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Silencing of 14-3-3ζ over-expression in hepatocellular carcinoma inhibits tumor growth and enhances chemosensitivity to cis-diammined dichloridoplatium
Cancer Letters 303 (2011) 99–107
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Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Silencing of 14-3-3f over-expression in hepatocellular carcinoma inhibits tumor growth and enhances chemosensitivity to cis-diammined dichloridoplatium Jung Eun Choi a, Wonhee Hur a, Chan Kwon Jung b, Lian Shu Piao a, KwangSoo Lyoo a, Sung Woo Hong a, Sung Woo Kim a, Hye-Yeon Yoon d, Seung Kew Yoon a,c,⇑ a
WHO Collaborating Center of Viral Hepatitis, The Catholic University of Korea, Seoul 137-701, Republic of Korea Department of Hospital Pathology, The Catholic University of Korea, Seoul 137-701, Republic of Korea Department of Internal Medicine College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea d Department of Life Science, The Ewha Womens University, Seoul 158-056, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 19 November 2010 Received in revised form 15 January 2011 Accepted 19 January 2011
Keywords: 14-3-3f Hepatocellular carcinoma RNA interference (RNAi) Cis-diammined dichloridoplatium (CDDP) Gene therapy
a b s t r a c t The 14-3-3f protein plays a key role in regulation of cellular processes. In the present study, we showed that 14-3-3f protein was significantly overexpressed in hepatoma cell lines and human tumorous tissues of hepatocellular carcinoma (HCC) patients. Knockdown with RNA interference in hepatoma cell lines with high 14-3-3f expression suppressed tumor cell proliferation via activation of c-Jun N-terminal kinase (JNK) and p38/MAPK. Furthermore, suppression of 14-3-3f enhanced the anti-cancer effect of cis-diammined dichloridoplatium (CDDP) in hepatoma cell lines. These results suggest that silencing of 14-3-3f may be an attractive target for HCC therapeutic development. Ó 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Human hepatocellular carcinoma (HCC) is the fifth most common type of cancer and the third leading worldwide cause of cancer death [1]. Despite recent advances in treatment of HCC, options for curing the disease are limited. In cases of advanced HCC, anti-cancer chemotherapeutic drugs, such as cis-diammined dichloridoplatium (cisplatin; CDDP) and 5-fluorouracil (5-FU) have been used; however, data demonstrating their efficacy in patients is limited [2–5]. The 14-3-3 proteins are a family of highly conserved cellular proteins that play important roles in various cellular processes [6–8]. Proteins in this family contain a ⇑ Corresponding author at: Department of Internal Medicine, Seoul St. Mary’s Hospital, #505 Banpo-Dong, Seocho-gu, Seoul 137-040, Republic of Korea. Tel.: +82 2 2258 7534; fax: +82 2 536 9559. E-mail address: [email protected] (S.K. Yoon). 0304-3835/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2011.01.015
pS/pT-binding motif and regulate cellular processes, including signal transduction, cell cycle, apoptosis, cellular metabolism, stress responses, cytoskeleton organization and malignant transformation [7–9]. To date, seven mammalian 14-3-3 isoforms (b, c, e, r, g, s, and f) that interact with other key cellular proteins during tumor development and progression have been identified [6,7]. Recently, 14-33f expression has been reported in various cancer cells [10–13]. However, until now, the possible association between 14-3-3f expression and hepatocarcinogenesis has not been investigated. RNA interference (RNAi) represents a natural mechanism by which small interfering RNA (siRNA) molecules can specifically and potently down-regulate expression of target genes [14]. Advances in siRNA technology in recent years have allowed the targeting of oncogenes with high specificity and efficiency in various cancers [15]. In previous study, we identified a 14-3-3f gene that is
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up-regulated in human HCC through genome-wide differential expression analysis and randomly-primed Annealing Control Primer™ (ACP)-based reverse transcription-polymerase chain reaction (RT-PCR) [16,17]. In this study, we investigated the possible association between 14-3-3f expression and HCC, and demonstrated that down-regulation of 14-3-3f expression by RNAi not only inhibits tumor cell growth but also enhances the chemotherapeutic effects of CDDP in HCC. This is the first report to demonstrate the co-therapeutic potential of 14-3-3f and CDDP in HCC. 2. Materials and methods 2.1. Clinical samples Specimens of tumor tissue and surrounding non-cancerous tissues were obtained from the livers of 135 HCC patients who had undergone a successful partial hepatectomy at Seoul St. Mary’s Hospital (Seoul, South Korea). Sample collection was approved by the Institutional Review Board of Kangnam St. Mary’s Hospital at the Catholic University of Korea. Informed consent for the use of specimens for research purposes only was obtained from all patients. Specimens were frozen in liquid nitrogen and stored until use. 2.2. Immunohistochemical staining For immunohistochemical staining, after deparaffinization of specimens, antigen retrieval was performed by microwave vacuum histoprocessor (RHS-1, Milestone, Bergamo, Italy) at 121 °C for 15 min. After peroxidase blocking, sections were incubated with anti-14-3-3f (Santa Cruz, San Diego, CA) diluted 1:2000 in Antibody Diluent (Glolen Bridge, Mukilteo, WA) at 4 °C overnight. The sections were developed with Polymer Detection System (Golden Bridge) and DAB. Slides were counterstained with hematoxylin (ScyTek, Logan, UT) after immunostaining. Intensity of reactivity was scored using a four-tier system: 0, indicated no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The percentage of cells expressing 14-3-3f of all of the normal control tissues was <10%. Therefore, the score of 2 and 10% as a cutoff value was chosen for continuation of analyses and to define higher or lower expression of 14-3-3f in tumor tissues. The pathologist reviewed all biopsies. 2.3. siRNA production To investigate the role of 14-3-3f in the growth of human hepatoma cells, specific siRNAs targeted to 14-33f were purchased from Santa Cruz Biotechnology. Four DNA sequences were chosen as candidate 14-3-3f siRNAs: siRNA-1 (50 -GGUACAUUGUGGCU-UCAAATT-30 ), siRNA-2 (50 -GCUUCCAUGUCUAAGCAAATT-30 ), siRNA-3 (50 -CC-AGU CACAGGUGU AGUAATT-30 ), and siRNA-4 (50 -GGUUGCU AAACUUCUCUA-ATT-30 ). A scrambled siRNA, whose sequence did not match that of any mammalian sequences listed in online databases, was synthesized by Ambion
