Author Affiliations: Department of Hepatobiliary and Pancreatic Surgery, the First Af?liated Hospital, Zhejiang University School of Medicine (Yu J, Zhang W, Gao F, Liu YX, Chen ZY and Zheng SS); Key Laboratory of Multi-Organ Transplantation of Ministry of Public Health (Cheng LY, Xie SF and Zheng SS), Hangzhou 310003, China

Corresponding Author: Shu-Sen Zheng, MD, PhD, FACS, Department of Hepatobiliary and Pancreatic Surgery, the First Affiliated Hospital, Zhejiang University School of Medicine; Key Laboratory of Multi-organ Transplantation of Ministry of Public Health, Hangzhou 310003, China (Tel/Fax: 86-571-87236601; Email: zyzss@zju.edu.cn)

 

© 2014, Hepatobiliary Pancreat Dis Int. All rights reserved.

doi: 10.1016/S1499-3872(14)60029-1

Contributors: ZSS proposed the study. YJ, ZW, GF, LYX, CZY, CLY and ZSS performed research and wrote the first draft. YJ and XSF collected and analyzed the data. All authors contributed to the design and interpretation of the study and to further drafts. ZSS is the guarantor.

Funding: This study was supported by grants from National High-Tech Research and Development Projects (863 Program) (2012AA021002), Organ Transplantation Key Technology Project (863 Program) (2012AA022409) and Zhejiang Province Natural Science Foundation (LQ12H03002 and LY12H03010).

Ethical approval: Not needed.

Competing interest: No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

BACKGROUND: FBW7 is a tumor suppressor which regulates a network of proteins with central roles in cell division, cell growth and differentiation. This study aimed to evaluate the role of FBW7 in chemosensitivity and epithelial-mesenchymal transition (EMT) in different hepatocellular carcinoma (HCC) cell lines and to investigate the relevant underlying mechanisms.

 

METHODS: Different human HCC cell lines (Hep3B, Huh-7, and SNU-449) were cultured. The cell viability was evaluated by cell counting kit-8, and FBW7 mRNA transcription and protein expression were quantitated by real-time PCR and Western blotting. Expressions of vimentin (mesenchymal biomarker) and E-cadherin (epithelial biomarker) were evaluated by Western blotting and immunocytochemistry. Cell invasion was assayed by Transwell migration, and FBW7 plasmid or siRNA was used to evaluate the effect of FBW7 overexpression or silencing on cell chemosensitivity.

 

RESULTS: FBW7 expression affected tumor cell chemosensitivity to doxorubicin and tumor cell invasive capacity in different HCC cell lines. FBW7^hi (high FBW7 expression) Hep3B and FBW7^mi (median FBW7 expression) Huh-7 cells were more sensitive to doxorubicin and lower in invasive capacity than FBW7^lo (low FBW7 expression) SNU-449 cells. Silencing of FBW7 in Huh-7 and Hep3B cells induced the resistance to doxorubicin and enhanced cell invasion, whereas overexpression of FBW7 in SNU-449 cells restored the sensitivity to doxorubicin and significantly reduced invasive capacity. Furthermore, doxorubicin induced EMT toward mesenchyme in HCC cells. Downregulation of FBW7 in Huh-7 and Hep3B cells or upregulation of FBW7 in SNU-449 cells altered the direction of EMT.

 

CONCLUSIONS: The level of FBW7 expression impacted the tumor resistance to doxorubicin and the invasion capability of HCC cells. FBW7 therefore may be a potential target for the chemotherapy of HCC through the regulation of EMT.

 

(Hepatobiliary Pancreat Dis Int 2014;13:184-191)

 

KEY WORDS: FBW7; hepatocellular carcinoma; doxorubicin; epithelial-mesenchymal transition; drug resistance

Hepatocellular carcinoma (HCC) is the sixth most common solid tumor worldwide. Based on the GLOBOCAN2008 estimates, there were 10.8 cases and 9.9 deaths per 100 000 people.^[1] About 80%-90% of HCCs were not resectable when diagnosed and therefore, chemotherapy might be the only option for some of these patients. However, the chemotherapy is far from satisfaction due to the development of multidrug resistance (MDR). Some proteins, such as F-box and WD repeat domain-containing 7 (FBW7), play important roles in chemosensitivity.

