SAHA modulates cell proliferation, colony forming and epithelial-mesenchymal transition in CCA cells

Background: Therapeutic options for advanced cholangiocarcinoma (CCA) are limited and ineffective due to the largely incomplete understanding of the molecular pathogenesis of this deadly tumor. So that, we planned to investigate epigenetic regulation of epithelial-mesenchymal transition (EMT) in cholangiocarcinoma cell line by applying Suberoylanilide hydroxamic acid (SAHA). We studied the effect of SAHA on cell proliferation, colony forming, migration and protein level of E-cadherin (E-cad) as an epithelial EMT marker, N-cadherin (N-cad) and Vimentin (Vim), as a mesenchymal markers of EMT, in human CCA cell line. Materials and methods: Cell proliferation and migration measurements were performed by flow cytometry and wound healing assay, respectively. E-cad, N-cad and Vim protein levels were determined by Western blot analysis. Results: It was found that SAHA significantly inhibits cell viability, proliferation and migration of TFK-1 cells, accompanied by reversing of EMT markers. SAHA, upregulated protein level of E-cad, while downregulated the protein levels of N-cad and Vimentin. Conclusions: SAHA treatment may bebeneficial for CCA patients and SAHA might be a potential therapeutic agent for the treatment of CCA. However, future studies are needed to evaluate the clinical applicability of SAHA as a part of the chemotherapeutic regimen for CCA.


Introduction
Cholangiocarcinoma (CCA) is the second most common primary hepatic cancer, after hepatocellular carcinoma. It is common in elderly patients. The peak incidence is in the seventh decade of life [1]. The majority of patients with cholangiocarcinoma are diagnosed with advanced stage of diseases. Curative surgery is the only treatment modality in early stage cases unfortunately tumor progresses insidiously in most cases and the majority of the patients lose the chance of curative surgery because of meta stasis. In advance stage of the CCA chemotherapy is another therapeutic option nevertheless, chemoresistance is an important problem in CCA [2]. Mechanisms of metastasis has not been clearly defined yet but it has been revealed that cancer cells must have acquire migratory and invasive properties by a cell plasticity-promoting phenomenon known as epithelial-mesenchymal transition (EMT) to metastasize from the primary origin [3].
EMT is a biological process that allow a polarized epithelial cell, which normally interacts with basement membrane via its basal surface, to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype, which includes enhanced migratory capacity, invasiveness, elevated resistance to apoptosis, and greatly increased production of extracellular matrix components (ECM) [4][5][6].
Pathogenesis of CCA is not well defined. Mesenchymal transition of epithelial tumor cells may contribute to the invasion ability and chemoresistancy of CCA [7,8]. Changes in gene expression levels occur during EMT including downregulation of epithelial markers such as E-cadherin (E-cad) and up-regulation of mesencyhmal markers such as vimentin (Vim) and N-cadherin (N-cad). EMT is fundamental in many physiological processes, such as embryogenesis and wound healing. Many clinical studies have been reported that EMT expressions correlated with an increased metastatic potential and poor clinical outcome [4,5].
The neoplastic transformation of biliary epithelial cells and malignant progression of CCA has genetic and epigenetic alternations. The concept of epigenetic therapy is now well-established in the field of oncology, with the successful clinical application of histone deacetylases (HDACs) described for many types of cancers [9][10][11]. Suberoylanilide hydroxamic acid (SAHA) is a known HDAC inhibitor that shows anticancer activity by relieving gene silencing against hematologic and solid tumors [12]. SAHA is currently being after investigated a number of clinical trials. SAHA is approved as HDAC inhibitor by FDA to treat cutaneous T-cell lymphoma Cell culture studies have shown that SAHA inhibits cell proliferation and induces apoptosis and cell cycle arrest in CCA cell lines [13]. In other study has been shown to HDAC inhibitor of SAHA reduce cell proliferation in CCA cells (M213, M214 and KKU-100) [14]. Although SAHA is widely accepted as an HDAC inhibitor, it has been reported to suppress EZH2 expression in cancer cells and to exert an anticancer effect, indicating that SAHA also functions as an EZH2 inhibitor [15]. Overexpression of EZH2 positively correlates with the progression of multiple malignancies including breast cancer, lymphoma, myeloma, colorectal cancer, endometrial cancer, bladder cancer and prostate cancer [16]. However, the potential effect for in vitro SAHA on EMT has not been investigated in CCA yet.
Therapeutic options for advanced CCA are limited and ineffective due to the largely incomplete understanding of the molecular pathogenesis of this deadly tumor. So that, we planned to investigate epigenetic regulation of EMT in cholangiocarcinoma cell line by applying SAHA. We studied the effect of SAHA on cell proliferation, colony forming, migration and protein level of E-cad as an epithelial EMT marker, N-cad and Vim, as a mesenchymal markers of EMT, in human CCA cell line.