(Austin, TX) and used as a negative control. Lyophilized siRNAs were resuspended in RNase-free water at room temperature and stored at 20 °C prior to use. 2.4. Cell culture and siRNA transfection HepG2, Huh7, and Hep3B cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 100 lg/ml penicillin, and 0.25 lg/ml streptomycin, and then maintained in a humidified incubator at 37 °C with 5% CO2. siRNA transfection was performed using the NucleofectorÒ electroporation system (Amaxa Biosystems, Cologne, Germany). 2.5. Generation of stable short-hairpin siRNA (shRNA) 14-3-3f infectants To establish a stable Huh7 cell line expressing sh14-33f, cells were infected using a shRNA-lentiviral infection system (Sigma, St. Louis, MO). Three coding regions from the human 14-3-3f gene (starting positions 232, 349, and 637 [GenBank Acc. No. NM_003406]) were selected as shRNA target sequences. We constructed three 14-3-3f shRNA-carrying lentiviral vectors: LV-sh14-3-3f-1, LVsh14-3-3f-2, and LV-sh14-3-3f-3. The shRNA negative control-lentiviral particle (LV-cont) was used as a negative control. To generate stable cells, Huh7 cells were plated in twelve-well plates (1 105 cells per well), transduced with 5 MOI lentiviral particles (using 8 lg/ml hexadimethrine bromide [Sigma]), and incubated at 37 °C with 5% CO2. Suppression of 14-3-3f in stable cells was confirmed by western blot analysis. 2.6. Western blot analysis Human hepatoma cells, liver tissue protein samples and a normal liver lysate (G-Biosciences, St. Louis, MO) were separated by 12% SDS–PAGE and transferred to a nitrocellulose membrane (Whatman, Maidstone, Kent, UK). Blots were then probed with monoclonal anti-b-actin (Sigma), polyclonal anti-14-3-3f (Santa Cruz), polyclonal anti-PCNA (Santa Cruz), monoclonal anti-phospho-JNK (Cell Signaling Technology, Beverly, MA), polyclonal anti-JNK1 (Santa Cruz), anti-phospho-p38 (Santa Cruz), anti-p38 (Santa Cruz), anti-phospho-ERK (Santa Cruz) and anti-ERK1 (Santa Cruz) antibodies overnight at 4 °C. Membranes were washed with TBS containing 0.05% Tween-20 (TBS-T) and then incubated with horseradish peroxidase-conjugated anti-mouse and-rabbit secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ; diluted 1:5000). Protein bands were visualized using an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. 2.7. Cell proliferation assays, apoptosis assays and cell cycle analysis To measure cell growth, cultured cells were harvested and counted using a Vi-Cell automated cell viability analyzer (Beckman-Coulter, Fullerton, CA). In addition,
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following transfection with 14-3-3f-specific siRNA, MTS assay was performed using the CellTiter 96 AQueous Assay System (Promega, Madison, WI). Absorbances were measured at 490 nm using a SpectraMax 250 Microplate Reader (Molecular Devices, Sunnyvale, CA). Apoptosis was identified using an Annexin-V apoptosis kit (BD BioSciences, San Diego, CA) according to the manufacturer’s instructions. A total of 10,000 cells were counted by flow cytometry using a fluorescence-activated cell sorter (FACS; Becton–Dickinson, San Jose, CA). Resulting data were analyzed using CellQuest software (BD BioSciences). For cell cycle analysis, stable Huh7 cells were seeded in a 100-mm culture dish the day before the synchronization experiment. The cells were incubated in DMEM without serum for 30 h and then the cell cycle was initiated with DMEM containing 10% FBS for 12 h. The cells were harvested and fixed in 70% ethanol for 30 min at 20 °C. Fixed cells were stained with 50 lg/ll propidium propidium idodie (PI; BD Biosceinces) and 100 lg/ml RNase (Sigma). Cell cycle profile of infected Huh7 cells was measured by FACS, and resulting data were analyzed using CellQuest software (BD BioSciences).
2.8. Soft agar colony assay shRNA-lentiviral particle-infected Huh7 cells (2 104 cells) were seeded onto 0.7% (w/v) microbiologygrade agarose prepared in complete medium. The upper layer was poured onto a layer of sterile 1% (w/v) agarose prepared in complete medium that had been allowed to solidify in 35-mm culture dishes. Dishes were incubated at 37 °C under 5% CO2 for 3–4 weeks. Resulting colonies were stained for >2 h with 0.005% Crystal Violet. The number of colonies with a diameter >20 lm in randomly selected fields (50 magnification) was counted.
2.9. Statistical analysis All experiments were performed at in least triplicate. Statistical comparisons were made by analysis of variance (ANOVA) using SPSS 13.0 software (Chicago, IL). A p-value of <0.05 was considered statistically significant.
3. Results 3.1. Over-expression of 14-3-3f in HCC To determine whether 14-3-3f was expressed in HCC, human hepatoma cells (HepG2, Huh7, and Hep3B) and human HCC specimens were analyzed immunohistochemically and by western blot analysis. Compared to normal liver lysates, three hepatoma cell lines displayed enhanced 14-3-3f expression (Fig. 1A). 14-3-3f was expressed at higher levels in 72 of 135 (53.3%) HCC tissues analyzed (Fig. 1B). Although 143-3f expression was observed in biliary duct epithelial cells in nontumorous tissues, most 14-3-3f expression was shown in tumorous tissues. In addition, western blot analysis of 20 paired HCC samples showed that 14-3-3f was more highly expressed in tumor tissues than in noncancerous liver tissues (Fig. 1C). The optical density of each band from tumorous (T) and non-tumorous (NT) tissues in the same patient was measured using TINA image analysis software. The ratio between the optical density of 14-3-3f and b-actin in the same sample was calculated as the relative content. As shown in Fig. 1C, the 14-3-3f/b-actin ratio was
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high, greater than 1.5-fold, in 12 of 20 (60%) T compared with corresponding NT. These results indicated involvement of 14-3-3f in HCC development. 3.2. Silencing of 14-3-3f suppresses cell proliferation To elucidate the role of 14-3-3f in HCC, RNAi studies using 14-3-3f specific siRNAs were performed to eliminate expression of endogenous 14-3-3f. Transfection of HepG2 cells with 50 nM si14-3-3f markedly inhibited 14-3-3f protein expression by more than 50% within 1 day, whereas scrambled siRNA (50 nM) did not suppress 14-3-3f expression (data not shown). Transfection of HepG2 cells with si14-3-3f effectively and specifically silenced transcription of 14-3-3f. Next, we investigated whether 14-3-3f siRNA transfection affected cell proliferation and survival. As illustrated in Fig. 2A, cell growth was reduced in si14-3-3f-transfected HepG2 cells compared to scrambled siRNA-transfected cells. This inhibitory effect was confirmed by MTS proliferation assay and was especially prominent at 4 days post-transfection (p = 0.009) (Fig. 2B). These results suggest that up-regulation of 14-33f in HCC cells enhances cell growth and survival. To test the hypothesis that silencing of 14-3-3f suppresses HCC cell proliferation, we assessed expression of PCNA in si14-3-3f-transfected HepG2 cells during the first 4 days following transfection. Levels of PCNA in si14-3-3f-transfected HepG2 cell lysates were determined by western blot analysis (a representative blot is shown in Fig. 2C). Expression of PCNA in si14-3-3f-transfected cells declined markedly in a dose-dependent manner. These results demonstrate that 14-3-3f over-expression is associated with enhanced tumor cell growth in