- Email: [email protected]
AKT pathway in human hepatocellular carcinoma
Accepted Manuscript Irisin stimulates cell proliferation and invasion by targeting the PI3K/AKT pathway in human hepatocellular carcinoma Guangjun Shi, Nan Tang, Jiantao Qiu, Deguo Zhang, Fei Huang, Yayu Cheng, Kun Ding, Weisheng Li, Ping Zhang, Xueying Tan PII:
S0006-291X(17)31682-0
DOI:
10.1016/j.bbrc.2017.08.148
Reference:
YBBRC 38423
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 6 August 2017 Accepted Date: 25 August 2017
Please cite this article as: G. Shi, N. Tang, J. Qiu, D. Zhang, F. Huang, Y. Cheng, K. Ding, W. Li, P. Zhang, X. Tan, Irisin stimulates cell proliferation and invasion by targeting the PI3K/AKT pathway in human hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.08.148. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Irisin stimulates cell proliferation and invasion by targeting the PI3K/AKT
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pathway in human hepatocellular carcinoma
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Guangjun Shia, Nan Tanga, Jiantao Qiub, Deguo Zhanga, Fei Huanga, Yayu Chengc, Kun Dingc,
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Weisheng Lic, Ping Zhangc,*, Xueying Tana,*
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a
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University, Qingdao, 266000, China
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b
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266000, China
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Department of Hepatobiliary Surgery, The Affiliated Qingdao Municipal Hospital of Qingdao
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Department of Cardiovascular Surgery, The Affiliated Hospital of Qingdao University, Qingdao,
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c
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Dao, China
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* Corresponding authors1: Xueying Tan, Department of Hepatobiliary Surgery, The Affiliated
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Qingdao Municipal Hospital of Qingdao University, Qing Dao, China; Telephone:
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86-532-18562692818; E-mail address: [email protected]
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and
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Ping Zhang, Department of Gynecology, The Affiliated Qingdao Municipal Hospital of Qingdao
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University, Qing Dao, China; Telephone: 86-532-17685506626; E-mail address:
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[email protected]
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Department of Gynecology, The Affiliated Qingdao Municipal Hospital of Qingdao University, Qing
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Abbreviations: FNDC5, fibronectin type III domain-containing protein 5; HCC, hepatocellular carcinoma; IM,
modified irisin; INM: nonmodified irisin; Dox, doxorubicin.
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ACCEPTED MANUSCRIPT Abstract
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Irisin is a newly identified myokine that may be cancer-associated, and its impact on liver cancer is
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unclear. To understand the roles of irisin in liver cancer, we investigated its effect in HepG2 and
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SMCC7721 hepatocellular carcinoma cells, and the underlying mechanisms. We determined irisin
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levels in liver tissues and serum samples obtained from patients by using real-time polymerase chain
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reaction and enzyme-linked immunosorbent assay. Irisin levels in cancerous livers were significantly
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upregulated compared with those in control livers, but serum irisin levels remained unchanged.
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Additionally, we evaluated the effects of different concentrations of human recombinant modified and
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active (glycosylated) irisin (IM) or human recombinant nonmodified irisin (INM) on cell migration,
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proliferation, viability, and invasiveness. CCK8, transwell, and scratching assays demonstrated that
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irisin significantly increased cell proliferation, invasion, and migration through activation of the
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PI3K/AKT pathway. Irisin-induced cell proliferation, migration, and invasion were blocked by a PI3K
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inhibitor (LY294002). Irisin also decreased the cytotoxicity of doxorubicin in HepG2 cells. These data
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indicate that increased irisin levels may have protective roles in liver cancer cells through partial
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activation of the PI3K/AKT pathway, which may facilitate liver cancer progression and decrease the
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sensitivity to chemotherapy.
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Keywords: Irisin, proliferation, doxorubicin, phosphorylated AKT
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ACCEPTED MANUSCRIPT 1. Introduction
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Irisin (FNDC5) is a recently identified adipokine/myokine [1] that has been widely studied worldwide.
