title: Breast cancer cells are arrested at different phases of the cell cycle following the re-expression of ARHI.
abstract: ARHI is a maternally imprinted tumor suppressor gene that is expressed in normal breast epithelial cells but not in most breast cancer cells. Aberrant methylation and hypernomic histone deacetylation have been implicated in the silencing of ARHI. To investigate the mechanism of ARHI induction, MDA-MB-231 breast cancer cells were either transfected with the eukaryotic expression vector, pcDNA3.1(+)-ARHI, or were simultaneously treated with a histone deacetylase inhibitor, [trichostatin A, (TSA)] and the methyltransferase inhibitor, 5-aza-2'-deoxycytidine (DAC). The latter treatment group also included the targeting of ARHI by small interfering RNA (siRNA) to further examine interactions between ARHI and the drugs applied. Levels of ARHI were detected by western blotting, MTT assays were used to evaluate cell proliferation, and both cell cycle progression and apoptosis were detected using flow cytometry. Both the transfection of pcDNA3.1(+)‑ARHI and the application of TSA+DAC induced the expression of ARHI. Furthermore, reduced cell proliferation, cell cycle arrest and enhanced apoptosis were observed for both groups compared to controls. However, a G1/S cell cycle arrest was observed for the pcDNA3.1(+)-ARHI group, while a G2 cell cycle arrest was observed for the TSA+DAC group. The latter effect was reversed with the introduction of ARHI-targeted siRNA in combination with TSA+DAC treatment. To further clarify these observations, expression levels of several key cell cycle regulators were analyzed by western blotting. The pcDNA3.1(+)-ARHI group exhibited higher expression levels of p53, p21 and p27, and lower levels of cyclin D1, CDK4 and CDK6 when compared to the control group (P<0.05). For the TSA+DAC group, higher levels of p53, p21, cyclin B1 and Chk1 were detected, concomitant with lower levels of CDK1, when compared to the control group. Taken together, these results suggest that ARHI acts as a tumor suppressor gene in MDA-MB-231 cells and, although TSA+DAC can block the cells at different cell cycle phage, the antitumor effect is ARHI-dependent.
authors.affiliation: Department of Oncology, The First Affiliated Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Xiaoxiao
authors.initials: X
authors.lastname: Zuo
authors.affiliation: Hematology Department, Xi'an Yang Central Hospital, Xianyang, Shaanxi 712000, P.R. China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Qin
authors.affiliation: Department of Oncology, The First Affiliated Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Xiaojin
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Respiratory Department, The First Affiliated Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Ning
authors.affiliation: Department of Oncology, The First Affiliated Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Shan
authors.initials: S
authors.lastname: Shao
authors.affiliation: Department of Oncology, The First Affiliated Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Minna
authors.initials: M
authors.lastname: Luo
authors.affiliation: Department of Oncology, The First Affiliated Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Na
authors.initials: N
authors.lastname: Yuan
authors.affiliation: Department of Oncology, The First Affiliated Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Shangke
authors.initials: S
authors.lastname: Huang
authors.affiliation: Department of Oncology, The First Affiliated Hospital, College of Medicine, Xi'an Jiaotong University, Xi'an, Shaanxi 710061, P.R. China.
authors.forename: Xinhan
authors.initials: X
authors.lastname: Zhao
mesh_terms: D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001374:Azacitidine / Q000031:analogs & derivatives* / Q000494:pharmacology; D001943:Breast Neoplasms / Q000188:drug therapy / Q000235:genetics*; D016203:CDC2 Protein Kinase; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects / Q000235:genetics; D000071877:Checkpoint Kinase 1; D056744:Cyclin B1 / Q000096:biosynthesis; D019938:Cyclin D1 / Q000096:biosynthesis; D051358:Cyclin-Dependent Kinase 4 / Q000096:biosynthesis; D051361:Cyclin-Dependent Kinase 6 / Q000096:biosynthesis; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000096:biosynthesis; D050760:Cyclin-Dependent Kinase Inhibitor p27 / Q000096:biosynthesis; D018844:Cyclin-Dependent Kinases / Q000096:biosynthesis; D000077209:Decitabine; D005260:Female; D059565:G2 Phase Cell Cycle Checkpoints / Q000187:drug effects / Q000235:genetics; D005822:Genetic Vectors / Q000235:genetics; D056572:Histone Deacetylase Inhibitors / Q000494:pharmacology; D006801:Humans; D006877:Hydroxamic Acids / Q000494:pharmacology*; D008780:Methyltransferases / Q000037:antagonists & inhibitors; D011494:Protein Kinases / Q000096:biosynthesis; D034622:RNA Interference; D034741:RNA, Small Interfering; D059807:S Phase Cell Cycle Checkpoints / Q000187:drug effects / Q000235:genetics; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000096:biosynthesis; D020741:rho GTP-Binding Proteins / Q000096:biosynthesis / Q000235:genetics*
chemical_list: C534699:CCNB1 protein, human; C530104:CCND1 protein, human; D056744:Cyclin B1; D050759:Cyclin-Dependent Kinase Inhibitor p21; C117386:DIRAS3 protein, human; D056572:Histone Deacetylase Inhibitors; D006877:Hydroxamic Acids; D034741:RNA, Small Interfering; D016159:Tumor Suppressor Protein p53; D019938:Cyclin D1; D050760:Cyclin-Dependent Kinase Inhibitor p27; C012589:trichostatin A; D000077209:Decitabine; D008780:Methyltransferases; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D016203:CDC2 Protein Kinase; C528899:CDK1 protein, human; C495900:CDK4 protein, human; C495911:CDK6 protein, human; D051358:Cyclin-Dependent Kinase 4; D051361:Cyclin-Dependent Kinase 6; D018844:Cyclin-Dependent Kinases; D020741:rho GTP-Binding Proteins; D001374:Azacitidine