Antibodies against Cbl-b (mouse monoclonal catalog no., sc-8006; dilution, 1:250) and actin (rabbit polyclonal; catalog no., H-196; dilution, 1:1,000) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Antibodies against Akt (rabbit polyclonal; catalog no., 4691; dilution, 1:1,000), phosphorylated (p)-Akt (rabbit polyclonal; catalog no., 4060; dilution, 1:500), FAK (rabbit polyclonal; catalog no., 3285; dilution, 1:500), anti-p-FAK (rabbit polyclonal; catalog no., 8556; dilution, 1:500) and ubiquitin (rabbit polyclonal; catalog no., 3936; dilution, 1:500) were obtained from Cell Signaling Technology, Inc. (Danvers, MA, USA). Dimethyl sulfoxide was obtained from Sigma-Aldrich (St. Louis, MO, USA).

The human gastric adenocarcinoma MGC803, lung adenocarcinoma A549, colon carcinoma RKO, breast adenocarcinoma MCF-7, breast carcinoma MDA-MB-231 and gastric epithelial cell GES-1 cell lines were obtained from the Academy of Military Medical Sciences (Beijing, China). The cells were cultured in RPMI-1640 medium or L-15 medium containing 10% fetal calf serum (all Gibco^®; Thermo Fisher Scientific, Inc., Waltham, MA, USA) at 37°C in a humidified incubator with an atmosphere of 5% CO₂.

The percentage of detached cells was analyzed using MTT assay. The cells (2×10⁵ cells/well) were treated with 2.5 mg/l trypsin for various times (0, 1, 3 and 5 min) and washed four times with ice-cold phosphate-buffered saline. The MTT assay was performed as described in a previous study by the present authors (15).

The cells (2×10⁵ cells/well) were seeded in 6-well plates and incubated overnight. The cells were treated with 2.5 mg/l trypsin for various times (0, 1, 3 and 5 min). The medium was removed and the cells were added to a 1.5 ml Eppendorf tube followed by transient centrifugalization. Western blot analysis was performed as described in a previous study (15). Western blot images were analyzed using National Institutes of Health image software (rsb.info.nih.gov/nih-image/) for further statistical analysis.

The antibodies against FAK and Cbl-b, protein G-agarose beads (Cell Signaling Technology, Inc.) and cell lysate were incubated overnight at 4°C. Subsequently, immunoprecipitates were washed four times with lysis buffer. Immunoprecipitation was performed as described in a previous study (16).

Plasmid construction was performed as described in a previous study (15). Briefly, MGC803 and MDA-MB-231 cells were transfected with short hairpin (sh) RNA targeting Cbl-b using Lipofectamine 2000 reagent (Invitrogen™; Thermo Fisher Scientific, Inc), according to the manufacturer's protocol. One set of synthetic oligonucleotides involved the sense and antisense target sequences of human Cbl-b: Sense, 5′-GGATCCCGGATGTGTTTGGGACTAATTTGATATCCGATTAGTCCCAAACACATCCTTTTTTCCAAAAGCTT-3′ and antisense, 5′-AAGCTTTTGGAAAAAAGGATGTGTTTGGGACTAATCGGATATCAAATTAGTCCCAAACACATCCGGGATCC-3′ were annealed and ligated into the BamHI/HindIII-cleaved backbone of pRNA-U6.1/Neo (Genescript, Piscataway, NJ, USA). Stably transfected cell lines were selected according to the methods described in a previous study (17). The cDNA of Flag-tagged ubiquitin (Flag-Ub) was provided by Dr Kiyonao Sada (Division of Genome Science and Microbiology, University of Fukui, Fukui, Japan). All cDNAs were then subcloned into the pSVL expression vector (GE Healthcare, Piscataway, NJ, USA). MGC803/NC and MGC803/shCbl-b, MDA-MB-231/NC and MDA-MB-231/shCbl-b cells transfected with shRNA-Cbl-b were used for the following experiments.

