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ORIGINAL ARTICLE
Year : 2020  |  Volume : 16  |  Issue : 5  |  Page : 1112-1118

Insufficient radiofrequency ablation promotes epithelial–mesenchymal transition mediated by interleukin-6/signal transducer and activator of transcription 3/Snail pathway in the H22 cells


1 Department of Interventional Medicine, The Second Hospital of Shandong University; Interventional Oncology Institute of Shandong University, Jinan, Shandong Province, CN–250033, PR of China
2 Department of Oncology, The Third Hospital of Qinhuangdao, Qinhuangdao, Hebei Province, China

Date of Submission03-Jan-2020
Date of Decision20-Mar-2020
Date of Acceptance11-May-2020
Date of Web Publication29-Sep-2020

Correspondence Address:
Yuliang Li
Department of Interventional Medicine, The Second Hospital of Shandong University, 247 Beiyuan Road, Jinan 250033, Shandong Province; Interventional Oncology Institute of Shandong University, Jinan 250033, Shandong Province
PR of China
Zhaomin Song
Department of Oncology, The Third Hospital of Qinhuangdao, 222 Jianguo Road, Qinhuangdao, Hebei Province
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrt.JCRT_12_20

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 > Abstract 


Context: Radiofrequency ablation (RFA), an established and minimally invasive therapy for hepatocellular carcinoma, has become an important treatment strategy. However, tumor aggressiveness remains a common problem. The epithelial–mesenchymal transition (EMT) is thought to play an important role in this process.
Design and Aims: Due to limited sample volumes harvested from patients, we established a heat-treated cell line and a mouse model to investigate the mechanisms of incomplete ablation in EMT.
Materials and Methods: We heat-treated H22 and HepG2 cells using a water bath to determine a suitable temperature for incomplete RFA. Male BALB/c mice were orthotopically transplanted with H22 cells and then subjected to incomplete ablation. Changes in the EMT biomarkers were detected by real-time polymerase chain reaction, western blotting, and immunofluorescence.
Statistical Analysis: The experimental results are expressed as means ± standard deviations.
Results: Incomplete RFA promoted EMT, downregulated E-cadherin, upregulated vimentin and Snail, and enhanced the phosphorylation of signal transducer and activator of transcription 3 (STAT3) both in vivo and in vitro. Moreover, interleukin (IL)-6 secretion increased after heat treatment in the H22 cells. AG490, an IL-6 inhibitor, inhibited the occurrence of EMT.
Conclusions: Insufficient ablation performed at low temperature successfully induces EMT and promotes tumor aggressiveness, which is mediated by the IL-6/STAT3/Snail pathway in both cell and mouse models.

Keywords: Epithelial–mesenchymal transition, hepatocellular carcinoma, interleukin-6/signal transducer and activator of transcription 3/Snail, insufficient ablation, radiofrequency ablation


How to cite this article:
Zhou T, Liu B, Wang Y, Wang W, Chang H, Li D, Li Y, Song Z. Insufficient radiofrequency ablation promotes epithelial–mesenchymal transition mediated by interleukin-6/signal transducer and activator of transcription 3/Snail pathway in the H22 cells. J Can Res Ther 2020;16:1112-8

How to cite this URL:
Zhou T, Liu B, Wang Y, Wang W, Chang H, Li D, Li Y, Song Z. Insufficient radiofrequency ablation promotes epithelial–mesenchymal transition mediated by interleukin-6/signal transducer and activator of transcription 3/Snail pathway in the H22 cells. J Can Res Ther [serial online] 2020 [cited 2020 Oct 23];16:1112-8. Available from: https://www.cancerjournal.net/text.asp?2020/16/5/1112/296429