HCC. 3.3. Effects of RNAi targeting of 14-3-3f on the cell cycle and apoptosis To address the mechanisms underlying RNAi-mediated inhibition of proliferation in HCC cells, we established Huh7 cells in which 14-3-3f was depleted through stable expression of shRNAs directed against 143-3f mRNA. Western blot analysis of LV-sh14-3-3f-1 and LV-sh14-3-3f3 stable infectants revealed that expression of 14-3-3f was decreased markedly relative to the control, whereas that of LV-sh14-3-3f-2 stable infectants was decreased slightly (Fig. 3A). We further investigated the influence of LV-sh14-3-3f stable infectants on apoptosis and cellular DNA content using Annexin-V apoptosis kit and PI staining. As shown in Fig. 3B, the rates of apoptosis in Huh7 cells infected with LV-sh14-33f-3 were comparable with those of control cells. Moreover, we determined cell cycle profile using synchronized and released cells. The distribution of LV-sh14-3-3f-3 infectants in G1 phase was significantly higher than that of LV-cont infectants (Fig. 3C). All experiments were performed more than three times independently. These results suggest that RNAimediated stable depletion of 14-3-3f expression in Huh7 cells induces G1 arrest, but not apoptosis. 3.4. 14-3-3f suppression inhibits tumorigenicity To determine whether suppression of 14-3-3f inhibits tumorigenicity, we performed a soft agar assay using a shRNA-lentiviral infection system. LV-sh14-3-3f-1- and LV-sh14-3-3f-3-transfected and control Huh7 cells were seeded onto medium containing soft agar and incubated for 4 weeks. Colonies greater than 20 lm in diameter were then counted. As shown in Fig. 4, colony formation was significantly inhibited as a result of the absence of 14-3-3f. These results indicate that suppression of 14-33f results in significant inhibition of HCC cell proliferation in vitro and, thus, that 14-3-3f may be involved in hepatocarcinogenesis. 3.5. 14-3-3f suppression synergistically enhances CDDP induced cytotoxicity in vitro To evaluate the effect of 14-3-3f knockdown on chemosensitivity, si14-3-3f-transfected HepG2 cells were exposed to 1 nM CDDP and subjected to cell viability and proliferation analyses. We used a minimal dose of CDDP to avoid excessive cell death due to the concern that higher doses of CDDP might be highly damaging to cells electroporated with si14-3-3f. We also examined the effect of CDDP on expression of PCNA, a marker of cell proliferation, in si14-3-3f-transfected cells. As shown in Fig. 5A, western blot analysis showed that PCNA expression in CDDP-treated HepG2 transfectants was significantly lower than in untreated HepG2
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Fig. 1. 14-3-3f expression in hepatocellular carcinoma. (A) Western blot analysis for detection of 14-3-3f in different HCC cell lines HepG2, Huh7, Hep3B and normal liver. Mouse monoclonal b-actin was used as the loading control. (B) Immunohistochemical staining pattern of 14-3-3f in tumorous tissues (a– c) and non-tumorous tissue (d). The tumor cells show weak (a), moderate (b), strong (c) 14-3-3f expression while the biliary duct epithelial cells in nontumorous portion also express 14-3-3f protein. Original magnification 400. (C) Expression of 14-3-3f by western blot analysis in matched tumorous (T) and non-tumorous tissues (NT) from 20 HCC patients. The results shown are from one representative experiment among three replicates. The band densities were quantified with TINA imaging analysis software, using b-actin as a reference. The data are expressed relative to the density of the control.
transfectants. Moreover, CDDP exerted a stronger inhibitory effect on cells treated with the maximum tested concentration of si14-3-3f. Next, to determine whether 14-3-3f knockdown enhanced the apoptotic effects of CDDP, Huh7 cells harboring LV-sh14-3-3f-3 or LV-cont were exposed to 100 lM CDDP for 72 h and then evaluated by flow cytometry using an Annexin-V apoptosis kit. After treatment with CDDP, the rate of apoptosis was higher in LV-sh14-3-3f-3-infected Huh7 cells than in control cells (Fig. 5B). Collectively, these results indicate that CDDP induces apoptotic cell death and reduces cell proliferation in 14-3-3f-knockdown cells in a dose- and time-dependent manner.
3.6. 14-3-3f suppression activates JNK and p38 pathway but not ERK MAP kinase To delineate the molecular mechanisms underlying the enhanced chemosensitivity of 14-3-3f-knockdown HCC cells, we initially hypothesized that 14-3-3f suppresses activation of ERK, JNK, and p38/MAPK. To address this hypothesis, we investigated the silencing of 14-3-3f on the downstream activation of the MAPK pathways by detecting phosphorylated ERK, JNK and p38/MAPK in si14-3-3f-transfected HepG2 cells. The levels of JNK and p38/MAPK phosphorylation in si14-3-3f-transfected cells were increased as compared this level in control and scrambled siRNA-transfected cells. However, the phosphorylation of ERK was not changed in si14-3-3f-transfected cells. These results imply that enhanced chemosensitivity by suppression of 14-3-3f seems to be associated with the activation of the JNK and p38/MAPK pathway.
4. Discussion We successfully identified several genes that show aberrant expression in human HCC, using an ACP-based RT-PCR approach to profile differential expression [16]. Of the differentially expressed genes identified, 14-3-3f was selected for further functional characterization due to the strong over-expression of its protein product in hepatoma cell lines. To date, the role of 14-3-3f in HCC remains unclear. In this study, we demonstrated the over-expression of 14-33f in human HCC specimens by western blot and immunohistochemical analyses. 14-3-3f was found to be overexpressed in more than 53.3% of the cancerous HCC tissues tested, but in none of the surrounding non-cancerous tissues. These results suggest involvement of 14-3-3f in hepatocarcinogenesis. To best of our knowledge, this is the first report on 14-3-3f characterization in human HCC. This finding encouraged us to investigate the functional regulation of 14-3-3f in HCC cells in order to determine whether its over-expression contributes to development of HCC.
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Fig. 2. Suppression of 14-3-3f inhibits HepG2 cells proliferation. Cell proliferation of si14-3-3f-transfected HepG2 cells determined by the trypan blue exclusion (A) and MTS assay (B). (A) Transfected HepG2 cells were trypsinized and stained using trypan blue each 1 day. Viable cells were counted by ViCell™ Automated Cell Viability Analyzer. (B) Cell proliferation of transfected HepG2 cells were determined by MTS assay. The absorbance was read at 490 nm using spectrophotometer microplate reader. Data represent the mean and standard deviation of three independent experiments. (C) Cell extracts were prepared and analyzed by Western blot analysis using the antibody against 14-3-3f, PCNA, and b-actin. C, HepG2; S, scrambled siRNA (50 nM); f25, si14-3-3f (25 nM); f50, si14-3-3f (50 nM). ⁄p < 0.05.
