliferation and DNA damage repair ability of the breast cancer cells.In the experiment of clone formation,the clones conforming to the standard were counted and the clone formation rates were calculated.In the comet experiment,nuclear fluorescence staining was performed to observe the migration length of the DNA migration part(comet tail),which could determine the DNA damage degree in individual cells.In immunofluorescence test of?-H2AX,breast cancer cells were seeded on the glass wafers in six-well cell culture plate.Cells in the experimental groups were pretreated with 4?M palbociclib and the exposed to X-ray irradiation(4 Gy).At different time points(0 h,0.5 h,4 h and 12 h),cells were treated with fluorescent staining.Finally,formation and persistent state of?-H2AX focuses in breast cancer cells were observed under fluorescent microscope following their exposure to X-ray irradiation,so as to determine the phenotypic changes in DNA damage repair ability of breast cancer cells.The changes of cell cycle ratios in breast cancer cells were measured by flow cytometry to detect the influence of palbociclib pretreatment on breast cancer cells'cell cycle changes after exposure to irradiation.The breast cancer cells were divided into the radiation subgroup and the palbociclib combining radiation subgroup(pretreated with DMSO or 4 ?M palbociclib for 24h,respectively).The cells of the two groups were collected 12 hours after receiving 4 Gy X-ray irradiation.Cell cycle was detected by flow cytometry after stained with PI according to the reagent instructions,and the results were calculated and with the cell cycle fitting software ModFitFurthermore,three datasets were downloaded from the GEO database(https://www.ncbi.nlm.nih.gov/geo/),respectively,including GSE117742 and GSE117743(breast cancer cells treated by palbociclib),and GSE102798(breast cancer cells treated by radiotherapy).Then,we determined the differential expressed genes with the online analytical tools GEO2R.Functional enrichment analysis was performed on these differentially expressed genes to identify the significant enrichment pathways.Furthermore,the candidate genes regulated by both palbociclib and X-ray were identified via taking intersections.Moreover,the third-level sequencing data of breast cancer patients were downloaded from TCGA database,and the candidate genes were identified through bioinformatics analysis and linear gene co-expression analysis,which might show a correlation with the expression of CDK4 or CDK6,the direct targets of palbociclib.To verify the expression correlation between the predicted candidate gene ARL4C and palblciclib target CDK6,we designed a pair of specific primer sequences for ARL4C,and the expression level of ARL4C in different breast cancer lines were validated by Polymerase Chain Reaction(PCR)experiment.At the same time,we constructed an overexpression vector system of the candidate target gene ARL4C,which was further transfected into breast cancer cell lines by liposome inclusion.The expression correlation between ARL4C and CDK6 were further validated by western-blot experimentThen we tried to verify the DNA damage related and cell cycle related pathways enriched from the functional enrichment analysis.The effects of palbociclib on expression of DNA damage repair markers and cell cycle related protein molecules were further detected by western-blot experiment.Breast cancer cells were pretreated with 4 ?M palbociclib for 24h and then exposed to X-ray irradiation in gradient doses 48 hours later,proteins of breast cancer cells in both experimental and control groups were collected to detect the expression of Ku70,Ku86 and RAD51.In addition,breast cancer cells were pretreated with 4 ?M palbociclib for 24h before X-ray irradiation,and cells in the control group,palbociclib treatment group,X-ray irradiation group and palbociclib combining X-ray irradiation group were collected after incubation of 48 hours,and cell proteins of the four groups were all collected to detect the expression of cell cycle related pathway proteins.Results Through observation and counting of standard formed clones in the clone formation experiment,we found that palbociclib had a significant radiosensitization effect on ER+breast cancer cells(MCF-7 and T47D cells).Compared with cells in the control group,cells in the experimental group cells pretreated by palbociclib showed significantly reduced proliferation ability and significantly lower survival fraction(SF)when exposed to the same dose of X-ray irradiation(p<0.01).Similar results were observed in both MCF-7 and T47D cell lines.The quadratic model was used to fit the cell survival curve with further data analysis,and there were significant statistical differences between the two curves in the linear regression model(p<0.001).DNA damage occurs when breast cancer cells were exposed to X-ray irradiation,and palbociclib can synergically enhance the damage of X-ray on the DNA and further delay the process of DNA damage repair by targeting breast cancer cells.In the comet experiment,compared with the control group,tumor cells pretreated by palbociclib showed significantly prolonged migration path of DNA fragments after X-ray exposure,and there were still prolonged comet tails after 20 hours of cell autonomous DNA damage repairment,which indicating a higher damage level of DNA and the recovery rate and degree of cancer cells were blocked.?-H2AX is a sensitive protein marker indicating DNA damage degrees.Compared with the control group treated with radiotherapy alone,the palbociclib combined with radiotherapy group showed delay and hysteresis in the focal clearance of the ?