Tumor suppressor effect of follistatin-like 1 in ovarian and endometrial carcinogenesis—a differential expression and functional analysis

Abstract

Endometrial and ovarian cancers are the most common and the most lethal gynecologic malignancies worldwide, respectively. By performing differential expression analysis using annealing control primer-based reverse transcription (RT)-polymerase chain reaction (PCR) on pooled complementary DNA (cDNA) from 45 endometrial and 36 ovarian cancers and their non-tumor samples, reduced expression of the follistatin-like 1 (FSTL1) was identified. Downregulation of FSTL1 was further confirmed on individual samples and cell lines by quantitative real-time RT-PCR and western blotting. For in vitro functional study, full-length cDNA of FSTL1 was cloned and transiently transfected into the ovarian cancer cell line Ovca420 and endometrial cancer cell line AN3CA. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay and cell count demonstrated significantly slower proliferation rate. By terminal uridine deoxynucleotidyl transferase dUTP nick end labeling and flow cytometric analysis, higher apoptotic activity and a remarkable increase in sub-G(1) cell population were observed in transfected cells, suggesting that FSTL1 induced apoptosis in cancer cells. Subsequent messenger RNA and protein expression analysis on downstream apoptotic molecules revealed upregulation and/or activation of FAS, FASLG, TRADD, Caspase-3, Caspase-7 and PARP by FSTL1 transfection, suggesting that FSTL1-induced apoptosis may be initiated mainly by FAS/FASLG death receptor-ligand binding. Cell migration and invasion assays demonstrated a remarkably lower cell migration and invasion capability in FSTL1-transfected cells in relation to downregulation of matrix metallopeptidase-2. Our findings suggested that a tumor suppressor role of FSTL1 may be important in ovarian and endometrial carcinogenesis.

Differential expression and phosphorylation of Pak1 and Pak2 in ovarian cancer: effects on prognosis and cell invasion

“…15 The cells were transfected with 3 lg DNA constructs using Lipofectamine 2000 (Invitrogen) per manufacturer's instructions. 16,17 Transfected cells were plated for migration and invasion assays at 72 hr after transfection. For SB 203580 treatment, caPak1-overexpressing cells were counted at 72 hr after transfection and plated for 6 h before treating with the p38 inhibitor SB 203580 (10 lM; Calbiochem, San Diego, CA) or vehicle (DMSO) for migration and invasion assays and immunoblot analyses.…”

“…Real-time quantitative PCR (qPCR) was performed on the ABI Prism 7700 sequence detection system as previously described. 16,17,23 The primer sequences are listed in Table 2.…”

“…The protein (20 lg) was separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane, and hybridized with corresponding antibodies. 14,16,17 The antibodies used in this study are listed in Supporting Information Table 1.…”

“…14,16,17 Briefly, 1.25 Â 10 5 cells were plated on the upper compartment of a Transwell chamber. For migration assays, cells migrated through an 8-lm pore membrane.…”

Activins and follistatins: Emerging roles in liver physiology and cancer

“…Fstl1 itself was identified as a TGF-β inducible gene [133] and has been implicated in inflammation and cardioprotection [134][135][136] . It has been suggested to act as a potential tumor suppressor in epithelial cancers [137][138][139][140] but is over-expressed in astrocytic brain tumors [141] . Considering hepatoma cells, we recently demonstrated that the expression of fstl1 is low in HepG2 cells, which show an epithelial morphology/proteome pattern and high in Hep3B cells with fibroblastoid characteristics.…”