 > Introduction Top


Hepatocellular carcinoma (HCC) is the second-leading cause of cancer-related deaths, worldwide.[1] Further, liver cancer is the fifth and seventh most common cancer in men and women, respectively. The main risk factors for HCC are hepatitis B or C viral infection, alcoholic liver cirrhosis, nonalcoholic fatty liver disease, and nonalcoholic steatohepatitis. Due to improved surveillance in identifiable high-risk patients (those with hepatitis B or C virus infection) and surgical intervention (resection or transplantation) for patients with early-stage disease, the 5-year survival rates of patients with HCC have improved.[2] Although hepatic resection and transplantation are considered essential in HCC therapy, radiofrequency ablation (RFA) also plays a key role. Percutaneous RFA was introduced in 1998.[3] Patients with hepatic function are graded as Child-Pugh Class A or B, and three or fewer tumors measuring 3 cm or less in diameter are the candidates for RFA therapy. However, despite these treatment options, the annual recurrence rate of resected HCC is 79.4% and 92.5% at 3 and 5 years, respectively.[4] Most importantly, because of low remaining liver volumes and insufficient hepatic function, repeated surgical operation is impossible. Thus, for patients with early-stage disease or recurrence after surgical treatment who do not qualified for liver resection and transplantation, RFA might be an effective alternative.

However, many cases of HCC show rapid and aggressive progression after RFA,[5] and it is possible that incomplete RFA is partly responsible for this. The epithelial–mesenchymal transition (EMT), which mediates tumor aggressiveness, has been implicated in this phenomenon. During EMT, epithelial cells lose their cell–cell adhesions and apical–basal polarity. This is associated with the reprogramming of mesenchymal markers, including loss of E-cadherin expression and gain of vimentin and Snail expression.[6]

Signal transducer and activator of transcription (STAT) proteins were first discovered in 1993 by James Darnell et al. and were found to be activated by cytokines and growth factors.[7] STAT3, a member of the STAT family, is clustered on chromosome 17 together with STAT5a and STAT5b. STAT3 encodes a protein that plays a key role in liver damage and oncogenesis by controlling cell cycle progression and apoptosis through intracellular signal transduction pathways or oncogenic signaling pathways. Interleukin (IL)-6, a pleiotropic cytokine, is important for proliferation and EMT induction in breast cancer cells.[8] Moreover, IL-6 activates the Janus kinase (JAK)/STAT3 signaling pathway by activating a specific IL-6 receptor that couples with transmembrane signal transducer gp130. This induces the phosphorylation of STAT3 at Tyr705 (Y705) (phospho-STAT3Y705).[9] Increased phospho-STAT3 enhances the expression of Snail, triggers EMT, and promotes carcinoma progression and metastasis.[10]

In this study, we established a mouse model of HCC by orthotopic transplantation. The mice were subjected to incomplete RFA to simulate this treatment in the clinical setting. We aimed to investigate the underlying mechanisms of EMT, specifically focusing on the IL-6/STAT3/Snail pathway. In addition, we collected several pathological sections from patients with liver cancer before and after RFA.


 > Materials and Methods Top


Experimental animals and cell lines

The mouse and human HCC cell lines, H22 and HepG2, respectively, were purchased from the Culture Collection of Chinese Academy of Sciences (Shanghai, China) and cultured in RPMI-1640 with 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5% CO2 at 37°C. Eleven male BALB/c mice were purchased from Jinan Pengyue Experimental Animal Center (mean weight, 28 g). All animals were housed in a specific pathogen-free environment at the Second Hospital of Shandong University, Jinan, China. The experiments were authorized by the Animal Care and Ethics Committee of Shandong University.

Heat treatment of H22 and HepG2 cells

H22 and HepG2 cells were cultured in 25-cm 2 flasks with serum-free medium for 24 h. Then, the flasks were covered with a plastic film and submerged in a 45°C water bath for 5 min; these samples were named H22-H or HepG2-H. Subsequently, all treated cell lines were maintained in a 37°C incubator with complete medium and 5% CO2 for 24 h.

Incubation with interleukin-6 or AG490

After serum starvation for 24 h, H22 cells were incubated with IL-6 (100 ng/mL) or AG490 (50 μM) for 4 h. The cells were then exposed to heat treatment as previously described at 45°C for 5 min. Subsequently, the cells were cultured in a 37°C incubator with 5% CO2 for 24 h.