Cell lines and culture conditions
We used cholangiocarcinoma cell line TFK-1. TFK-1 cell line was purchased from DSMZ. Those cells were seeded in RPMI-1640 medium. Medium was supplemented with 10% FBS and 1% penicilin-streptomycin. The stock concentration of SAHA was determined as 1000 μM and dimethyl sulfoxide (DMSO; BioChemical) was used as the negative control. Cell line was treated with 2 μg/mL of the HDAC inhibitor SAHA for 48 h. Ethics committee approval is not needed.

Cell viability analysis
Cells were cultured in a six-well plate and treated with different doses of SAHA (1-10 μM). After 48 h of incubation, cells were harvested by trypsin. Then cells were washed with sufficient PBS. The samples were then stained with 450 μL Count and Viability Assay kit at room temperature for 5 min in darkness. Using a flowcytometry. IC 50 value was calculated by Graph Pad Prism Software. The IC 50 value was determined as to be 2 μM according to this formula; 50 IC 100 control treated group /control 100 control is cell number in nontreated group, treated group is the number of cells in the SAHA-treated group at different concentr

Colony forming assay
Cells were cultured in a six-well plate and treated with 2 μM SAHA (500 cells per well) plates. The culture medium was replaced every 2 days. After 10 days colonies were fixed in 10% formaldehyde, stained with crystal violet solution and then counted.

Wound healing assay
TFK-1 cells were seeded in six wells and grown in duplicate as 2.5 × 10 5 cells in each well. When the cells reached 80-90% density, normal medium was added to the control group and SAHA at the dose of 2 μM was added to the SAHA group. After 48 h in this way, a 200 μL pipette tip was wound on the cells. When the wound was created, it was taken as 0 h and the image was taken with a camera microscope. Then, the cells were left in normal medium until the wound in the control group was closed, and images were taken every 24 h with a camera microscope. The wound widths pixel were then measured using the Image J (National Institute of Mental Health, MA, USA).

Western blot analysis
The culture medium was removed from the TFK-1 cells.

Effect of SAHA on cell viability
We first investigated effective concentration of SAHA on TFK-1 cell line. The cells were treated with SAHA for 48 h at concentrations of 1, 2.5, 5 and 10 μM. We showed that SAHA treatment markedly reduced TFK-1 cell viability in a dose-dependent manner. Two micromole SAHA at IC 50 was selected as application dosage for the study (Figure 1).

Effect of SAHA on colony forming assay
We next examined the effects of SAHA on colony formation in TFK-1 [14]. In this assay, SAHA treated cells are contact inhibited and do form statistically less colonies than non-SAHA treated cells; thus, the assay evaluate the neoplastic tendency of cancer cells. Colony forming assay of TFK-1 cells showed that 48 h treatment with 2 μM SAHA markedly decreased the colony numbers compared with control ( Figure 2).

Effect of SAHA on migration of TFK-1 cells
To assess whether SAHA is involved in cell migration of TFK-1 cells, we performed an in vitro scratch wound healing assay. Two micromole SAHA was used to demonstrate the effect of SAHA on the migration of TFK-1 cells. Images were taken every 24 h with a camera microscope ( Figure 3A). Since there was no agent inhibiting cell migration in the control group, it was seen that the wound was closed at the end of 144 h. In the SAHA group, cell migration was inhibited and the wound was not closed. Wound widths were measured and the significant change in wound width with time was plotted in Figure 3B. In conclusion, SAHA has been shown to suppress migration ability of TFK-1 cells.