 

FBW7, the substrate recognition component of the SKP1-CUL1-F-box (SCF) ubiquitin ligase complex, is an evolutionarily conserved member of the F-box protein family. FBW7 regulates a network of signal proteins functioning in cell division, cell growth and differentiation.^[2] FBW7 binds to substrates after they have been phosphorylated within conserved phospho-degron motifs, called the Cdc4 phospho-degrons (CPD). SCF complexes bind to protein substrates, targeting them for ubiquitylation and subsequent degradation by proteasomes.^[3] Fully phosphorylated FBW7 substrates may facilitate cells to rapidly and coordinately downregulate cell growth and division in response to antiproliferative signals.^[2] Although FBW7 mutation, which results in dysregulation of apoptosis and chemosensitivity, may be associated with MDR,^[4] the mechanism is still unclear.

 

Epithelial-mesenchymal transition (EMT) is also associated to the development of MDR. EMT describes the process by which cells gradually lose typical epithelial characteristics and acquire mesenchymal traits.^[5] EMT can pathologically lead to carcinoma progression, invasion and metastasis, and may thereby play a key role in enabling carcinoma cells to evade chemotherapy.^[6] Epithelial functions that are lost during EMT include disordered expression and distribution of proteins, such as β-catenin and E-cadherin, which mediate loss of cell-cell, cell-matrix contacts and cytoskeletal organization responsible for normal epithelial polarity. The concurrent gain of mesenchymal characteristics includes the ability to migrate and invade the surrounding matrix, through expression of proteins such as vimentin and fibronectin.^[5,7] Epithelial-type HCC cells are more chemosensitive than mesenchymal cells;^[8] when HCC cells undergoing EMT re-express E-cadherin, they regain chemosensitivity.^[5] The present study was to evaluate the relationship between doxorubicin sensitivity and FBW7 expression in HCC cells, to investigate whether FBW7 mutation interfered with EMT, and to explore the underlying mechanisms.

 

 

Human HCC cell lines (Hep3B, Huh-7, and SNU-449) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and maintained according to ATCC guidelines. Hep3B cells were cultured in minimum essential medium (Gibco), Huh-7 cells in Dulbecco's modified Eagle's medium (Gibco) and SNU-449 cells in RPMI 1640 medium (Gibco). All of these media were supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells were maintained at 37 �� with 5% CO₂. Doxorubicin was purchased from Sigma (St. Louis, MO, USA)

 

HCC cells were seeded into 6-well plates (2×10⁵ cells/well) and incubated overnight until they were adherent. After 72-hour doxorubicin treatment, the cells were harvested in 100 µL of cell lysis buffer (Cell Signaling, Danvers, MA, USA) containing protease inhibitors (Sigma). Then, cell lysates were separated by 10% SDS-PAGE and transferred electrophoretically to polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). Membranes were then blocked by 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.1% Tween-20 and followed by incubation overnight with primary antibodies (E-cadherin, vimentin and FBW7; Cell Signaling). After incubation with secondary antibody conjugated to goat anti-mouse horseradish peroxidase (GE Healthcare, Piscataway, NJ, USA) for 2 hours, membranes were washed, and immunoreactive bands were detected by ECL (GE Healthcare) and visualized by autoradiography (X-ray film; Kodak, Rochester, NY, USA). Protein loading was normalized against GAPDH (Cell Signaling).

 

The HCC cell lines were cultured as described above. Total RNA was extracted with Trizol (Invitrogen), and cDNA was synthesized using a cDNA synthesis kit (Takara, Japan) following the manufacturer's instructions. The mRNA levels of FBW7 were measured by real-time PCR using the following gene-specific primers (Takara): forward, 5'-CAC TCA AAG TGT GGA ATG CAG AGA C-3'; reverse, 5'-GCA TCT CGA GAA CCG CTA ACA A-3'. The relative expression of FBW7 mRNA was normalized to β-actin using the β-actin gene-specific primers (Takara): forward, 5'-GGA GAT TAC TGC CCT GGC TCC TA-3'; reverse, 5'-GAC TCA TCG TAC TCC TGC TTG CTG-3'.

