Forced Trefoil Factor Family Peptide 3 (TFF3) Expression Reduces Growth, Viability, and Tumorigenicity of Human Retinoblastoma Cell Lines

Abstract

Trefoil factor family (TFF) peptides have been shown to effect cell proliferation, apoptosis, migration and invasion of normal cells and various cancer cell lines. In the literature TFF peptides are controversially discussed as tumor suppressors and potential tumor progression factors. In the study presented, we investigated the effect of TFF3 overexpression on growth, viability, migration and tumorigenicity of the human retinoblastoma cell lines Y-79, WERI-Rb1, RBL-13 and RBL-15. As revealed by WST-1 and TUNEL assays as well as DAPI and BrdU cell counts, recombinant human TFF3 significantly lowers retinoblastoma cell viability and increases apoptosis levels. Transient TFF3 overexpression likewise significantly increases RB cell apoptosis. Stable, lentiviral TFF3 overexpression lowers retinoblastoma cell viability, proliferation and growth and significantly increases cell death in retinoblastoma cells. Blockage experiments using a broad-spectrum caspase inhibitor and capase-3 immunocytochemistry revealed the involvement of caspases in general and of caspase-3 in particular in TFF3 induced apoptosis in retinoblastoma cell lines. Soft agarose and in ovo chicken chorioallantoic membrane (CAM) assays revealed that TFF3 overexpression influences anchorage independent growth and significantly decreases the size of tumors forming from retinoblastoma cells. Our study demonstrates that forced TFF3 expression exerts a significant pro-apoptotic, anti-proliferative, and tumor suppressive effect in retinoblastoma cells, setting a starting point for new additive chemotherapeutic approaches in the treatment of retinoblastoma.

Reduction of the tumorigenic potential of human retinoblastoma cell lines byTFF1overexpression involves p53/caspase signaling and miR-18a regulation

“…For the generation of a TFF1 expression vector under the control of the ubiquitin C promoter (pCS2+_UBQ_TFF1), the human TFF1 cDNA sequence was excised by digestion with SalII /BamHI and ligated into the pCS2+_UBQ vector (for details see Ref. ), digested with the same restriction enzymes.…”

“…For transient TFF1 overexpression, Y79 RB cells were transfected as previously described by our group in Ref. . Instead of the pCS2+_UBQ_ TFF3 the pCS2+_UBQ_ TFF1 vector was used for transfection.…”

“…For stable TFF1 overexpression or TFF1 knockdown, GFP‐labeled Y79, RB355 and RBL30 RB cell lines were used, generated as described previously . For lentiviral transductions, the experimental procedure used was the same as previously described, but instead of TFF3 ‐coding lentiviral particles, TFF1 ‐ or sh TFF1 ‐coding particles were used.…”

“…Seven days after grafting (E10–17) tumors which formed from the grafted cells were excised, measured, photographed and ( i ) shock frozen for quantitative real‐time PCR or ( ii ) cryo‐embedded as described in Ref. . Six days after injection (E12‐E18) tissue punches of each ventral “lower CAM” were isolated for scanning GFP‐positive RB cells by fluorescence microscopy and cryoconserved for RNA extraction …”

Extracellular production of recombinant sus scrofa trefoil factor 3 by Brevibacillus choshinensis

“…The interaction between TFF3 and mucins has also been reported to protect intestinal epithelia from injuries, maintaining mucosal barrier function (26). Furthermore, mitogenic effects of TFF3 have been identified in vitro in primary cell culture, and TFF3 has been demonstrated to inhibit apoptosis, induce cellular invasion, act as an inflammatory modulator and exhibit pro-angiogenic activity (27)(28)(29)(30)(31)(32). A protective or healing effect of TFF3 has also been reported in a series of experiments, such as cells in vitro, mice, rats and human (12)(13)(14)(15)(16)(17).…”