Effect of SAHA on E-cadherin, N-cadherin and Vim protein levels
As indicated by the data in Figures 3 and 4, SAHA plays a critical role in EMT in TFK-1 cell line. We examined whether inhibition of SAHA leads to inhibition of signaling pathways of EMT in vitro. Cell lysates were collected 48 h after treatment with SAHA. Lysates were subjected to western blot analysis. SAHA, upregulated protein level of E-cad, while downregulated the protein levels of N-cad, Vimentin ( Figure 4A-C).

Effect of SAHA on EZH2
It has been reported that SAHA suppressed EZH2 protein level in human cancer cells [15]. EZH2 enhances tumorigenesis and is commonly overexpressed in several types of cancer [16]. So, we further wondered the effects of SAHA on EZH2 protein level in TFK-1 cell line. SAHA did not affect EZH2 protein level ( Figure 5).

Discussion
HDACs have been reported as one of the epigenetic mechanisms associated with tumorigenesis. Many of signaling pathways which regulate critical processes such as cell growth, survival, differentiation and migration are altered in cancer cells. Understanding of the molecular changes that accompany cell transformation led to the development of anticancer targeted therapy [10][11][12].
SAHA is used in clinical research for cancer treatment. The ability of SAHA to inhibit HDAC activity and thereby regulate gene transcription has been linked to its ability to inhibit growth and induce apoptosis in several types of malignant cells in vitro. However, many of the mechanisms underlying the effects of this agent on cell growth, differentiation, and death have not been fully clarified [12].
Effect of SAHA treatment on EMT features of cholangiocarcinoma has not been studied yet. We researched the effects of SAHA on CCA cell viability, proliferation and migration in vitro. The molecular mechanisms underlying SAHA activities were also explored. It was found that SAHA could significantly inhibit cell viability, proliferation and migration of TFK-1 cells, accompanied by reversing of EMT markers.
The ability of tumor cells to express some mesenchymal properties at different levels is largely reported [23][24][25][26]. Clinical studies correlated the expression of EMT features with an increased metastatic potential and a poor clinical outcome in many cancers, including colorectal [27], and gastric cancer [28]. Effect of SAHA on EMT is contraversial in different cancer types. It has been reported that SAHA promotes the epithelial mesenchymal transition of triple negative breast cancer cells via HDAC8/ FOXA1 signals [29]. In another study on this topic, it has been published that HDAC inhibitors induce epithelialto-mesenchymal transition in prostate cancer cells [30]. In addition, It has been suggested that HDAC inhibitor induction of epithelial-mesenchymal transitions via upregulation of Snail facilitates cancer progression [31]. We found that SAHA changed protein pattern of EMT markers by decreasing level of mesenchymal-associated molecules such as Vimentin and N-cadherin [24,32]. In contrast, SAHA increased the level of epithelial junctional protein E-cadherin. Thus, these results suggest that SAHA treatment may reverse EMT.
The CFU assay has been used extensively to determine the proliferation of cancer cells and to determine the effects of drugs [33,34]. CFU assay was used to investigate the effect of SAHA on TFK-1 cell colonization. CFU assay revealed that SAHA treatment clearly decreased colony formation of TFK-1 cells. We do not know how SAHA repressed colony formation of cancer cells. In the literature EZH2 inhibition of SAHA has been accepted as SAHA's anti-proliferative mechanism [15]. However, in this study, SAHA treatment did not affect EZH2 protein levels.
Migration capability of TFK-1 cells was showed by wound healing experiment. SAHA treatment clearly decreased migration capability of TFK-1 cells as reported in different cancer lines. Suppression or prevention of EMT process by SAHA treatment may be the factor in stopping migration of cancer cells. Decreasing cancer cells viability, stopping cancer cells proliferation and preventing cancer cell migration are important steps in cancer treatment. SAHA is an epigenetic drug which shows antitumor affect by inhibiting HDAC. HDAC inhibition by SAHA express those positive affects in different cancer models.
In this study, SAHA treatment clearly decreased migration and proliferation of CCA, which is highly metastatic. SAHA also leads to inhibition of signaling pathways of EMT in vitro. Thus, our findings suggest that SAHA treatment may be beneficial for CCA patients and SAHA might be a potential therapeutic agent for the treatment of CCA. However, future studies are needed to evaluate the clinical applicability of SAHA as a part of the chemotherapeutic regimen for CCA.
Ethics committee approval: Ethics committee approval is not needed.