Immunofluorescence

E-cadherin, vimentin, Snail, and phospho-STAT3 levels in the H22 cells with or without heat treatment were evaluated by immunofluorescence staining. After heat treatment and incubation for 24 h at 37°C, 1 × 106 cells were seeded onto slides, dried, and fixed with 4% paraformaldehyde at room temperature for 10 min. Cells were then washed with phosphate-buffered saline (PBS) three times and were fixed with 0.4% triton X-100 for 10 min. The washing process was repeated. Slides were blocked with goat serum for 30 min at 37°C. Subsequently, cells were incubated with primary antibodies targeting E-cadherin (1:100; Abcam, Cambridge, UK), vimentin (1:100; Abcam), Snail (1:75; Abcam), and phospho-STAT3 (1:75; Cell Signaling Technology, Danvers, MA, USA) at 4°C overnight and 37°C for 1 h. The slides were then rinsed in PBS three times and incubated with fluorescently labeled goat anti-rabbit or anti-mouse IgG secondary antibodies (1:100 dilution; GenScript, China) for 1 h at room temperature. After repeating the washing processes, the nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI) (Solarbio, Beijing, China) for 5 min. The slides were observed with an inverted fluorescence microscope (Nikon, Japan).

Quantification of soluble interleukin-6 protein by enzyme-linked immunosorbent assays

Culture supernatants from H22 cells with or without heat treatment were harvested after 24 h for the determination of IL-6 by an enzyme-linked immunosorbent assay (ELISA), according to the manufacturer's instructions (JiangLai, Shanghai, China). Following the addition of internal standards and samples (50 μL/well), 100 μL of horseradish peroxidase-conjugated antibody was added to each well, and the plates were incubated for 1 h at 37°C, followed by five washes with buffer. Next, 50 μL each of reagents A and B was added to all wells, and plates were incubated for 15 min at 37°C in the dark. Color development was stopped by adding 50 μL of stop solution, and the absorbance was recorded at 450 nm using a microplate reader. A standard curve was constructed to calculate the concentration of IL-6 in the samples.

Animal model

A total of 2 × 106 H22 cells were subcutaneously injected into the left hind-limb of male BALB/c mice. About 10 days later, tumors reached approximately 1 cm in diameter and mice were sacrificed by a fatal injection of chloral hydrate. The subcutaneous tumors were harvested, soaked in physiological saline solution, and cut into cubes (1 mm in diameter). Tumors with no macroscopic signs of necrosis were used for transplantation under Glisson's capsules (n = 10). Seven days later, five of the mice were treated using a RF generator at 45°C for 5 min. Tumors in the other five mice were harvested for subsequent analyses.

Radiofrequency ablation

Five animals were anesthetized via an intraperitoneal injection of 5% chloral hydrate (0.08 ml/10 g), and the abdominal area was shaved and disinfected. The mice were then placed in the supine position on an operating pad. For RFA treatment, an 18-gauge RFA probe with an active length of 2 cm (Celon ProSurge 100T-09; Olympus, Tokyo, Japan) was inserted into the liver tumor. Ablation was performed using an Olympus Winter and Ibe GmbH Radiofrequency-Induced Thermotherapy System. Previous studies have shown that maintaining human HCC cell lines (HepG2 and Huh7) at 45°C for 10 min results in active cells without signs of apoptosis. When cultured for several generations, changes in cell phenotypes are observed. Thus, we ablated the tumors at 45°C for 5 min and closed the abdominal cavities. After the operation, animals were observed for adverse reactions, and the abdominal area was palpated every other day to measure the growth of residual tumors. As stated above, five animals were sacrificed 7 days later, and tumor tissue was harvested.