 

A cell counting kit-8 kit (CCK-8; Dojindo, Kumamoto, Japan) was used to evaluate the relative cell viability. In brief, the cells were seeded into a 96-well microplate (5×10³ cells/well) and incubated overnight until they were adherent. After 24 hours of culture in a serum-free medium, HCC cells were treated with doxorubicin at different concentrations for 72 hours. Then, 10 µL CCK-8 solution was added into the medium and incubated for another 2 hours. The absorbance of the medium was measured at 450 nm using an MRX II microplate reader (Dynex, Chantilly, VA, USA).

 

HCC cell lines were transfected with FBW7 siRNA (Santa Cruz Biotechnology) diluted in lipofectamine 2000 (10 µmol/mL; Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. After 12-hour transfection, the medium was replaced with a complete medium and HCC cells were incubated for specified times. The effects of transfection on cell viability were tested by CCK-8.

 

Recombinant overexpression plasmid designed in our lab (named as "pLVX-FBW7-IRES-ZsGreen1") was transfected into the HCC cell lines using lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. All experiments were performed in 12 hours after transfection, and repeated for three times. The effects of transfection on cell viability were tested by CCK-8.

 

The HCC cell lines were seeded into 24-well plates and treated as described above. HCC cells were fixed with 4% formaldehyde for 15 minutes, washed with PBS, and incubated with 5% BSA for 30 minutes at room temperature (RT). Then, the HCC cells were stained with mouse anti-human vimentin or anti-human E-cadherin primary antibodies (Cell Signaling) at 4 �� overnight. The cells were subsequently incubated with goat anti-mouse IgG-FITC (vimentin) or IgG-Cy5 (E-cadherin) secondary antibodies (Abcam, Cambridge, MA, USA) in PBS for 2 hours at 4 ��. The cells were incubated for 2 minutes at RT with 4, 6-diamidino-2-phenylindole (DAPI) (Sigma) after staining, washed twice with phosphate buffered saline (PBS), and visualized under an inverted fluorescence IX81 microscope (Olympus, Tokyo, Japan).

 

HCC cells seeded into the upper chambers of polycarbonate membrane treated Transwell® compartment (Corning Incorporated, Corning, NY, USA) pre-coated with basement membrane matrix (BD Bioscience, Bedford, MA, USA) were cultured in serum-free medium. The lower receiver well was added with 10% FBS as chemoattractant. After 48-hour incubation, the invaded cells were fixed and stained by crystal violet. The number of stained cells was counted in five randomly selected fields within each insert under an inverted fluorescence IX81 microscope, and the invasive rates were calculated by dividing the number of invaded cells and the number of cells seeded in each insert.

 

Statistical analysis was made using Prism 5 software (GraphPad, San Diego, CA, USA). Data were presented as the mean±SD. Two-way ANOVA was used to compare the difference in the inhibitory effects of doxorubicin and combined therapy after the Bonferroni post-hoc test. For all the tests, statistical significance was set at P<0.05.

 

 

FBW7 protein and mRNA expressions were significantly higher in Hep3B and Huh-7 cells compared with those in SNU-449 cells (FBW7^lo) (Fig. 1A, B). Hep3B cells had the highest expression of FBW7 (Hep3B: FBW7^hi; Huh-7: FBW7^mi). CCK8 assay after 72-hour treatment with doxorubicin in three HCC cells revealed that increased doses of doxorubicin reduced cell viability in the three HCC cell lines. However, FBW7^hi Hep3B and FBW7^mi Huh-7 cells were more sensitized than FBW7^lo SNU-449 cells (Fig. 1C). Mean IC50 values for doxorubicin in Hep3B, Huh-7 and SNU-449 cells were 0.1267 µg/mL (0.1136-0.1398 µg/mL; 95% CI), 0.8160 µg/mL (0.7185-0.9648 µg/mL; 95% CI), and 1.2700 µg/mL (1.1110-1.4280 µg/mL; 95% CI), respectively (Table).