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After intense exercise, PGC1-α, a transcriptional coactivator of the PPARc nuclear receptor,
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stimulates the expression of FNDC5, FNDC5 N-terminal domain (1-31 amino-acids (aa)) is
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proteolytically cleaved, and irisin (32-143 aa) is formed and released into the blood. Irisin was
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initially identified in skeletal muscles and adipose tissues [2], but its expression has been observed in
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the brain, pancreas, liver, stomach [3], heart [4], spleen, and skin [5]. Additionally, the expression of
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irisin was determined to increase in patients with metabolic disorder [6], cervical cancer, endometrial
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cancer [7],cancer cachexia [8], and prior gestational diabetes mellitus [9], and determined to decrease
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in type 2 diabetes patients [10, 11] and non-alcoholic fatty liver disease [12]. Irisin serum levels have
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been shown to be decreased in breast cancer [13], and irisin was shown to have considerable
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suppressive effects on malignant breast cancer cells [14]. Primary liver cancer was the sixth most
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frequent cancer and the second leading cause of cancer-related death worldwide in 2015, accounting
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for 810,500 deaths [15]. However, the detailed molecular mechanisms underlying liver cancer
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development and progression remain unclear. Although a significant increase in irisin levels has been
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detected in hepatocellular carcinoma (HCC) [16], no previous in vitro or in vivo experiments have
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confirmed the potential role of irisin in HCC.
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Here, we aimed to determine the expression levels of irisin in HCC tissues and serum samples,
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and, for the first time, to characterize the effects of irisin on liver cancer and molecular mechanisms
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underlying these effects.
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2. Materials and Methods 3
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This study has been approved by the Ethics Committee of Qing Dao municipal hospital. Twenty
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patients with HCC were recruited at the Qing Dao Municipal Hospital in 2016 and 2017.
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Corresponding non-tumor liver specimens were used as the controls. The study was conducted in
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collaboration with the Liver Center of the Qingdao Municipal Hospital. Only adult patients were
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enrolled, and informed consent was obtained from all patients. Exclusion criteria were as follows: (1)
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acute liver failure; (2) non-HCC liver neoplasms (i.e., cholangiocarcinoma, adenomatosis, hepatic
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hemangioma); (3) acute and/or chronic kidney disease (defined as serum creatinine 1.5 mg/dL at
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transplantation); (4) body-mass index (BMI) > 30 kg/m2; (5) autoimmune liver disease on active
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steroid treatment; (6) hepatitis C virus (HCV)-related disease treated with interferon; (7) a history of
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major cardiovascular events, such as myocardial infarction, unstable angina, or stroke; (8) diabetes; (9)
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history of chemotherapy and/or radiotherapy; (10) occurrence of transfer; (11) polycystic ovary
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syndrome; or (12) mental impairment.
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2.2. Determination of human serum FNDC5/irisin level
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The serum FNDC5/irisin level was determined using an enzyme-linked immunosorbent assay (ELISA)
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kit (USCN Life Science, Wuhan, China) according to the manufacturer’s instructions. The absorbance
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of the samples was measured at 450 nm (Bio-Tek ELX800, Winooski, USA).
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2.3. Real-time quantitative polymerase chain reaction (qPCR)
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Total RNA was extracted from tissues using Trizol (Invitrogen, California, USA). RNA was
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reverse-transcribed using reverse transcriptase (Takara, Dalian, China). To determine irisin mRNA
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levels, real-time qPCR was performed using an ABI 7500 Fast Real-Time PCR system (Applied
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glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
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2.4. Cell culture and treatments
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HepG2 and SMMC7721 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM,
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Hyclone Laboratories, Utah, USA) supplemented with 10% fetal bovine serum (FBS; Excell Bio,
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Shanghai, China) and penicillin (100 U/mL) and streptomycin (100 U/mL). The cells were transferred
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into culture flasks and cultured at 37°C in a humidified atmosphere with 5% CO2. The cells were
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treated for 24 h with either human recombinant modified (glycosylated) irisin (IM) from PlexBio (San
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Francisco, CA, USA) or human recombinant nonmodified irisin (INM; Cayman Chemical, Ann Arbor,
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MI, USA) at concentrations of 0.625 nM, 1.25 nM, 2.5 nM, 5 nM, 10 nM, or 20 nM, dissolved in
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culture media.