FAK siRNA was obtained from Shanghai GeneChem Co. Ltd., (Shanghai, China). MGC803 cells were transfected with siRNAs using Lipofectamine 2000, according to the manufacturer's protocol. FAK siRNA sequences were synthesized as follows: FAK siRNA sequences were synthesized as follows: siFAK-1, CAGGUGAAGAGCGAUUAU Att; siFAK-2, CUCCAGUCUACAGAUUUGAtt; siFAK-3, CCCAGGUUUACUGAACUUAtt. After 48 h of transient transfection, the cells were analyzed by western blotting to determine the effect of FAK siRNA on FAK expression.

All experiments were performed in triplicate. Data were expressed as the mean ± standard deviation. Statistical significance was determined using the Student's t-test. Statistical analysis was performed using SPSS version 18.0 software (SPSS, Inc., Chicago, IL, USA). P<0.05 was considered to indicate a statistically significant difference.

To investigate the effect of FAK in cell detachment, the human gastric cancer MGC803 cell line was treated with trypsin for 0, 1, 3 and 5 min. The percentage of detached cells was significantly increased in a time-dependent manner (Fig. 1A; P<0.05). Furthermore, western blot analysis revealed that FAK was gradually reduced in a time-dependent manner (Fig. 1B). Similar results were observed in human breast cancer MCF-7, lung cancer A549, colon cancer RKO and gastric epithelial GES-1 cells (Fig. 1C; P<0.05). These results indicate that FAK is downregulated in the process of cell detachment.

To further clarify the function of FAK in the process of cell detachment, MGC803 cells were transfected with FAK-specific siRNAs for 48 h. Knockdown of FAK expression was detected by western blotting (Fig. 2A). siRNA 3 was selected for further analysis, which revealed that knockdown of FAK significantly increased cell detachment compared with controls in a time-dependent manner (P<0.05; Fig. 2B). In addition, p-Akt levels were decreased in the siRNA/FAK group (Fig. 2C). These data indicate that FAK inhibits cell detachment in cancer cells and human gastric epithelial cells.

A previous study demonstrated that ubiquitin-dependent protein degradation is critical in the regulation of FAK (14). However, whether ubiquitination degradation mediates trypsin-induced degradation of FAK is currently unknown. In the present study, MGC803 cells were transfected with plasmids expressing Flag-Ub. Mono-ubiquitination of FAK was increased in a time-dependent manner following trypsin treatment of the transfected cells (Fig. 3A). In the NH₄Cl-pretreated group, FAK degradation was suppressed compared with the control (Fig. 3B). However, similar results were not observed in cells pretreated with the proteasome inhibitor PS-341 (Fig. 3C). To further investigate whether the degradation of FAK occurs via the ubiquitin-lysosome system, MGC803 cells were transfected with plasmid expressing Flag-Ub for 48 h, in the absence or presence of 50 nM NH₄Cl for 12 h. Mono-ubiquitination of FAK was enhanced in the NH₄Cl pretreated group (Fig. 3D). These results indicate that the ubiquitin-lysosome system mediates trypsin-induced degradation of FAK.

A previous study demonstrated that the E3 ubiquitin protein ligase c-Cbl is involved in the ubiquitination of FAK (14). Therefore, the involvement of Cbl-b in ubiquitination and degradation of FAK requires further investigation. In the present study, MGC803 cells were treated with trypsin for 1 and 5 min and the level of Cbl-b protein was analyzed. The expression of Cbl-b increased in a time-dependent manner (Fig. 4A). Furthermore, Cbl-b interacted with FAK in the absence and presence of trypsin treatment, as demonstrated by co-immunoprecipitation (Fig. 4B). In order to further investigate the function of Cbl-b in cell detachment, previously established shRNA plasmids targeting Cbl-b, which were stably transfected into MGC803 cells (16), were used for further experiments. The shRNA plasmids targeting Cbl-b were also stably transfected into MDA-MB-231 cells and Cbl-b expression was evaluated by western blotting (Fig. 4C). Knockdown of Cbl-b increased the expression of FAK (Fig. 4D). These results indicate that Cbl-b accelerates trypsin-induced cell detachment via degradation of FAK.