 

To further confirm the relationship between FBW7 expression and doxorubicin chemosensitivity in HCC cells, FBW7 siRNA was used to silence FBW7 expression in all three HCC cell lines and the interference efficiency was measured by Western blotting (Fig. 2). The dose-dependent viability assay showed that silencing FBW7 expression led to downregulation of doxorubicin sensitivity in the cell lines (Fig. 2). FBW7 siRNA co-treatment significantly attenuated doxorubicin cytotoxicity in Hep3B and Huh-7 cells (two-way ANOVA; P<0.001) with concurrent increases in IC50 values of doxorubicin 0.5440 µg/mL (0.4230-0.6650 µg/mL; 95% CI), and 3.0230 µg/mL (1.6990-4.3460 µg/mL; 95% CI), respectively. These values were similar to those in SNU-449 cells. The IC50 value of doxorubicin was 7.6350 µg/mL (2.9990-12.2700 µg/mL; 95% CI) in FBW7 siRNA transfected cells (Table).

 

Transfection with FBW7 plasmid (pLVX-FBW7-IRES-ZsGreen1) was used to upregulate FBW7 expression in the HCC cell lines. Expression levels were also measured by Western blotting (Fig. 2), and the effect on chemosensitivity was verified by cell viability assays as described above. The results showed that FBW7 plasmid transfection significantly increased doxorubicin-induced cytotoxicity in Hep3B, Huh-7 and SNU-449 cells (Fig. 2), with concurrent decreases in IC50 values of doxorubicin 0.0828 µg/mL (0.0729-0.0928 µg/mL; 95% CI), 0.4514 µg/mL (0.3980-0.5049 µg/mL; 95% CI), and 1.0950 µg/mL (0.8816-1.3090 µg/mL; 95% CI), respectively (Table).

 

To ascertain whether the disparity in response to doxorubicin between Huh-7/Hep3B cells and SNU-449 cells was phenotype dependent, Western blotting and immunocytochemistry were used to measure the expression of phenotype markers in HCC cells. Western blotting revealed that doxorubicin-resistant FBW7^lo SNU-449 cells showed the lowest E-cadherin expression, but the highest vimentin expression. In contrast to FBW7^hi Hep3B and FBW7^mi Huh-7 cells (Fig. 3A), the results were similar in immunocytochemistry, with FBW7^lo SNU-449 cells displaying an almost complete absence of E-cadherin signal (red), but high level of vimentin signal (green), compared to FBW7^hi Hep3B and FBW7^mi Huh-7 cells (Fig. 3B).

 

After doxorubicin treatment (IC50 concentration) for 72 hours, the three HCC cell lines showed reduced expression of FBW7 (Fig. 4), accompanied with significantly increased levels of vimentin and decreased levels of E-cadherin. The effect was more obvious in FBW7^hi Hep3B and FBW7^mi Huh-7 cells (Fig. 5A, C, E). Immunocytochemistry showed similar results, i.e., lower expression of E-cadherin and higher expression of vimentin in doxorubicin treated cells (Fig. 5B, D, F)

 

To confirm whether FBW7 is involved in the doxorubicin-induced EMT in HCC cells, we transfected mesenchymal FBW7^lo SNU-449 cells with FBW7 plasmid and epithelial FBW7^hi Hep3B and FBW7^mi Huh-7 cells with siRNA, respectively. Western blotting analysis showed that SNU-449 cells showed a higher expression of E-cadherin and a lower expression of vimentin after transfection with FBW7 plasmid (Fig. 6A), suggesting that these cells were transformed to epithelial phenotype after FBW7 upregulation. Immunocytochemistry showed that SNU-449 cells displayed a higher expression of E-cadherin (red) and a lower expression of vimentin (green) after FBW7 plasmid transfection (Fig. 6B).

 

In contrast, after transfection of FBW7^hi Hep3B and FBW7^mi Huh-7 cells with FBW7 siRNA to silence FBW7 expression, both Western blotting analysis and immunocytochemistry revealed a decreased expression of epithelial marker protein E-cadherin and an increased expression of mesenchymal marker protein, vimentin (Fig. 6C-F).

