The Inhibitory Effect Of CDK4/6 Inhibitor PD-0332991on ESCC Cells And Its Related Molecular Mechanism

Background:Esophageal carcinoma(EC),a tumor of the digestive tract,is one of the most aggressive cancers and has been ranked as the fifth leading cause of cancer-related deaths worldwide.Approximately 70% of global ESCC cases occur in China,where it is ranked as the fourth most lethal cancer,and about 95% of the cases are esophageal squamous cell carcinoma(ESCC).Despite the multi-therapeutic strategies such as chemotherapy,radiotherapy,or combinations,the overall 5-year survival rate of this disease is only 10-20%.Therefore,there is an urgent need to explore novel therapeutic approaches to improve the clinical treatment among ESCC patients.PD-0332991 is an orally available,highly specific and reversible inhibitor for both CDK4 and CDK6 kinase activity.Increasing evidences show that PD-0332991 can exert an anti-proliferative effect in breast and ovarian cancer cells,myeloma cells and glioblastoma cells in vitro and in mouse models.More importantly,recently PD-0332991 has been approved by the FDA(2015)in combination with letrozole as first-line treatment of oestrogen receptor-positive(ER+),human epidermal growth factor receptor-2-negative(HER-2-),advanced breast cancer or in combination with fulvestrant for the treatment of women with hormone receptor-positive,HER-2-negative advanced or metastatic breast cancer.Whether PD-0332991 could be used to treat ESCC is currently unknown.Objective:In this present study,we will examine the antineoplastic effect of PD-0332991 against ESCC cells in vitro and in vivo,and elucidate its related molecular mechanism.Methods:The in vitro effects of PD-0332991 on ESCC cell proliferation were determined in 7 cell lines(EC109,EC9706,CE-81 T,KYSE30,KYSE140,KYSE150 and KYSE410)using the MTS assay.Soft agar assay was used to assess the inhibitory effect of PD-0332991 on tumorigenicity of ESCC cells in vitro.PI staining by flow cytometry was used to evaluate the effect of PD-0332991 on ESCC cell cycle distribution.Annexin V-FITC/PI double staining by flow cytometry was used to examine the effect of PD-0332991 on ESCC cell apoptosis.The dual CMXRos and MTGreen staining by flow cytometry was used to examine the mitochondrial transmembrane potential of ESCC cells.Western blot analysis was used to assay the effect of PD-0332991 on apoptosis-related proteins(PARP,Caspase-9,Caspase-8,Caspase-3,Active Caspase-3,Survivin,Bcl-XL,Bid,XIAP,Bcl-2,Mcl-1,Bax).Wound healing assay was used to detect the effect of PD-0332991 on ESCC cell migration.Transwell assay was utilized to evaluate the effect of PD-0332991 on ESCC cell migration and invasion.lentiviruses encoding shRNA against CDK4 and/or CDK6 were used to determine the effects of CDK4/6 inhibition.Cell viability and possible synergy between PD-0332991 and 5-flurouracil(5-FU)/Cisplatin was measured by MTS assay and CalcuSyn software.the effect of PD-0332991 on ESCC cell senescence was assayed via measurement of senescence associated β-galactosidase(SA-β-gal)activity.The specific plasmid and siRNA were used to determine whether FOXM1 was involved in PD-0332991-mediated cellular senescence.Changes in protein levels were assessed with western blot analysis.Furthermore,the effects of PD-0332991 on ESCC cell growth were determined by subcutaneously injecting EC109 cells(5 × 106 in PBS)in the flank of nude mice.The related proteins in tumor tissues were measured by immunohistochemistry or western blotting.The effects of PD-0332991 on metastasis of ESCC cells were determined by injecting KYSE150 cells(1 × 106 in PBS)through the lateral tail vein of nude mice.Results:MTS assay demonstrated that PD-0332991 inhibited the proliferation of all 7 cell lines in a dose-dependent manner,with IC50 values ranging from 11.42 to 20.15 μM,after 72 h of incubation.Furthermore,ESCC cells treated with PD-0332991 showed dose-dependent decrease in colony forming ability.Flow cytometric analysis of cell cycle distribution showed that PD-0332991(2.5 μM)induced significant G1 arrest in ESCC cells after 