-H2AX protein fluorescence.At 0.5h,both types of cells showed a large number of the ?-H2AX protein fluorescence foci.Compared with the control group,McF-7 breast cancer cells pretreated by palbociclib showed no significant decrease in the foci of the y-H2AX cells at 4h,and the fluorescence intensity had not returned to the background level at 12h.All T47D breast cancer cells pretreated by palbociclib showed significant ?-H2AX fluorescence intensity constantly from Oh to 12h,which was still significantly higher than that of the control group at 12h.The results of the comet experiment suggested that palbociclib combined with X-ray could cooperatively enhance the DNA damage of X-ray irradiation,whereas results of the immunofluorescence experiment showed that palbociclib combined with X-ray could hinder the repair process of DNA double-strand fracture of breast cancer cells caused by radiotherapy,which could further inhibit proliferation and progression of breast cancer.There was a cell cycle stagnation from G1 to S phase or G2 phase to M phase in breast cancer cells after exposure to X-ray irradiation,which contributed to inhibition on the proliferation and progression process of cancer cells.Breast cancer cells(McF-7 and T47D cells)pretreated with palbociclib showed significant cell cycle changes compared with the control group,showing a significant increase in the proportion of cancer cells in G0/G1 phase(p?0.05),while the ratio of cancer cells in S phase was lower significantly.Meanwhile,on contrast to the control group treated with radiotherapy alone,there is a significant increase in G1/S block in the Palbociclib combined radiotherapy subgroup(p?0.05).Furthermore,differential expression analysis was performed on three GEO sequencing data and enrichment analysis was further used on these genes.It was figured out that both the DNA damage related pathway and the cell cycle related pathway were enriched by the differential expression genes significantly.In addition,a total of 10 candidate genes were identified among the intersections.These genes were regulated by both factors of palbociclib and X-ray irradiation,which may serve as potential targets contributing to the sensitization effect of palbociclib.Through gene linear co-expression analysis,we found that the expression of the breast cancer prognostic related gene ARL4C was correlated with the expression of CDK6(r=0.60,p<0.001).In molecular biology experiments,we found that ARL4C showed low expression level in breast cancer cell lines with PCR experiments,and overexpression of ARL4C in estrogen-receptor-positive breast cancer cell lines could increase the level of CDK6 protein to some extent.These results confirmed the expression correlation between ARL4C and CDK6.However,the specific function and underlying molecular mechanism of ARL4C in breast cancer still need further studies.DNA damage is mainly repaired by homologous recombination and non-homologous recombination.Rad51 is the main protein involved in homologous recombination repair,and Ku70 and Ku86 are the main proteins involved in non-homologous recombination repair.The protein expression of the two breast cancer cells was identified by western-blot experiment.In both MCF-7 and T47D cell lines,palbociclib continuously decreased the expression level of RAD51 protein with an increase of radiation dose.At the same time,the level of RAD51 in the palbociclib combined with radiotherapy subgroup was significantly decreased compared with that in the radiotherapy alone subgroup,suggesting that palbociclib could inhibit homologous repair of DNA damage in estrogen receptor-positive breast cancer cells and enhance the killing effect of X-ray on breast cancer cells.Meanwhile,in the control group treated with radiotherapy alone,expression levels of Ku70,Ku86 and RAD51 had no significant correlation with the radiation dose,while in the palbociclib combined with radiotherapy group,there was a decrease trend in the protein expression level of RAD51 with the increasing dose of X-ray radiation,suggesting that palbociclib could enhance the sensitivity of breast cancer cells to X-ray via regulating RAD51 expression.In addition,palbociclib is a kind of CDK4/6 targeted inhibitor and we further detected the expression of cell cycle checkpoint protein related pathways through the western-blot experiment.In both breast cancer cell lines,compared with palbociclib alone subgroups and radiation treatment alone subgroup,palbociclib combining radiotherapy subgroups showed a significant decrease in expression levels of p-Rb,p-erk and p21,which prompts that palbociclib might sensitize radiation therapy via blocking the cell cycle,and palbociclib might have synergistic effect with radiotherapy in killing breast cancer cellsConclusionPalbociclib coud enhance the sensitivity of estrogen receptor-positive breast cancer cells to radiotherapy by blocking cell cycle,and increase the killing effect of X-ray on cancer cells.The main mechanism might focus on delay of DNA damage repair by inducing G1/S block and inhibiting the expression of DNA damage repair protein.In addition,ARL4C was confirmed to have expression correlation with CDK6,which may be a potential target of radiotherapy sensitization of palbociclib.In conclusion,this study provides a theoretical basis for the clinical application of palbociclib as a sensitizer in combination with radiotherapy for breast cancer patients,and its application in radiotherapy sensitization could provide a new thinking for the clinical comprehensive treatment of breast cancer patients. |
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