Real-time polymerase chain reaction

Gene expression of E-cadherin, vimentin, and Snail was determined using quantitative real-time polymerase chain reaction (PCR). Total mRNA was isolated from tumors and heat-treated H22 cells using TRIzol (TIANGEN, China) according to the manufacturer's instructions. cDNA was synthesized from 1.0 μg RNA using a FastQuant (FQ) RT Kit (TIANGEN). The mixture (5× g DNA buffer [2 μL]; total RNA [1 μg]; RNase-free ddH2O [7 μL]) was incubated at 42°C for 3 min, chilled on ice, and then reverse-transcribed into cDNA using 10× fast RT buffer (2 μL), RT enzyme mix (1 μL), FQ-RT primer mix (2 μL), and RNase-free ddH2O (5 μL). The mixture was incubated at 42°C for 15 min and 95°C for 3 min and then chilled on ice. The reverse transcription (RT)-PCR was performed in a total volume of 20 μL containing 10 μL 2× SuperReal PreMix Plus, 0.6 μL forward primer, 0.6 μL reverse primer, 1 μL cDNA, and 7.8 μL RNase-free ddH2O under the following conditions: one cycle of 15 min at 95°C followed by 40 cycles of 10 s at 95°C and 32 s at 60°C. Sequences for RT-PCR oligonucleotide primers were as follows: E-cadherin: forward 5rwCAGGTCTCCTCATGGCTTTGC-3G and reverse 5evCTTCCGAAAAGAAGGCTGTCC-3T; vimentin: forward 5rwCGTCCACACGCACCTACAG-3T and reverse 5evGGGGGATGAGGAATAGAGGCT-3G; Snail: forward 5rwCACACGCTGCCTTGTGTCT-3C and reverse 5evGGTCAGCAAAAGCACGGTT-3T; and β-actin: forward 5rwGGCTGTATTCCCCTCCATCG-3C and reverse 5evCCAGTTGGTAACAATGCCATGT-3A.

Western blotting

Total protein was extracted from fresh-frozen tissues and cells using TRIzol (TIANGEN) according to the manufacturer's instructions and stored in a freezer at −20°C. Equal quantities of protein were separated by electrophoresis on 10% sodium dodecyl sulfate polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Bio-Rad, CA, USA). The membranes were blocked with 5% nonfat milk for 1 h and incubated overnight at 4°C with primary antibodies, including anti-E-cadherin (1:1000; Abcam), anti-vimentin (1:1000; Abcam), anti-phospho-STAT3Y705 (1:1000; Abcam), anti-Snail (1:1000; Abcam), and anti-β-actin (1:2000; GenScript). The membranes were then incubated with secondary antibodies for 1 h at room temperature and analyzed using a FluorChem Q system (ProteinSimple, USA).

Immunohistochemistry

Seven days after RFA, we collected liver lobes bearing primary tumors and ablation lesions, as well as adjacent healthy liver tissues and relapsed tumors. These tissues were then fixed in 4% paraformaldehyde for up to 24 h, embedded in paraffin, serially sectioned (5 mm thick), and placed on glass slides for immunohistochemistry. After the sections were deparaffinized in xylene and rehydrated through a graded series of alcohol solutions, we performed antigen retrieval by boiling the samples in ethylene diamine tetra acetic acid (pH = 8.0) for 20 min in a microwave oven. Endogenous peroxidase was blocked with 3% hydrogen peroxide for 10 min at room temperature, and nonspecific binding was performed with goat serum for 30 min. The sections were incubated overnight at 4°C with primary antibodies, including anti-E-cadherin (1:200; Abcam), anti-vimentin (1:200; Abcam), anti-phopsho-STAT3Y705 (1:100; Abcam), and anti-Snail (1:200; Abcam). Finally, the samples were incubated in 3,3-diaminobenzidine tetrahydrochloride and observed under a microscope.

Statistics

The experimental results are expressed as means ± standard deviations. All experiments were independently repeated three times. T-tests were used for comparisons between groups, and variance analysis was used for comparisons between multiple groups. P < 0.05 was considered statistically significant.