 

In addition, matrigel invasion assay showed that mesenchymal FBW7^lo SNU-449 cells had higher invasive capacity than epithelial FBW7^hi Hep3B and FBW7^mi Huh-7 cells (Fig. 7). The transfection of FBW7 plasmid significantly reduced invasion in SNU-449 cells. In line with this, transfection of FBW7 siRNA significantly increased invasive capacity in Hep3B and Huh-7 cells (Fig. 7).

 

 

As a serious problem in carcinoma treatment,^[9] MDR is hindering the clinical use of common chemotherapeutic agents such as doxorubicin and cisplatin.^{[10, 11]} Resistance to doxorubicin leads to a lower response rate and the use of high doses of drugs, giving rise to severe adverse effects.^[12] In our study, FBW7 was associated with doxorubicin sensitization in HCC cells. FBW7^hi Hep3B cells and FBW7^mi Huh-7 cells displayed a higher level of chemosensitivity to doxorubicin than FBW7^lo SNU-449 cells. However, FBW7 siRNA inhibited the sensitivity to doxorubicin in all three cell lines. In comparison, the upregulation of FBW7 expression by transfecting FBW7 plasmid enhanced the sensitivity of doxorubicin. This finding indicated that FBW7 expression in HCC cells was correlated with the chemosensitivity to doxorubicin. Upregulation of FBW7 significantly enhanced the cytotoxicity of doxorubicin in HCC cells. As a tumor suppressor, FBW7 regulated a network of proteins with central roles in cell division, cell growth and differentiation.^[2] Studies^[13-17] reported FBW7 loss-of-function mutations in cell lines from several primary cancers as well as FBW7 mutations in approximately 6% of tumors.^[2] In recent studies,^{[2, 4]} FBW7 mutations were found to be linked to MDR. FBW7-deficient T-ALL cells were particularly resistant to the BCL2 antagonist ABT-737 and primary tumors enriched for FBW7 inactivation were resistant to anti-tubulin chemotherapeutics. Although these findings highlight the role of FBW7 in drug resistance and subsequent oncogenesis,^[16] its precise mechanisms are not clear. In the present study, doxorubicin resistance in FBW7^lo HCC cells was associated with EMT processes. EMT biomarker patterns demonstrated that FBW7^lo SNU-449 and FBW7^hi Hep3B or FBW7^mi Huh-7 cells belonged to two different phenotypes, and that FBW7^hi Hep3B and FBW7^mi Huh-7 cells displayed more epithelial characteristics (E-cadherin) than FBW7^lo SNU-449 cells. Similarly, the expression of mesenchymal biomarker (vimentin) was higher in FBW7^lo SNU-449 cells than in FBW7^hi Hep3B or FBW7^mi Huh-7 cells, indicating that epithelial HCC cells are more sensitive to doxorubicin therapy than mesenchymal cells. Furthermore, our study showed that knockdown of FBW7 altered EMT patterns in FBW7^hi Hep3B and FBW7^mi Huh-7 cells, namely, upregulation of mesenchymal biomarkers and downregulation of epithelial biomarkers.

 

In the present study, we evaluated the potential role of EMT in doxorubicin resistance associated with low FBW7 expression. In HCC cells, EMT is related to proliferation, invasion, and metastasis in HCC;^[18] and potent oncogenicity associated with EMT may be a contributory factor in inefficient drug responses and carcinoma recurrence in current systemic therapies.^[19] In addition, there is evidence that reverse EMT, referred to as mesenchymal-epithelial transition (MET), enables HCC cells to regain chemosensitivity.^[5]

 

In conclusion, our study showed that decreased expression of FBW7 leads to doxorubicin resistance in HCC cells, and that the underlying mechanism is associated with regulation of the EMT/MET process. Thus we propose that FBW7, a tumor suppressor, is potentially i mportant in searching for a future chemotherapy for HCC. Targeting FBW7 is capable of eliminating drug resistance in chemotherapy of HCC.

 

 

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