24 h treatment,with a dose-dependent downregulation of p-Rb,Cyclin D1,as well as the downstream targets of E2F(Cyclin A and Cyclin E).Annexin V-FITC/PI double staining showed that PD-0332991 treatent significantly induced ESCC cell apoptosis in a dose-and time-dependent manner.Western blotting results showed a concentration-and time-dependent cleavage of PARP and Caspase-3,downregulation of Caspase-3,Caspase-8,Caspase-9,as well as the pro-apoptotic protein Bid;In addition,PD-0332991 also inhibited the expression of the anti-apoptotic proteins Survivin,Bcl-XL,XIAP and Bcl-2,as well as increase of pro-apoptotic protein Bax in a dose-and time-dependent manner.Consistently,PD-0332991 treatment showed a time-dependent decrease in the mitochondrial transmembrane potential,as well as the increasing release of cytochrome c from mitochondria into the cytosol in EC109 and EC9706 cells.Wound healing and Boyden chamber Transwell assay also showed that the migration ability of both lines of ESCC cells was significantly impaired by PD-0332991 at the concentration of 5 μM.In addition,5 μM PD-0332991 could also strongly inhibit the invasion of EC109 and EC9706 cells.Western blotting analysis indicated that PD-0332991 dramatically inhibited the protein level of MMP-2 without changing the expression of MMP-9.Importantly,in comparison with scramble,specific knockdown of CDK4 alone just had a very weak inhibitory effect on ESCC cells.Silencing CDK6 on its own significantly suppressed the anchorage-independent growth,migration,invasion,as well as the expression of MMP-2 in both EC109 and EC9706 cells.In contrast,the inhibitory effect was markedly enhanced after knockdown of both CDK4 and CDK6.Furthermore,PD-0332991 drastically increased the activity of SA-β-gal,a marker for senescent cells in EC109 and EC9706 cells.Moreover,western blotting analysis revealed that the protein level of FOXM1 was decreased by PD-0332991 in a concentration-dependent manner.Enforced expression of FOXM1 by plasmid attenuated PD-0332991-induced cell senescence in EC109 and EC9706 cells;In contrast,silencing FOXM1 with two different FOXM1 siRNA duplexes resulted in a substantial increase in basal senescence.Moreover,knockdown of FOXM1 obviously potentiated PD-0332991-induced cell senescence,as indicated by a significant increase in the percentage of SA-β-gal positive cells,compared with the negative control RNA duplex(si-con)-transfection.The results of MTS assay and CalcuSyn software showed that there was a synergism between PD-0332991 and the conventional chemotherapeutic agents(5-FU and Cisplatin).In addtiion,trypan blue exclusion assay indicated that PD-0332991 promoted the cell death of 5-FU or Cisplatin in ESCC cells;Furthermore,western blotting analysis showed that PD-0332991 could enhance 5-FU-or Cisplatin-mediated PARP cleavage in both EC109 and EC9706 cells.Importantly,PD-0332991 could also inhibit ESCC cell growth in an EC109 xengraft nude mouse model.Moreover,the immunohistochemistry analysis demonstrated that PD-0332991 significantly suppressed the level of phosphorylated-Rb as well as the proliferation marker Ki67,but promoted the expression of Active Caspase-3,compared with the vehicle control group.Western blot analysis also showed that PD-0332991 inhibited the protein level of p-Rb,but not the total Rb.In addition,PD-0332991 treatment resulted in downregulation of the expression of FOXM1,and upregulation of the senescence marker p16 protein.An experimental metastasis model showed that PD-0332991 significantly attenuated the ability of lung metastasis in nude mice,compared with that of the vehicle group.Conclusions:In conclusion,We identified PD-0332991,a potent inhibitor of CDK4 and CDK6,that potently inhibits cell growth,migration and invasion,as well as induces apoptosis and senescence of ESCC cells in vitro and in vivo.Moreover,PD-0332991 could significantly sensitize ESCC cell lines to conventional chemotherapeutic agents 5-FU and Cisplatin.Therefore,PD-0332991 may be a promising agent for treating patients with ESCC,that warrants further clinical investigation.