To explore the possibility that 14-3-3f may be an effective therapeutic target, we used RNAi to silence endogenous 14-3-3f expression in human hepatoma cells, and analyzed the characteristics of transiently or stably transfected hepatoma cells. As shown in Fig. 2C, transfection of HepG2 cells with si14-3-3f dose-dependently inhibited 14-3-3f protein expression, indicating that transfection of these cells with si14-3-3f specifically and effectively blocks 14-3-3f transcription. These observations are consistent with those of previous studies utilizing 14-3-3f siRNA-mediated gene silencing in lung and breast cancer cells [18,19]. Next, we examined the effect of si14-3-3f on hepatoma cell proliferation. We found cell growth to be significantly reduced in si14-3-3f-transfected HepG2 cells compared to cells transfected with scrambled siRNA (Fig. 2), suggesting that increased 14-3-3f expression in HCC cells might enhance their growth and survival. Moreover, transfection with si14-3-3f dose-dependently reduced expression of PCNA. These results indicated that 14-3-3f over-expression probably enhances tumor cell growth in HCC. To further address the mechanisms underlying RNAi-mediated inhibition of proliferation in HCC cells, we established a stable 14-3-3f-depleted cell line using lentiviral vectors. In this study, we showed that stable 14-3-3f depletion lead to G1 arrest, but not apoptosis in
hepatoma cell (Fig. 3). That down-regulation of 14-3-3f did not induce apoptotic cell death in hepatoma cell is intriguing. This observation may be explained by the findings of a previous study, which showed that silencing of 14-3-3f inducing G1 arrest by stimulating p38/MAPK in breast cancer [20]. In the present study, we observed that si14-3-3f significantly inhibited anchorage-independent growth in vitro (Fig. 4). Taken together, these results suggest that 14-33f is an important promoter of cell proliferation and a potential target for suppression of proliferation in HCC. Though commonly used in treatment of HCC, the chemotherapeutic agent CDDP does not, as a result of drug resistance, greatly improve survival rates among HCC patients [21]. Chemosensitivity to CDDP may be highly dependent on its intracellular concentration: reduced accumulation in cancer cells results in increased resistance to CDDP [22]. To overcome this problem, a number of studies have explored the potential of combining anti-cancer drugs with gene therapy, and have demonstrated that suppressing the expression of oncogenes and anti-apoptotic genes enhances the ability of anti-cancer drugs to inhibit tumor cell growth and decreases tumor cell viability, respectively. In a recent study, suppression of 14-3-3f in A549 lung cancer cells increased sensitivity to CDDP in vitro and in vivo and was
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Fig. 3. 14-3-3f depletion causes cell cycle arrest but not apoptosis. (A) Infection of Huh7 cells with 14-3-3f shRNA-carrying lentiviral vectors. Protein lysates from the shRNA infected cells were analyzed by Western blot analysis using a 14-3-3f antibody. Mouse monoclonal b-actin was used as the loading control. (B) Apoptosis in infected Huh7 cells was measured by FACS analysis using Annexin-V PI double-staining. Less than 1% of LV-sh14-3-3f-3 and LVcont-infected cells were apoptosis. (C) Infected Huh7 cells were synchronized by serum starvation for 30 h and then the cells were released for 12 h by adding serum. The distribution of infected Huh7 cells in the cell cycle was determined by FACS analysis using PI staining. Data represent the mean and standard deviation of three independent experiments. Asterisks indicate a significant difference in comparison with LV-cont infected cells. LV-cont, LVcont-infected Huh7 cells; LV-sh14-3-3f-3, LV-sh14-3-3-f-3-infected Huh7 cells.
Fig. 4. Depletion of 14-3-3f inhibits tumorigenicity in a soft agar assay. LV-sh14-3-3f-3- and LV-cont-infected Huh7 cells were seeded into medium containing soft agar and incubated for 4 weeks. Colonies were stained with Crystal Violet and counted. (A) Both types of infectants yielded stained colonies at 4 week after seeding. Black bar indicated 50 lm. Original magnification 50. (B) The graph illustrated the number of colonies that were greater than 20 lm in diameter. Asterisks indicate a significant difference in comparison with LV-cont infected cells. Experiments were performed in triplicate. ⁄p < 0.05 vs. controls.
associated with inhibition of cell proliferation, additive G2-M arrest, and increased apoptosis [18]. Based on these results, we decided to treat si14-3-3f-transfected cells with
CDDP as a novel approach to inhibition of cell proliferation and increase of chemosensitivity to CDDP in hepatoma cells. Our results showed that CDDP and si14-3-3f synergistically
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Fig. 5. Down-regulation of 14-3-3f enhances chemosensitivity to CDDP. (A) One day after transfection with si14-3-3f or scrambled siRNA, HepG2 cells were treated with 1 nM CDDP. Cell extracts were prepared and analyzed by Western blot analysis using the antibody against 14-3-3f, PCNA and b-actin. Western blot analysis revealed si14-3-3f more decreased expression of PCNA in cells treated with CDDP than without CDDP. (B) LV-cont or LV-sh14-3-3f-3 infected cells were treated 100 lM CDDP to measure chemosensitivity to CDDP. Apoptotic cell death was measured by FACS analysis using Annexin-V PI doublestaining. (upper panel; 48 h, middle panel; 72 h).
suppressed the growth of hepatoma cells, with the response being significantly more pronounced than those observed when the same agents were applied individually. These results suggest that combined use of anti-cancer drugs and RNAi may improve therapeutic outcomes without increasing side effects. Next, we investigated how suppression of 14-3-3f protein expression increased the chemosensitivity of hepatoma cells to CDDP. 14-3-3f binds to a variety of sig-
nal transduction, checkpoint control, and cytoskeletal proteins [23,24]. Notably, its interaction with the kinase ASK1 inhibits apoptosis by inactivation of its catalytic activity, and inactivates JNK and p38/MAPKs [25,26]. Previous studies have shown that CDDP induces c-Jun and JNK/p38 signaling in certain cancer cells, leading to apoptosis [27,28]. To delineate the molecular mechanisms underpinning the enhancement of hepatoma cell chemosensitivity by si14-3-3f, we analyzed expression of the active forms of
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Fig. 6. Activation of JNK and p38/MAPK induce in 14-3-3f-knockdown cells with 1 nM CDDP. One day after transfection with si14-3-3f or scrambled siRNA, HepG2 cells were treated with 1 nM CDDP. Cell extracts were determined expression of active JNK, ERK and, p38/MAPK by Western blot analysis. Expression of active theses kinases was normalized corresponding inactive kinases level. These results shown in the panels are representative of three independent experiments.
ERK, JNK, and p38/MARK in si14-3-3f-transfected HepG2 cells. Our results demonstrated increased activation of JNK and p38/MARK, but not ERK, in si14-3-3f-transfected cells (Fig. 6), suggesting that 14-3-3f influences chemosensitivity to CDDP through regulation of JNK and p38/MAPK. In conclusion, we demonstrated over-expression of 143-3f in human HCC samples and showed that suppression of 14-3-3f inhibits tumor cell growth and cause cell cycle arrest. Our findings suggest that control of 14-3-3f expression might not only control tumorigenicity but also increase the chemosensitivity of HCC cells to CDDP by altering the activation of JNK and p38/MAPK. Therefore, combining CDDP treatment with RNAi-mediated suppression of 14-3-3f expression may constitute a useful therapeutic approach for treatment of HCC. Acknowledgments This study was supported by the 21C Frontier Functional Human Genome Project from the Ministry of Science & Technology in Korea (FG-08-12-05) and the Nuclear R&D Program of the Ministry of Science and Technology, the Republic of Korea (2010-0017595). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.canlet.2011. 01.015. References [1] T.M. Block, A.S. Mehta, C.J. Fimmel, R. Jordan, Molecular viral oncology of hepatocellular carcinoma, Oncogene 22 (33) (2003) 5093–5107.