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2.5. Cell proliferation
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Cell proliferation was assessed using the Cell Counting Kit 8 (CCK 8; 7-sea, Haimen, China). HepG2
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and SMCC7721 cells were seeded into 96-well plates at 2000 cells/well in 200 µL of medium, and
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treated with different concentrations of IM or INM (0.625, 1.25, 2.5, 5, 10, and 20 nM) or without
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these molecules (N=8) for 24 h in a humidified atmosphere with 5% CO2. Subsequently, the cells
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were then incubated with 10 µL of CCK8 per well at 37°C for 1 h. An ELISA reader was then used to
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measure the optical density at 450 nm.
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Furthermore, HepG2 and SMCC7721 cells were treated with IM (2.5 nM) for 0, 12, 24, 48, and
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72 h (N=8), 10 µL of CCK8 solution was added to each well, and the cultures were incubated at 37°C
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for 1 h. The optical density of each well was determined at 450 nm.
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ACCEPTED MANUSCRIPT 2.6. Cell migration and invasion
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Cell migration was assessed by scratching monolayers of cells cultured in six-well plates. HepG2 cells
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were treated with or without 2.5 nM IM for 24 h prior to the introduction of the scratch. Subsequently,
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the cells were washed three times with phosphate-buffered saline (PBS), and the medium was
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removed from the wells and replaced with serum-free medium (N=5). Migration was observed after
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24 h and imaged under a light microscope (Olympus, Tokyo, Japan). Cell invasion was evaluated
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using Transwell insert chambers with an 8-µm pore size (Corning, Maine, USA) coated with Matrigel
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(BD Biosciences, Bedford, MA), and 5×104 cells were seeded in the serum-free medium in the upper
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chamber. The lower chamber was filled with DMEM with 10% FBS. After 24 h, the cells that had
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traversed the membrane were fixed, stained in 0.1% crystal violet solution for 30 min, and counted
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(N=5).
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2.7. Western blot
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Total protein was extracted from HepG2 cells. The protein concentration was determined using the
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BCA method, as recommended by the manufacturer (Beyotime Biotechnology, China). The collected
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proteins were boiled for 5 min and separated using SDS-PAGE (10%), after which they were
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transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The
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PVDF membranes were blocked with 5% bovine serum albumin (BSA) for 2 h at 25°C and then
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incubated with primary antibodies against AKT (1:1000) and phospho (p)-AKT (1:2000) (CST, Santa
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Cruz Biotechnology, CA, USA), at 4°C overnight. After washing with phosphate-buffered saline with
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Tween 20 (PBST) three times, the membranes were incubated with the secondary rabbit polyclonal
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antibody (1:2000) (Abgent, Suzhou, China) for 1 h at 25°C. Labeled protein bands were revealed by
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ECL detection reagent (Millipore, MA, USA). The obtained bands were quantified by determining the
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2.8. Annexin V/propidium iodide (PI) staining
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HepG2 cells were treated with medium or Dox (10 µM) with and without IM (2.5 nM) for 24 h. The
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efficiency of Dox against HepG2 cells was determined by using annexin V/PI staining. HepG2 cells
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were trypsinized, harvested, and washed with PBS two times, after which they were harvested by
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centrifugation at 1000 rpm for 3 min. Apoptotic cells were detected using an Annexin V-FITC
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apoptosis detection kit (Beyotime Biotechnology, Shanghai, China), according to the manufacturer’s
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instructions. Briefly, 1×105 treated cells were collected and incubated with Annexin V-FITC and PI,
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and analyzed using flow cytometry (BD FACS Calibur, BD, USA).
