title: DNA promoter methylation-dependent transcription of the double C2-like domain β (DOC2B) gene regulates tumor growth in human cervical cancer.
abstract: Double C2-like domain β (DOC2B) gene encodes for a calcium-binding protein, which is involved in neurotransmitter release, sorting, and exocytosis. We have identified the promoter region of the DOC2B gene as hypermethylated in pre-malignant, malignant cervical tissues, and cervical cancer cell lines by methylation-sensitive dimethyl sulfoxide-polymerase chain reaction and bisulfite genome sequencing; whereas, it was unmethylated in normal cervical tissues (p < 0.05). The promoter hypermethylation was inversely associated with mRNA expression in SiHa, CaSki, and HeLa cells and treatment with demethylating agent 5-aza-2-deoxycytidine restored DOC2B expression. The region -630 to +25 bp of the DOC2B gene showed robust promoter activity by a luciferase reporter assay and was inhibited by in vitro artificial methylation with Sss1 methylase prior to transient transfections. Overexpression of the DOC2B gene in SiHa cells when compared with controls showed significantly reduced colony formation, cell proliferation, induced cell cycle arrest, and repressed cell migration and invasion (p < 0.05). Ectopic expression of DOC2B resulted in anoikis-mediated cell death and repressed tumor growth in a nude mice xenograft model (p < 0.05). DOC2B expressing cells showed a significant increase in intracellular calcium level (p < 0.05), impaired AKT1 and ERK1/2 signaling, and induced actin cytoskeleton remodeling. Our results show that promoter hypermethylation and silencing of the DOC2B gene is an early and frequent event during cervical carcinogenesis and whose reduced expression due to DNA promoter methylation may lead to selective cervical tumor growth.
authors.affiliation: Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, India.
authors.forename: Shama Prasada
authors.initials: SP
authors.lastname: Kabekkodu
authors.affiliation: Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, India.
authors.forename: Samatha
authors.initials: S
authors.lastname: Bhat
authors.affiliation: Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, India.
authors.forename: Raghu
authors.initials: R
authors.lastname: Radhakrishnan
authors.affiliation: Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, India.
authors.forename: Abhijit
authors.initials: A
authors.lastname: Aithal
authors.affiliation: Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, India.
authors.forename: Roshan
authors.initials: R
authors.lastname: Mascarenhas
authors.affiliation: Department of OBGYN, Kasturba Medical College, Manipal University, Manipal 576104, India.
authors.forename: Deeksha
authors.initials: D
authors.lastname: Pandey
authors.affiliation: Department of OBGYN, Kasturba Medical College, Manipal University, Manipal 576104, India.
authors.forename: Lavanya
authors.initials: L
authors.lastname: Rai
authors.affiliation: Department of OBGYN, Kasturba Medical College, Manipal University, Mangalore 575001, India.
authors.forename: Pralhad
authors.initials: P
authors.lastname: Kushtagi
authors.affiliation: Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, India.
authors.forename: Gopinath Puthiya
authors.initials: GP
authors.lastname: Mundyat
authors.affiliation: Division of Biotechnology, Manipal Life Sciences Centre, Manipal University, Manipal 576104, India. Electronic address: ksatyamoorthy@manipal.edu.
authors.forename: Kapaettu
authors.initials: K
authors.lastname: Satyamoorthy
mesh_terms: D000199:Actins / Q000378:metabolism; D000818:Animals; D017209:Apoptosis; D002118:Calcium / Q000737:chemistry / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D018899:CpG Islands; D019175:DNA Methylation; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D005838:Genotype; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009361:Neoplasm Invasiveness; D009368:Neoplasm Transplantation; D009419:Nerve Tissue Proteins / Q000235:genetics* / Q000378:metabolism; D027383:Papillomaviridae / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D015398:Signal Transduction; D013447:Sulfites / Q000737:chemistry; D002583:Uterine Cervical Neoplasms / Q000378:metabolism*
chemical_list: D000199:Actins; D002135:Calcium-Binding Proteins; C490420:DOC2B protein, human; D009419:Nerve Tissue Proteins; D013447:Sulfites; C042345:hydrogen sulfite; D002118:Calcium
keywords: Actin Remodeling; Akt; Cell Growth; Cell Invasion; Cell Motility; Cervical Cancer; DNA Promoter Methylation; DOC2B; ERK; Human Papillomavirus