 > Results Top


Insufficient heat treatment induces epithelial–mesenchymal transition in the H22 cells and HepG2 cells

After heat-treating the cells, we detected the expression of various EMT biomarkers. Western blotting and RT-PCR revealed decreased E-cadherin expression (P < 0.05) and increased vimentin and Snail expression (P < 0.01) following exposure to 45°C for 5 min [Figure 1]a, [Figure 1]c, [Figure 1]e and [Figure 2].
Figure 1: Epithelial–mesenchymal transition after insufficient heat treatment. (a) E-cadherin, vimentin, and Snail in H22 and H22-H cells. (b) Interleukin-6 expression changes after heat treatment in H22 cells. (c-e) E-cadherin, vimentin, Snail, and phosphorylation of signal transducer and activator of transcription 3 at Tyr705 in H22 and H22-H groups, and HepG2 and HepG2-H groups

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Figure 2: E-cadherin, vimentin, Snail and phospho-STAT3 levels in H22 cells with or without heat treatment, evaluated by immunofluorescence staining

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Heat treatment promotes the secretion of interleukin-6

To assess whether cytokines were responsible for the observed phospho-STAT3 at Y705, we assessed the activation of molecules upstream of STAT3. STAT3, an effector of IL-6 signaling, is phosphorylated by IL-6. The expression of IL-6 in heat-treated H22 cells increased dramatically compared to control cells [P < 0.05; [Figure 1]b.

Heat treatment stimulates phosphorylation of signal transducer and activator of transcription 3 at Tyr705 expression

Analysis of the aforementioned markers showed that H22 cells could undergo EMT after treatment at 45°C for 5 min. The expression of Snail is reportedly regulated by phospho-STAT3Y705. Notably, levels of phospho-STAT3 increased significantly after heat treatment [P < 0.05; [Figure 1]d and [Figure 2].

IL-6 combined with heat treatment enhances epithelial–mesenchymal transition

Because heat treatment promoted the secretion of IL-6, we then treated the cells with IL-6 (100 ng/mL) for 4 h and performed heat treatment. Western blotting indicated that in IL-6-treated H22 cells, E-cadherin expression decreased significantly, and vimentin, Snail, and phospho-STAT3Y705 were highly overexpressed. Thus, IL-6 enhanced the phospho-STAT3, which was related to high Snail expression, a core promoter of the EMT; thereafter, other EMT markers changed accordingly (P < 0.05). Similar changes were observed in the cells treated with both heat and IL-6 in comparison to untreated H22 cells [P < 0.05; [Figure 3]a.
Figure 3: Stimulation of IL-6 induced a mesenchymal phenotype and AG490 reversed the observed EMT-like changes. (a) IL-6 combined with heat treatment enhanced EMT. (b) Inhibition of IL-6 by AG490 reversed the observed changes in EMT biomarkers

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Inhibition of interleukin-6 by AG490 reversed the observed epithelial–mesenchymal transition-like changes

AG490, a specific inhibitor of JAK2, can block the IL-6/STAT3 signaling pathway, resulting in decreased STAT3 phosphorylation. Thus, we incubated H22 cells with AG490 (50 μM) after cultivating cells in serum-free medium overnight for 4 h and then performed heat treatment. Western blotting results supported the effects of AG490 on IL-6/STAT3 signaling and reversed the EMT-like changes. In AG490-treated cells, E-cadherin was upregulated and vimentin, Snail, and phospho-STAT3Y705 levels were decreased compared to those in heat-treated H22 cells [P < 0.05; [Figure 3]b.