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2.9. Statistical analyses
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Data are presented as the mean ± standard deviation. Comparisons between the groups were
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performed using one–way analysis of variance (ANOVA). Student’s t-test was performed to determine
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the differences between two groups. P<0.05 was considered statistically significant.
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3. Results
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3.1. Irisin expression in HCC tissues
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Serum irisin levels did not differ between HCC patients and controls (Fig. 1A). Real-time qPCR
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analysis showed a significant increase in FNDC5 expression (over seven fold) in HCC tissues
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compared with that in the controls (Fig. 1B).
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3.2. Effects of irisin on cell proliferation, migration, and invasion
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The viability of HepG2 and SMCC7721 cells was shown to increase following the application of all
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Furthermore, the CCK8 assay demonstrated that IM treatment (2.5 nM) led to a significant increase in
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cell proliferation, compared with that of the control cells (Fig. 2B). Additionally, HepG2 cells treated
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with irisin (2.5 nM) displayed significantly increased cell migration (Fig. 2C) and invasiveness (Fig.
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2D), compared with those of the control cells.
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3.3. Irisin-mediated activation of PI3K/AKT signaling
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As shown in Figure 3A, compared with that in the control cells, p-AKT levels were shown to be
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significantly increased at 5, 15, and 30 min following irisin treatment. AKT phosphorylation induced
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by irisin was shown to be gradually suppressed by the PI3K inhibitor (40 µM) (Fig. 3B), whereas total
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AKT levels did not decrease. To further examine this effect, HepG2 cells were treated with LY294002
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(PI3K inhibitor) or without it in the presence of irisin. Irisin-induced HepG2 cell proliferation,
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migration, and invasiveness were shown to be significantly reduced following LY294002 treatment
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(Fig. 3C-3E).
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3.4. Effect of irisin on cellular sensitivity to Dox
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Dox treatment (10 µM) alone induced cell apoptosis, whereas addition of 2.5 nM IM significantly
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inhibited Dox cytotoxicity. To investigate the potential mechanism underlying this effect, we treated
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cells with LY294002 in the presence of irisin and Dox. Irisin significantly decreased the cytotoxicity
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of Dox, whereas the PI3K inhibitor inhibited the effects of irisin (Fig. 4).
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4. Discussion
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Recent studies found that overexpression of irisin in the hepatic tissue of HCC patients is associated
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proliferation and apoptosis have not been investigated. Consistent with the previously obtained results,
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we found that FNDC5 levels increased in the liver of patients with HCC. Irisin has been reported to
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significantly decrease the proliferation, viability, and migration of MDA-MB-231 and MCF-7 breast
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cancer cells [14]. However, in endometrial, colon, thyroid, and esophageal cancer cells in vitro, irisin
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was shown to have no effect on cell proliferation, adhesion, or colony formation relative to the
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findings in the control samples [17]. In contrast to the previous findings [14, 17], the findings
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obtained in this study suggest that post-translational modification of irisin and INM promote liver
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cancer cell proliferation, migration, and invasion. The discrepancy between our findings and those
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obtained previously may be due to differences in the cell lines used, and suggest that the effects of
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irisin may be tissue- and cell-specific.
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Many studies have shown that the PI3K/AKT pathway is involved in the development and
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progression of several cancers, including glioblastoma and ovarian, pancreatic, and liver cancers.
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PI3K/AKT has also been shown to regulate the expression of downstream target proteins, including
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mTOR [18, 19], NF-κB [20, 21], P70S6K [22], and Bax to regulate cell proliferation and migration.
