Fragile histidine triad (FHIT) is a tumor suppressor protein that regulates cancer cell proliferation and apoptosis. However, its exact mechanism of action is poorly understood. Phosphatidylinositol 3-OH kinase (PI3K)-Akt-survivin is an important signaling pathway that was regulated by FHIT in lung cancer cells. To determine whether FHIT can regulate this pathway in cholangiocarcinoma QBC939 cells, we constructed an FHIT expression plasmid and used it to transfect QBC939 cells. Protein and mRNA expression were measured by western blotting and qRT-PCR, respectively. The viability and apoptosis of QBC939 cells were then assessed using MTT assays and flow cytometry. Our results revealed that the expression of survivin and Bcl-2 was downregulated, and caspase 3 was upregulated, in cells overexpressing FHIT. In addition, FHIT suppressed the phosphorylation of Akt. The changes in cell proliferation and apoptosis were obvious in cells overexpressing FHIT which parallels that of treatment with LY294002, a potent inhibitor of phosphoinositide 3-kinases. Treatment with LY294002 further decreased the expression of survivin and Bcl-2 and increased caspase-3 levels. These results suggest that FHIT can block the PI3K-Akt-survivin pathway by suppressing the phosphorylation of Akt and the expression of survivin and Bcl-2 and upregulating caspase 3.
Cholangiocarcinoma is a highly aggressive malignancy, with an extremely poor prognosis. Currently [
A growing body of evidence suggests that specific fragile sites in human chromosomes play roles in tumorigenesis. Research has focused on FHIT. FHIT is altered in many different kinds of primary or advanced carcinomas. Specifically, deletions within both FHIT alleles result in the loss of exons with a concomitant absence of full-length FHIT transcript and protein. Loss of expression of the FHIT gene with a loss of regulatory control is common in epithelial malignancies. The FHIT gene expression has been found in primary tumors and cell lines derived from lung, breast, head and neck, esophagus, stomach, colon and rectum, pancreas, kidney, cervix, and liver cancer [
Although data suggest that FHIT is a tumor suppressor gene, its exact mechanism of action and the signaling pathways it activates are poorly understood. PI3K-Akt pathway was an important pathway which could promote proliferation and inhibit apoptosis [
The human cholangiocarcinoma QBC939 cell line was obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). It was cultured in RPMI 1640 (Sigma, St. Louis, MO, USA) medium supplemented with 10% fetal bovine serum and penicillin and streptomycin (Sigma) at 37°C in a humidified 5% CO2 atmosphere. Rabbit polyclonal antibodies against FHIT were purchased from Life Technologies (USA). Rabbit polyclonal antibodies against survivin, Bcl-2, caspase-3, AKT and p-AKT were from Genscript Co. (USA).
Human FHIT cDNA was amplified from total RNA isolated from QBC939 cells (a human cholangiocarcinoma cell line) by RT-PCR using the primers 5′-ccaatggatccATGTCGTTCAGATTTGGCCA-3′ and 5′-ccaatctcgagTCACTGAAAGTAGACCCGCAGA-3. The PCR product was cloned into the pcDNA3.1 plasmid (Life Technologies). The sequences of all constructs were confirmed by sequencing. The transfection of plasmids into QBC939 cells was performed using Lipofectamine (Life Technologies), and positive clones were selected using neomycin. The cells were divided into four groups: a wild-type group (cell), a negative control group (NC; transfected with empty vector), an experimental group that was transfected with the FHIT overexpression plasmid (FHIT), and an LY294002 (PI3K-Akt signal pathway inhibitor) treatment group that was treated with 20
QBC939 cells were cultured and transfected with FHIT plasmid or empty vector. RT-PCR was performed to detect the expression of
Cells were harvested and lysed in radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM EDTA, 1 mM EGTA, 1% nonidet P-40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl sulfate). Protein concentrations were calculated using a BCA protein assay kit (Thermo Fisher Scientific, USA), and equal amounts of total protein per lane were separated by SDS gel electrophoresis and transferred to polyvinylidene fluoride membranes (Sigma, USA) using semidry transfer. Nonspecific binding of proteins to the membrane was blocked by incubation with TBS-T buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, and 0.1% Tween-20) containing 5% skimmed milk. Membranes were then incubated with the appropriate primary and horseradish peroxidase-conjugated secondary antibodies. The primary rabbit polyclonal antibodies were all purchased from Cell Signaling Technologies (Beverly, MA, USA). Western blot images were captured using an Epi Chemi II Darkroom and Sensicam imager with Labworks 4 software (UVP; PerkinElmer Life Science, Boston, MA, USA).