Orthotopic transplantation and insufficient radiofrequency ablation

Next, we established an orthotopic transplantation mouse model using H22 HCC cells. The incidence rate of HCC tumor-bearing mice was 100%, with no metastatic tumors observed in the abdomen or peritoneum. Seven days after transplantation, tumor sizes reached approximately 2 mm in diameter. We then performed low-temperature RFA at 45°C for 5 min. Seven days after ablation, we palpated abdominal masses. After sacrificing the animals, larger masses growing exophytically from the edge of the ablation lesion were observed, and large volumes of ascites were noted [Figure 4]A and [Figure 4]B.
Figure 4: Animal models and western blot results of animal groups. (A) a: We established an orthotopic transplantation mouse model using H22 HCC cells. b: 7 days after ablation, larger masses growing exophytically from the edge of the ablation lesion were observed. c: Ablation lesion. d: New mass after ablation (B) The control and treated groups. (C) Western blot results of epithelial–mesenchymal transition biomarkers in animal groups

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Insufficient radiofrequency ablation contributes to epithelial–mesenchymal transition in an animal model of liver cancer

We isolated proteins from untreated and treated tumors and performed western blotting and immunohistochemistry [Figure 4]C and [Figure 5]. In treated tumors compared to controls, E-cadherin expression was decreased, whereas vimentin, Snail, and phospho-STAT3Y705 levels were markedly higher, consistent with in vitro experiments. The insufficient RFA tumors also exhibited evidence of EMT.
Figure 5: Immunohistochemistry in animal models

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 > Discussion Top


Most patients with HCC cannot undergo surgical operation at the time of diagnosis, and only approximately 20% of patients have resectable tumors.[11] Therefore, RFA has been accepted as a curative technique for HCC. Two randomized controlled trials reported no significant difference in overall survival with RFA compared to surgical operation for HCC.[12],[13] However, RFA is associated with tumor aggressiveness due to large tumors beyond the range of the RF electrode, microscopic vascular invasion or satellites around the large tumor, irregular tumor shapes that are incompletely covered by the effective energy, the “heat-sink” effect when the tumor is adjacent to larger vessels that can absorb heat energy, and incomplete treatment to avoid unnecessary injury to the stomach, bile duct, gallbladder, diaphragm, and intestinal tract.[14] Many studies have shown that the aggressiveness of HCC after RFA is related to EMT and changes in the sarcomatous tissue,[15] and this has been supported by in vitro findings.[16],[17] Therefore, in this study, we used mouse liver cancer cells to verify the occurrence of EMT in vitro and developed an orthotopic mouse model of HCC to examine the effects of insufficient RFA in vivo.

To elucidate the mechanism through which heat treatment affects the occurrence of EMT, we investigated whether STAT3 plays a key role in EMT by upregulating Snail. According to our ELISA and western blotting results, IL-6 and phospho-STAT3Y705 levels were markedly increased. Moreover, activation of STAT3 promoted the expression of Snail by binding to the promoter of c-Myc, which is involved in the transcriptional activation of Snail.

In our animal experiment, only 7 days was required after transplantation to induce liver carcinoma, which shortened latency considerably compared to other transgenic mouse models of HCC.[18] Moreover, we successfully completed the RFA procedure in our mouse model. After RFA, we found a high aggressiveness rate and analyzed the biomarkers of EMT. Obvious changes were observed for several markers. E-cadherin is cleaved at the plasma membrane and subsequently degraded, and vimentin accumulation leads to a mesenchymal phenotype during the EMT.[19],[20] These points can help explain why our mouse model exhibited HCC aggressiveness, resulting from the induction of EMT after sublethal RFA in vivo, thereby confirming the in vitro findings of previous works. In summary, compared with other methods, this procedure required much less time and had a higher tumor incidence rate.

Further studies are needed to clarify the mechanisms underlying the rapid progression of residual HCC after RFA to confirm the therapeutic principles of RFA. Here, we provided evidence that the IL-6/STAT3/Snail pathway plays a vital role in tumor aggressiveness after RFA.

Financial support and sponsorship

The study was supported by National Natural Science Foundation of China (61671276) and the Natural Science Foundation of Shandong Province (ZR2014HM050, ZR2018PH 032, ZR201709250376).

Conflicts of interest

There are no conflicts of interest.



 
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Lau WY, Lai EC. The current role of radiofrequency ablation in the management of hepatocellular carcinoma: A systematic review. Ann Surg 2009;249:20-5.  Back to cited text no. 11
    
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