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Contents lists available at ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Silencing of 14-3-3f over-expression in hepatocellular carcinoma inhibits tumor growth and enhances chemosensitivity to cis-diammined dichloridoplatium Jung Eun Choi a, Wonhee Hur a, Chan Kwon Jung b, Lian Shu Piao a, KwangSoo Lyoo a, Sung Woo Hong a, Sung Woo Kim a, Hye-Yeon Yoon d, Seung Kew Yoon a,c,⇑ a
WHO Collaborating Center of Viral Hepatitis, The Catholic University of Korea, Seoul 137-701, Republic of Korea Department of Hospital Pathology, The Catholic University of Korea, Seoul 137-701, Republic of Korea Department of Internal Medicine College of Medicine, The Catholic University of Korea, Seoul 137-701, Republic of Korea d Department of Life Science, The Ewha Womens University, Seoul 158-056, Republic of Korea b c
a r t i c l e
i n f o
Article history: Received 19 November 2010 Received in revised form 15 January 2011 Accepted 19 January 2011
Keywords: 14-3-3f Hepatocellular carcinoma RNA interference (RNAi) Cis-diammined dichloridoplatium (CDDP) Gene therapy
a b s t r a c t The 14-3-3f protein plays a key role in regulation of cellular processes. In the present study, we showed that 14-3-3f protein was significantly overexpressed in hepatoma cell lines and human tumorous tissues of hepatocellular carcinoma (HCC) patients. Knockdown with RNA interference in hepatoma cell lines with high 14-3-3f expression suppressed tumor cell proliferation via activation of c-Jun N-terminal kinase (JNK) and p38/MAPK. Furthermore, suppression of 14-3-3f enhanced the anti-cancer effect of cis-diammined dichloridoplatium (CDDP) in hepatoma cell lines. These results suggest that silencing of 14-3-3f may be an attractive target for HCC therapeutic development. Ó 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Human hepatocellular carcinoma (HCC) is the fifth most common type of cancer and the third leading worldwide cause of cancer death [1]. Despite recent advances in treatment of HCC, options for curing the disease are limited. In cases of advanced HCC, anti-cancer chemotherapeutic drugs, such as cis-diammined dichloridoplatium (cisplatin; CDDP) and 5-fluorouracil (5-FU) have been used; however, data demonstrating their efficacy in patients is limited [2–5]. The 14-3-3 proteins are a family of highly conserved cellular proteins that play important roles in various cellular processes [6–8]. Proteins in this family contain a ⇑ Corresponding author at: Department of Internal Medicine, Seoul St. Mary’s Hospital, #505 Banpo-Dong, Seocho-gu, Seoul 137-040, Republic of Korea. Tel.: +82 2 2258 7534; fax: +82 2 536 9559. E-mail address: [email protected] (S.K. Yoon). 0304-3835/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2011.01.015
pS/pT-binding motif and regulate cellular processes, including signal transduction, cell cycle, apoptosis, cellular metabolism, stress responses, cytoskeleton organization and malignant transformation [7–9]. To date, seven mammalian 14-3-3 isoforms (b, c, e, r, g, s, and f) that interact with other key cellular proteins during tumor development and progression have been identified [6,7]. Recently, 14-33f expression has been reported in various cancer cells [10–13]. However, until now, the possible association between 14-3-3f expression and hepatocarcinogenesis has not been investigated. RNA interference (RNAi) represents a natural mechanism by which small interfering RNA (siRNA) molecules can specifically and potently down-regulate expression of target genes [14]. Advances in siRNA technology in recent years have allowed the targeting of oncogenes with high specificity and efficiency in various cancers [15]. In previous study, we identified a 14-3-3f gene that is
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up-regulated in human HCC through genome-wide differential expression analysis and randomly-primed Annealing Control Primer™ (ACP)-based reverse transcription-polymerase chain reaction (RT-PCR) [16,17]. In this study, we investigated the possible association between 14-3-3f expression and HCC, and demonstrated that down-regulation of 14-3-3f expression by RNAi not only inhibits tumor cell growth but also enhances the chemotherapeutic effects of CDDP in HCC. This is the first report to demonstrate the co-therapeutic potential of 14-3-3f and CDDP in HCC. 2. Materials and methods 2.1. Clinical samples Specimens of tumor tissue and surrounding non-cancerous tissues were obtained from the livers of 135 HCC patients who had undergone a successful partial hepatectomy at Seoul St. Mary’s Hospital (Seoul, South Korea). Sample collection was approved by the Institutional Review Board of Kangnam St. Mary’s Hospital at the Catholic University of Korea. Informed consent for the use of specimens for research purposes only was obtained from all patients. Specimens were frozen in liquid nitrogen and stored until use. 2.2. Immunohistochemical staining For immunohistochemical staining, after deparaffinization of specimens, antigen retrieval was performed by microwave vacuum histoprocessor (RHS-1, Milestone, Bergamo, Italy) at 121 °C for 15 min. After peroxidase blocking, sections were incubated with anti-14-3-3f (Santa Cruz, San Diego, CA) diluted 1:2000 in Antibody Diluent (Glolen Bridge, Mukilteo, WA) at 4 °C overnight. The sections were developed with Polymer Detection System (Golden Bridge) and DAB. Slides were counterstained with hematoxylin (ScyTek, Logan, UT) after immunostaining. Intensity of reactivity was scored using a four-tier system: 0, indicated no staining; 1, weak staining; 2, moderate staining; and 3, strong staining. The percentage of cells expressing 14-3-3f of all of the normal control tissues was <10%. Therefore, the score of 2 and 10% as a cutoff value was chosen for continuation of analyses and to define higher or lower expression of 14-3-3f in tumor tissues. The pathologist reviewed all biopsies. 2.3. siRNA production To investigate the role of 14-3-3f in the growth of human hepatoma cells, specific siRNAs targeted to 14-33f were purchased from Santa Cruz Biotechnology. Four DNA sequences were chosen as candidate 14-3-3f siRNAs: siRNA-1 (50 -GGUACAUUGUGGCU-UCAAATT-30 ), siRNA-2 (50 -GCUUCCAUGUCUAAGCAAATT-30 ), siRNA-3 (50 -CC-AGU CACAGGUGU AGUAATT-30 ), and siRNA-4 (50 -GGUUGCU AAACUUCUCUA-ATT-30 ). A scrambled siRNA, whose sequence did not match that of any mammalian sequences listed in online databases, was synthesized by Ambion