QBC939 cells were seeded into 96-well plates (3,000 per well) in a final volume of 200
Cells in the different groups were treated as indicated, and Annexin V-FITC/PI assays were performed following the manufacturer’s protocol (Roche Diagnostics, Pleasanton, CA, USA). Briefly, cultured cells were harvested, washed with binding buffer, and incubated in 200
All statistical analyses were performed using SPSS ver. 13.0 software (SPSS Inc., Chicago, IL, USA). Pearson’s chi-squared (
To investigate the function of FHIT, we constructed an FHIT overexpression vector and transfected it into QBC939 cells. The expression of FHIT was then confirmed using RT-PCR and western blotting. The mRNA expression of FHIT was increased 7.945-fold after transfection (Figure
The expression of FHIT in QBC939 cells after transfection. (a) The expression of
To determine the role of FHIT in the PI3K-Akt pathway, we investigated the levels of p-Akt in cells transfected with the FHIT expression vector. Western blotting revealed that the levels of p-Akt were suppressed in QBC939 cells treated with LY294002 (the PI3K inhibitor) or transfected with FHIT (Figure
The levels of p-Akt were assessed by western blotting. (a) Western blotting for p-Akt. (b) Quantification of p-Akt protein levels in each group, presented in bar graphs as fold-increase.
RT-PCR analysis revealed that
The expression of survivin, Bcl-2, and caspase-3 in QBC939 cells in each group after transfection or treatment with LY294002. (a) The mRNA expression of
Cell proliferation and apoptosis were determined by MTT assay and flow cytometry, respectively. MTT assay revealed that cell proliferation was reduced in cells overexpressing FHIT or treated with LY294002 compared with the NC group (Figure
Effect of FHIT overexpression and LY294002 treatment on cell proliferation. OD (optical density) was used to assess cell proliferation at 0, 24, 48, and 72 h after treatment with LY294002 or transfection.
Apoptosis in each group was assessed after transfection or treatment with LY294002. Apoptosis was assessed after 48 h treatment with 20
The FHIT gene spans the most common fragile site in the human genome, FRA3B (3p14.2) [
PI3K/Akt pathway was an important pathway which could promote proliferation and inhibit apoptosis. Meanwhile, survivin, Bcl-2, and caspase-3 were the downstream target of Akt, which were related with proliferation and apoptosis [
We investigate the potential pathway in which FHIT regulates cells proliferation and apoptosis in cholangiocarcinoma. We found that the expression of p-Akt was decreased after FHIT was overexpressed. These observations were comparable to the effects of LY294002, which blocked the PI3K/Akt pathway by combing with inactive PI3K. So we thought FHIT could inhibit the phosphorylation of Akt, but how FHIT block PI3K/Akt pathway was needed to explore.
To confirm the effect of FHIT on PI3K/Akt pathway, we measured the expression of survivin, Bcl-2, and caspase-3 which play a key role in the regulation of cell proliferation and apoptosis. Specially, survivin was one of the members of protein family which could inhibit apoptosis, regulate the cell cycle, and promote angiogenesis [
In conclusion, the PI3K-Akt pathway may play an important role in FHIT-induced proliferation and apoptosis in cholangiocarcinoma cells. Understanding the relationship between FHIT and PI3K-Akt pathway could provide useful information not only for the early detection of cholangiocarcinoma, but also for the development of novel therapeutic strategies.
The authors declare that there is no conflict of interests regarding the publication of this paper.
Sanyuan Hu, Qiang Huang, Zhen Liu, and Fang Xie designed the study and wrote the paper. Qiang Huang, Zhen Liu, and Fang Xie performed all experimental procedures. Sanyuan Hu, Qiang Huang, Fang Xie, and Chenhai Liu were involved in data interpretation and paper preparation. Feng Shao and Cheng-lin Zhu assisted in editing the paper.
The authors acknowledge the assistance given by Dr. Ren (Hepatic-Biliary-Pancreatic Key Laboratory of Anhui Province) for experimental support. This work was supported by Anhui Medical University Science Research Foundation (Grant no. 2011kj071).