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Further, AKT expression was found to be increased in liver cancer tissue and liver cancer cells in vitro
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[23], and was shown to be essential in the development and progression of liver cancer. Irisin has been
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shown to bind to as-yet unidentified receptors on the cell surface, activating p38 mitogen-activated
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protein kinase (p38MAPK), ERK mitogen-activated protein kinase (ERK-MAPK), and AKT to
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promote the browning of white adipocytes [24, 25], cell survival [26, 27], and pancreatic β cell
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proliferation [28]. In this study, we demonstrated that irisin significantly increases PI3K/AKT
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pathway activity in HepG2 cells. After 5 min, 15 min, or 30 min of treatment with 2.5 nM irisin, the
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addition of the PI3K inhibitor LY294002, the levels of AKT protein phosphorylation declined, as did
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cell proliferation, migration, and invasion. This indicates that the PI3K/AKT pathway plays an
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important role in irisin-induced cell proliferation, migration, and invasion.
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The PI3K/AKT pathway was previously shown to inhibit the initiation of apoptosis through the inhibition of caspase-9 phosphorylation [29], phosphorylation of Bad protein [30], inhibition of
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glycogen synthase kinase-3 β (p-GSK-3 β) activity [31], and regulation of the expression of
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transcription factors such as Forkhead [32] and NF-κB [33]. In addition, the PI3K/Akt signaling
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pathway participates in the multidrug resistance of human hepatocellular carcinoma [34]. Previous
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studies have demonstrated that irisin inhibits high glucose-induced apoptosis of endothelial cells [26]
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and pancreatic β cells [28]. Therefore, the present study examined whether irisin inhibited the
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apoptosis of HepG2 cells induced by Dox and whether the PI3K/AKT pathway was involved in the
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process. We demonstrated that the number of apoptotic cells increased significantly after intervention
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with Dox compared with that in the control group. HepG2 cell apoptosis significantly reduced in the
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irisin-treated group compared with that in the Dox group, and treatment with a PI3K inhibitor reduced
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this effect. Our results show that irisin can decrease the cytotoxicity of Dox in HepG2 cells through
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the PI3K/AKT pathway.
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In conclusion, the data presented in this study show that irisin regulates cell proliferation and
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migration of human liver cancer cells in vitro. We demonstrated that irisin activates the PI3K/AKT
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signaling pathway, promoting cell proliferation and decreasing the sensitivity to Dox. This indicates
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that the PI3K/AKT signaling pathway plays a central role in the irisin-induced proliferation of HepG2
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cells. Therefore, irisin may represent a novel potential target for future cancer therapies and its roles 10
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should be further investigated.
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Acknowledgments
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We would like to thank Editage [www.editage.cn] for English language editing.
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Funding
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This study was supported by research grants from the National Natural Science Fund of China (NO.
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81601617); Fundamental Research on Application of Qingdao Science and Technology Bureau (NO.
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12-1-4-16-（5）-jch).
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Figure legends
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Figure 1. Hepatic FNDC5 levels in hepatocellular carcinoma patients and controls. A: Plasma irisin
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levels did not different between HCC patients and controls. B: Heptic FNDC5/Irisin mRNA is
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over-expressed in HCC patients respect to the controls. *** P<0.001, compared with the controls.
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Figure 2. Effects of irisin on proliferation and migration of HepG2 and SMCC7721 cells. A: Viability
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of HepG2 and SMCC7721 cells treated with medium, or with different concentrations of modified
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irisin (IM) or nonmodified irisin (INM) ranging from 0.625 to 20 nM (N=8 in each group). B: Optical
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density at 450 nm, measured in the CCK8 assay, following treatment with IM (2.5 nM) at 24, 48, and
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72 h (N=8 in each group). C and D: Migration and invasiveness of the HepG2 cells treated as
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indicated (N=5 in each group). * P<0.05, ** P<0.01 and *** P<0.001, compared with the control. #
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P<0.05, compared with the control.
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Figure 3. Effects of irisin on adipocyte proliferation, migration, and invasion through the PI3K/AKT
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pathway. A: Phosphorylated and total AKT levels in HepG2 cells treated with phosphate-buffered
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saline (PBS; control) or r-irisin (2.5 nmol/L) at the indicated time points (N=3). B: Phosphorylated
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and total AKT levels in HepG2 cells pretreated with LY294002 at the indicated concentrations,
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followed by irisin treatment (N=3). C-E: Inhibition of the PI3K/AKT pathway reduces cell
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proliferation (N=8), migration (N=5), and invasion (N=5). * P<0.05, ** P<0.01, and *** P<0.001,
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compared with the control. # #P<0.01, and ### compared with the irisin group.