(Austin, TX) and used as a negative control. Lyophilized siRNAs were resuspended in RNase-free water at room temperature and stored at 20 °C prior to use. 2.4. Cell culture and siRNA transfection HepG2, Huh7, and Hep3B cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS), 100 lg/ml penicillin, and 0.25 lg/ml streptomycin, and then maintained in a humidified incubator at 37 °C with 5% CO2. siRNA transfection was performed using the NucleofectorÒ electroporation system (Amaxa Biosystems, Cologne, Germany). 2.5. Generation of stable short-hairpin siRNA (shRNA) 14-3-3f infectants To establish a stable Huh7 cell line expressing sh14-33f, cells were infected using a shRNA-lentiviral infection system (Sigma, St. Louis, MO). Three coding regions from the human 14-3-3f gene (starting positions 232, 349, and 637 [GenBank Acc. No. NM_003406]) were selected as shRNA target sequences. We constructed three 14-3-3f shRNA-carrying lentiviral vectors: LV-sh14-3-3f-1, LVsh14-3-3f-2, and LV-sh14-3-3f-3. The shRNA negative control-lentiviral particle (LV-cont) was used as a negative control. To generate stable cells, Huh7 cells were plated in twelve-well plates (1 105 cells per well), transduced with 5 MOI lentiviral particles (using 8 lg/ml hexadimethrine bromide [Sigma]), and incubated at 37 °C with 5% CO2. Suppression of 14-3-3f in stable cells was confirmed by western blot analysis. 2.6. Western blot analysis Human hepatoma cells, liver tissue protein samples and a normal liver lysate (G-Biosciences, St. Louis, MO) were separated by 12% SDS–PAGE and transferred to a nitrocellulose membrane (Whatman, Maidstone, Kent, UK). Blots were then probed with monoclonal anti-b-actin (Sigma), polyclonal anti-14-3-3f (Santa Cruz), polyclonal anti-PCNA (Santa Cruz), monoclonal anti-phospho-JNK (Cell Signaling Technology, Beverly, MA), polyclonal anti-JNK1 (Santa Cruz), anti-phospho-p38 (Santa Cruz), anti-p38 (Santa Cruz), anti-phospho-ERK (Santa Cruz) and anti-ERK1 (Santa Cruz) antibodies overnight at 4 °C. Membranes were washed with TBS containing 0.05% Tween-20 (TBS-T) and then incubated with horseradish peroxidase-conjugated anti-mouse and-rabbit secondary antibodies (Amersham Pharmacia Biotech, Piscataway, NJ; diluted 1:5000). Protein bands were visualized using an enhanced chemiluminescence (ECL) system (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. 2.7. Cell proliferation assays, apoptosis assays and cell cycle analysis To measure cell growth, cultured cells were harvested and counted using a Vi-Cell automated cell viability analyzer (Beckman-Coulter, Fullerton, CA). In addition,
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following transfection with 14-3-3f-specific siRNA, MTS assay was performed using the CellTiter 96 AQueous Assay System (Promega, Madison, WI). Absorbances were measured at 490 nm using a SpectraMax 250 Microplate Reader (Molecular Devices, Sunnyvale, CA). Apoptosis was identified using an Annexin-V apoptosis kit (BD BioSciences, San Diego, CA) according to the manufacturer’s instructions. A total of 10,000 cells were counted by flow cytometry using a fluorescence-activated cell sorter (FACS; Becton–Dickinson, San Jose, CA). Resulting data were analyzed using CellQuest software (BD BioSciences). For cell cycle analysis, stable Huh7 cells were seeded in a 100-mm culture dish the day before the synchronization experiment. The cells were incubated in DMEM without serum for 30 h and then the cell cycle was initiated with DMEM containing 10% FBS for 12 h. The cells were harvested and fixed in 70% ethanol for 30 min at 20 °C. Fixed cells were stained with 50 lg/ll propidium propidium idodie (PI; BD Biosceinces) and 100 lg/ml RNase (Sigma). Cell cycle profile of infected Huh7 cells was measured by FACS, and resulting data were analyzed using CellQuest software (BD BioSciences).
2.8. Soft agar colony assay shRNA-lentiviral particle-infected Huh7 cells (2 104 cells) were seeded onto 0.7% (w/v) microbiologygrade agarose prepared in complete medium. The upper layer was poured onto a layer of sterile 1% (w/v) agarose prepared in complete medium that had been allowed to solidify in 35-mm culture dishes. Dishes were incubated at 37 °C under 5% CO2 for 3–4 weeks. Resulting colonies were stained for >2 h with 0.005% Crystal Violet. The number of colonies with a diameter >20 lm in randomly selected fields (50 magnification) was counted.
2.9. Statistical analysis All experiments were performed at in least triplicate. Statistical comparisons were made by analysis of variance (ANOVA) using SPSS 13.0 software (Chicago, IL). A p-value of <0.05 was considered statistically significant.
3. Results 3.1. Over-expression of 14-3-3f in HCC To determine whether 14-3-3f was expressed in HCC, human hepatoma cells (HepG2, Huh7, and Hep3B) and human HCC specimens were analyzed immunohistochemically and by western blot analysis. Compared to normal liver lysates, three hepatoma cell lines displayed enhanced 14-3-3f expression (Fig. 1A). 14-3-3f was expressed at higher levels in 72 of 135 (53.3%) HCC tissues analyzed (Fig. 1B). Although 143-3f expression was observed in biliary duct epithelial cells in nontumorous tissues, most 14-3-3f expression was shown in tumorous tissues. In addition, western blot analysis of 20 paired HCC samples showed that 14-3-3f was more highly expressed in tumor tissues than in noncancerous liver tissues (Fig. 1C). The optical density of each band from tumorous (T) and non-tumorous (NT) tissues in the same patient was measured using TINA image analysis software. The ratio between the optical density of 14-3-3f and b-actin in the same sample was calculated as the relative content. As shown in Fig. 1C, the 14-3-3f/b-actin ratio was
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high, greater than 1.5-fold, in 12 of 20 (60%) T compared with corresponding NT. These results indicated involvement of 14-3-3f in HCC development. 3.2. Silencing of 14-3-3f suppresses cell proliferation To elucidate the role of 14-3-3f in HCC, RNAi studies using 14-3-3f specific siRNAs were performed to eliminate expression of endogenous 14-3-3f. Transfection of HepG2 cells with 50 nM si14-3-3f markedly inhibited 14-3-3f protein expression by more than 50% within 1 day, whereas scrambled siRNA (50 nM) did not suppress 14-3-3f expression (data not shown). Transfection of HepG2 cells with si14-3-3f effectively and specifically silenced transcription of 14-3-3f. Next, we investigated whether 14-3-3f siRNA transfection affected cell proliferation and survival. As illustrated in Fig. 2A, cell growth was reduced in si14-3-3f-transfected HepG2 cells compared to scrambled siRNA-transfected cells. This inhibitory effect was confirmed by MTS proliferation assay and was especially prominent at 4 days post-transfection (p = 0.009) (Fig. 2B). These results suggest that up-regulation of 14-33f in HCC cells enhances cell growth and survival. To test the hypothesis that silencing of 14-3-3f suppresses HCC cell proliferation, we assessed expression of PCNA in si14-3-3f-transfected HepG2 cells during the first 4 days following transfection. Levels of PCNA in si14-3-3f-transfected HepG2 cell lysates were determined by western blot analysis (a representative blot is shown in Fig. 2C). Expression of PCNA in si14-3-3f-transfected cells declined markedly in a dose-dependent manner. These results demonstrate that 14-3-3f over-expression is associated with enhanced tumor cell growth in HCC. 3.3. Effects of RNAi targeting