Moreover, Mena silencing attenuated the invasion-suppressive effects of TES about GC cells
Moreover, Mena silencing attenuated the invasion-suppressive effects of TES about GC cells. conserved protein of 421 amino acids comprising three C-terminal LIM domains [4]. LIM domains each consists of two zinc-finger motifs that mediate proteinCprotein relationships with transcription factors, cytoskeletal proteins, and signaling proteins [4C6]. TES has been identified as a putative TSG in many human cancers, such as breast and uterine cancers [7] and glioblastoma [8]. In these malignancy types, the manifestation of TES was decreased or totally lost by promoter hypermethylation [7, 8]. Overexpression of TES significantly inhibited tumor cell growth in vitro and reduced the tumorigenic potential of particular tumor cell BIRT-377 lines in vivo [7]. Moreover, knockout in mice resulted in improved susceptibility to carcinogen-induced GC [9]. However, the part of TES in GC has not been further investigated, and the molecular mechanism of TES underlying GC carcinogenesis and metastasis remains unfamiliar. Earlier studies have shown that TES localized to focal adhesions and cellCcell or cellCsubstratum contact sites, suggesting a role in cell adherence, migration, and motility [4, 10, 11]. In addition, it is an interacting partner of the known cell adhesion and cytoskeleton regulatory proteins, such as Zyxin, Talin, and Mena [4, 5]. Mena, a BIRT-377 member of the Ena/vasodilator-stimulated phosphoprotein (VASP) family, is usually involved in regulating the assembly of actin filaments and modulates cell adhesion and motility [5, 12C14]. Ena/VASP family proteins can recruit MRL proteins (consisting of Mig10, Rap1-interacting adapter molecule [RIAM], and Lamellipodin [Lpd]) to the leading edge of filopodia and lamellipodia to regulate cell lamellipodial distributing and motility [5, 15]. It has been reported that Mena is usually involved in cell migration and motility by its conversation with Lpd [15]. Therefore, we hypothesized that TES plays a role as tumor suppressor in GC through BIRT-377 interacting with Mena. In this study, we systematically explored the tumor suppressive functions of TES in GC both in vitro and in vivo and decided its conversation with Mena in GC. Materials and methods Cell lines All cell lines were authenticated by short-tandem repeat analysis. The human embryonic kidney cell collection HEK293A (obtained in November 2009, authenticated in June 2015) and GC cell lines MKN45, SGC7901, MGC803, AGS, and HGC27 (obtained in July 2011, authenticated in June 2015) were obtained from the Committee of Type Culture Collection of Chinese Academy of Sciences (Shanghai, China). All cells were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37?C in a humidified chamber containing 5% CO2. Patients and tissue samples The medical records of 172 GC patients treated at Sun Yat-sen University Malignancy Center (Guangzhou, China) between January 2003 and December 2005 were examined. The patient selection criteria were as follows: (1) the patient was pathologically diagnosed with gastric adenocarcinoma; (2) the patient experienced received gastrectomy with limited BIRT-377 or extended lymphadenectomy; (3) the patient did not receive any anticancer treatment before surgery; (4) the patient had complete clinical information, including follow-up data; (5) the patient had no other synchronous malignancies or familial malignancy; (6) the patient had no recurrent or remnant GC; and (7) the patient survived at least 3?months after TPT1 surgery. Follow-up data were obtained through on-site interview, telephone calling or medical chart review. Overall survival (OS) was defined as the time from surgery to death from any cause or last follow-up. The study was approved by the Ethics Committee of Sun Yat-sen University Malignancy Center (Guangzhou, China), and written knowledgeable consent was obtained from all participants. Recombinant adenoviral expression vector construction and transfection The TES recombinant adenoviral expression vector (Ad-TES) and control vector (Ad-Control) were constructed using the Gateway cloning system (Invitrogen, Carlsbad, CA, USA), according to the manufacturers protocol. After linearization by PacI enzyme, Ad-TES and Ad-Control were transfected into HEK293A cells.