of 14-3-3f on the cell cycle and apoptosis To address the mechanisms underlying RNAi-mediated inhibition of proliferation in HCC cells, we established Huh7 cells in which 14-3-3f was depleted through stable expression of shRNAs directed against 143-3f mRNA. Western blot analysis of LV-sh14-3-3f-1 and LV-sh14-3-3f3 stable infectants revealed that expression of 14-3-3f was decreased markedly relative to the control, whereas that of LV-sh14-3-3f-2 stable infectants was decreased slightly (Fig. 3A). We further investigated the influence of LV-sh14-3-3f stable infectants on apoptosis and cellular DNA content using Annexin-V apoptosis kit and PI staining. As shown in Fig. 3B, the rates of apoptosis in Huh7 cells infected with LV-sh14-33f-3 were comparable with those of control cells. Moreover, we determined cell cycle profile using synchronized and released cells. The distribution of LV-sh14-3-3f-3 infectants in G1 phase was significantly higher than that of LV-cont infectants (Fig. 3C). All experiments were performed more than three times independently. These results suggest that RNAimediated stable depletion of 14-3-3f expression in Huh7 cells induces G1 arrest, but not apoptosis. 3.4. 14-3-3f suppression inhibits tumorigenicity To determine whether suppression of 14-3-3f inhibits tumorigenicity, we performed a soft agar assay using a shRNA-lentiviral infection system. LV-sh14-3-3f-1- and LV-sh14-3-3f-3-transfected and control Huh7 cells were seeded onto medium containing soft agar and incubated for 4 weeks. Colonies greater than 20 lm in diameter were then counted. As shown in Fig. 4, colony formation was significantly inhibited as a result of the absence of 14-3-3f. These results indicate that suppression of 14-33f results in significant inhibition of HCC cell proliferation in vitro and, thus, that 14-3-3f may be involved in hepatocarcinogenesis. 3.5. 14-3-3f suppression synergistically enhances CDDP induced cytotoxicity in vitro To evaluate the effect of 14-3-3f knockdown on chemosensitivity, si14-3-3f-transfected HepG2 cells were exposed to 1 nM CDDP and subjected to cell viability and proliferation analyses. We used a minimal dose of CDDP to avoid excessive cell death due to the concern that higher doses of CDDP might be highly damaging to cells electroporated with si14-3-3f. We also examined the effect of CDDP on expression of PCNA, a marker of cell proliferation, in si14-3-3f-transfected cells. As shown in Fig. 5A, western blot analysis showed that PCNA expression in CDDP-treated HepG2 transfectants was significantly lower than in untreated HepG2
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Fig. 1. 14-3-3f expression in hepatocellular carcinoma. (A) Western blot analysis for detection of 14-3-3f in different HCC cell lines HepG2, Huh7, Hep3B and normal liver. Mouse monoclonal b-actin was used as the loading control. (B) Immunohistochemical staining pattern of 14-3-3f in tumorous tissues (a– c) and non-tumorous tissue (d). The tumor cells show weak (a), moderate (b), strong (c) 14-3-3f expression while the biliary duct epithelial cells in nontumorous portion also express 14-3-3f protein. Original magnification 400. (C) Expression of 14-3-3f by western blot analysis in matched tumorous (T) and non-tumorous tissues (NT) from 20 HCC patients. The results shown are from one representative experiment among three replicates. The band densities were quantified with TINA imaging analysis software, using b-actin as a reference. The data are expressed relative to the density of the control.
transfectants. Moreover, CDDP exerted a stronger inhibitory effect on cells treated with the maximum tested concentration of si14-3-3f. Next, to determine whether 14-3-3f knockdown enhanced the apoptotic effects of CDDP, Huh7 cells harboring LV-sh14-3-3f-3 or LV-cont were exposed to 100 lM CDDP for 72 h and then evaluated by flow cytometry using an Annexin-V apoptosis kit. After treatment with CDDP, the rate of apoptosis was higher in LV-sh14-3-3f-3-infected Huh7 cells than in control cells (Fig. 5B). Collectively, these results indicate that CDDP induces apoptotic cell death and reduces cell proliferation in 14-3-3f-knockdown cells in a dose- and time-dependent manner.
3.6. 14-3-3f suppression activates JNK and p38 pathway but not ERK MAP kinase To delineate the molecular mechanisms underlying the enhanced chemosensitivity of 14-3-3f-knockdown HCC cells, we initially hypothesized that 14-3-3f suppresses activation of ERK, JNK, and p38/MAPK. To address this hypothesis, we investigated the silencing of 14-3-3f on the downstream activation of the MAPK pathways by detecting phosphorylated ERK, JNK and p38/MAPK in si14-3-3f-transfected HepG2 cells. The levels of JNK and p38/MAPK phosphorylation in si14-3-3f-transfected cells were increased as compared this level in control and scrambled siRNA-transfected cells. However, the phosphorylation of ERK was not changed in si14-3-3f-transfected cells. These results imply that enhanced chemosensitivity by suppression of 14-3-3f seems to be associated with the activation of the JNK and p38/MAPK pathway.
4. Discussion We successfully identified several genes that show aberrant expression in human HCC, using an ACP-based RT-PCR approach to profile differential expression [16]. Of the differentially expressed genes identified, 14-3-3f was selected for further functional characterization due to the strong over-expression of its protein product in hepatoma cell lines. To date, the role of 14-3-3f in HCC remains unclear. In this study, we demonstrated the over-expression of 14-33f in human HCC specimens by western blot and immunohistochemical analyses. 14-3-3f was found to be overexpressed in more than 53.3% of the cancerous HCC tissues tested, but in none of the surrounding non-cancerous tissues. These results suggest involvement of 14-3-3f in hepatocarcinogenesis. To best of our knowledge, this is the first report on 14-3-3f characterization in human HCC. This finding encouraged us to investigate the functional regulation of 14-3-3f in HCC cells in order to determine whether its over-expression contributes to development of HCC.
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Fig. 2. Suppression of 14-3-3f inhibits HepG2 cells proliferation. Cell proliferation of si14-3-3f-transfected HepG2 cells determined by the trypan blue exclusion (A) and MTS assay (B). (A) Transfected HepG2 cells were trypsinized and stained using trypan blue each 1 day. Viable cells were counted by ViCell™ Automated Cell Viability Analyzer. (B) Cell proliferation of transfected HepG2 cells were determined by MTS assay. The absorbance was read at 490 nm using spectrophotometer microplate reader. Data represent the mean and standard deviation of three independent experiments. (C) Cell extracts were prepared and analyzed by Western blot analysis using the antibody against 14-3-3f, PCNA, and b-actin. C, HepG2; S, scrambled siRNA (50 nM); f25, si14-3-3f (25 nM); f50, si14-3-3f (50 nM). ⁄p < 0.05.
To explore the possibility that 14-3-3f may be an effective therapeutic target, we used RNAi to silence endogenous 14-3-3f expression in human hepatoma cells, and analyzed the characteristics of transiently or stably transfected hepatoma cells. As shown in Fig. 2C, transfection of HepG2 cells with si14-3-3f dose-dependently inhibited 14-3-3f protein expression, indicating that transfection of these cells with si14-3-3f specifically and effectively blocks 14-3-3f transcription. These observations are consistent with those of previous studies utilizing 14-3-3f siRNA-mediated gene silencing in lung and breast cancer cells [18,19]. Next, we examined the effect of si14-3-3f on hepatoma cell proliferation. We found cell growth to be significantly reduced in si14-3-3f-transfected HepG2 cells compared to cells transfected with scrambled siRNA (Fig. 2), suggesting that increased 14-3-3f expression in HCC cells might enhance their growth and survival. Moreover, transfection with si14-3-3f dose-dependently reduced expression of PCNA. These results indicated that 14-3-3f over-expression probably enhances tumor cell growth in HCC. To further address the mechanisms underlying RNAi-mediated inhibition of proliferation in HCC cells, we established a stable 14-3-3f-depleted cell line using lentiviral vectors. In this study, we showed that stable 14-3-3f depletion lead to G1 arrest, but not apoptosis in
hepatoma cell (Fig. 3). That down-regulation of 14-3-3f did not induce apoptotic cell death in hepatoma cell is intriguing. This observation may be explained by the findings of a previous study, which showed that silencing of 14-3-3f inducing G1 arrest by stimulating p38/MAPK in breast cancer [20]. In the present study, we observed that si14-3-3f significantly inhibited anchorage-independent growth in vitro (Fig. 4). Taken together, these results suggest that 14-33f is an important promoter of cell proliferation and a potential target for suppression of proliferation in HCC. Though commonly used in treatment of HCC, the chemotherapeutic agent CDDP does not, as a result of drug resistance, greatly improve survival rates among HCC patients [21]. Chemosensitivity to CDDP may be highly dependent on its intracellular concentration: reduced accumulation in cancer cells results in increased resistance to CDDP [22]. To overcome this problem, a number of studies have explored the potential of combining anti-cancer drugs with gene therapy, and have demonstrated that suppressing the expression of oncogenes and anti-apoptotic genes enhances the ability of anti-cancer drugs to inhibit tumor cell growth and decreases tumor cell viability, respectively. In a recent study, suppression of 14-3-3f in A549 lung cancer cells increased sensitivity to CDDP in vitro and in vivo and was
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Fig. 3. 14-3-3f depletion causes cell cycle arrest but not apoptosis. (A) Infection of Huh7 cells with 14-3-3f shRNA-carrying lentiviral vectors. Protein lysates from the shRNA infected cells were analyzed by Western blot analysis using a 14-3-3f antibody. Mouse monoclonal b-actin was used as the loading control. (B) Apoptosis in infected Huh7 cells was measured by FACS analysis using Annexin-V PI double-staining. Less than 1% of LV-sh14-3-3f-3 and LVcont-infected cells were apoptosis. (C) Infected Huh7 cells were synchronized by serum starvation for 30 h and then the cells were released for 12 h by adding serum. The distribution of infected Huh7 cells in the cell cycle was determined by FACS analysis using PI staining. Data represent the mean and standard deviation of three independent experiments. Asterisks indicate a significant difference in comparison with LV-cont infected cells. LV-cont, LVcont-infected Huh7 cells; LV-sh14-3-3f-3, LV-sh14-3-3-f-3-infected Huh7 cells.
Fig. 4. Depletion of 14-3-3f inhibits tumorigenicity in a soft agar assay. LV-sh14-3-3f-3- and LV-cont-infected Huh7 cells were seeded into medium containing soft agar and incubated for 4 weeks. Colonies were stained with Crystal Violet and counted. (A) Both types of infectants yielded stained colonies at 4 week after seeding. Black bar indicated 50 lm. Original magnification 50. (B) The graph illustrated the number of colonies that were greater than 20 lm in diameter. Asterisks indicate a significant difference in comparison with LV-cont infected cells. Experiments were performed in triplicate. ⁄p < 0.05 vs. controls.
associated with inhibition of cell proliferation, additive G2-M arrest, and increased apoptosis [18]. Based on these results, we decided to treat si14-3-3f-transfected cells with
CDDP as a novel approach to inhibition of cell proliferation and increase of chemosensitivity to CDDP in hepatoma cells. Our results showed that CDDP and si14-3-3f synergistically
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Fig. 5. Down-regulation of 14-3-3f enhances chemosensitivity to CDDP. (A) One day after transfection with si14-3-3f or scrambled siRNA, HepG2 cells were treated with 1 nM CDDP. Cell extracts were prepared and analyzed by Western blot analysis using the antibody against 14-3-3f, PCNA and b-actin. Western blot analysis revealed si14-3-3f more decreased expression of PCNA in cells treated with CDDP than without CDDP. (B) LV-cont or LV-sh14-3-3f-3 infected cells were treated 100 lM CDDP to measure chemosensitivity to CDDP. Apoptotic cell death was measured by FACS analysis using Annexin-V PI doublestaining. (upper panel; 48 h, middle panel; 72 h).
suppressed the growth of hepatoma cells, with the response being significantly more pronounced than those observed when the same agents were applied individually. These results suggest that combined use of anti-cancer drugs and RNAi may improve therapeutic outcomes without increasing side effects. Next, we investigated how suppression of 14-3-3f protein expression increased the chemosensitivity of hepatoma cells to CDDP. 14-3-3f binds to a variety of sig-
nal transduction, checkpoint control, and cytoskeletal proteins [23,24]. Notably, its interaction with the kinase ASK1 inhibits apoptosis by inactivation of its catalytic activity, and inactivates JNK and p38/MAPKs [25,26]. Previous studies have shown that CDDP induces c-Jun and JNK/p38 signaling in certain cancer cells, leading to apoptosis [27,28]. To delineate the molecular mechanisms underpinning the enhancement of hepatoma cell chemosensitivity by si14-3-3f, we analyzed expression of the active forms of
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Fig. 6. Activation of JNK and p38/MAPK induce in 14-3-3f-knockdown cells with 1 nM CDDP. One day after transfection with si14-3-3f or scrambled siRNA, HepG2 cells were treated with 1 nM CDDP. Cell extracts were determined expression of active JNK, ERK and, p38/MAPK by Western blot analysis. Expression of active theses kinases was normalized corresponding inactive kinases level. These results shown in the panels are representative of three independent experiments.
ERK, JNK, and p38/MARK in si14-3-3f-transfected HepG2 cells. Our results demonstrated increased activation of JNK and p38/MARK, but not ERK, in si14-3-3f-transfected cells (Fig. 6), suggesting that 14-3-3f influences chemosensitivity to CDDP through regulation of JNK and p38/MAPK. In conclusion, we demonstrated over-expression of 143-3f in human HCC samples and showed that suppression of 14-3-3f inhibits tumor cell growth and cause cell cycle arrest. Our findings suggest that control of 14-3-3f expression might not only control tumorigenicity but also increase the chemosensitivity of HCC cells to CDDP by altering the activation of JNK and p38/MAPK. Therefore, combining CDDP treatment with RNAi-mediated suppression of 14-3-3f expression may constitute a useful therapeutic approach for treatment of HCC. Acknowledgments This study was supported by the 21C Frontier Functional Human Genome Project from the Ministry of Science & Technology in Korea (FG-08-12-05) and the Nuclear R&D Program of the Ministry of Science and Technology, the Republic of Korea (2010-0017595). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.canlet.2011. 01.015. References [1] T.M. Block, A.S. Mehta, C.J. Fimmel, R. Jordan, Molecular viral oncology of hepatocellular carcinoma, Oncogene 22 (33) (2003) 5093–5107.
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