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Figure 4. Effects of irisin on HepG2 cell apoptosis induced by doxorubicin. HepG2 cells were treated
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with medium or doxorubicin (10 µM) with or without modified irisin (IM; 2.5 nM) or LY294002 for
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24 h (N=3). ** P<0.01, compared with the control. # P<0.05, compared with the Dox group. + P<0.05,
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compared with the Dox + irisin group.
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Highlights:
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Irisin levels are upgraded in patients with hepatocellular carcinoma. Irisin induces HEPG2 and SMCC7721 cell proliferation, migration, invasiveness, and inhibit appotosis of HEPG2 cell induced by Dox. Irisin induces the activation of PI3K/AKT pathway. Irisin has protective roles in liver cancer cells, inducing disease progression.
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Conflict of Interest Guangjun Shi declares that he has no conflict of interest. Nan Tang Shi declares that he has no conflict of interest. Jiantao Qiu declares that he has no conflict of interest. Deguo Zhang declares that he has no conflict of interest. Fei Huang declares that he has no conflict of interest. Kun Ding declares that she has no conflict of interest. Weisheng Li declares that he has no conflict of interest. Ping Zhang declares that she has no conflict of interest. Xueying Tan declares that he has no conflict of interest.
S0006-291X(17)31682-0
DOI:
10.1016/j.bbrc.2017.08.148
Reference:
YBBRC 38423
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 6 August 2017 Accepted Date: 25 August 2017
Please cite this article as: G. Shi, N. Tang, J. Qiu, D. Zhang, F. Huang, Y. Cheng, K. Ding, W. Li, P. Zhang, X. Tan, Irisin stimulates cell proliferation and invasion by targeting the PI3K/AKT pathway in human hepatocellular carcinoma, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/j.bbrc.2017.08.148. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Irisin stimulates cell proliferation and invasion by targeting the PI3K/AKT
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pathway in human hepatocellular carcinoma
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Guangjun Shia, Nan Tanga, Jiantao Qiub, Deguo Zhanga, Fei Huanga, Yayu Chengc, Kun Dingc,
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Weisheng Lic, Ping Zhangc,*, Xueying Tana,*
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a
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University, Qingdao, 266000, China
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b
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266000, China
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Department of Hepatobiliary Surgery, The Affiliated Qingdao Municipal Hospital of Qingdao
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Department of Cardiovascular Surgery, The Affiliated Hospital of Qingdao University, Qingdao,
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c
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Dao, China
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* Corresponding authors1: Xueying Tan, Department of Hepatobiliary Surgery, The Affiliated
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Qingdao Municipal Hospital of Qingdao University, Qing Dao, China; Telephone:
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86-532-18562692818; E-mail address: [email protected]
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and
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Ping Zhang, Department of Gynecology, The Affiliated Qingdao Municipal Hospital of Qingdao
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University, Qing Dao, China; Telephone: 86-532-17685506626; E-mail address:
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[email protected]
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Department of Gynecology, The Affiliated Qingdao Municipal Hospital of Qingdao University, Qing
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Abbreviations: FNDC5, fibronectin type III domain-containing protein 5; HCC, hepatocellular carcinoma; IM,
modified irisin; INM: nonmodified irisin; Dox, doxorubicin.
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ACCEPTED MANUSCRIPT Abstract
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Irisin is a newly identified myokine that may be cancer-associated, and its impact on liver cancer is
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unclear. To understand the roles of irisin in liver cancer, we investigated its effect in HepG2 and
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SMCC7721 hepatocellular carcinoma cells, and the underlying mechanisms. We determined irisin
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levels in liver tissues and serum samples obtained from patients by using real-time polymerase chain
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reaction and enzyme-linked immunosorbent assay. Irisin levels in cancerous livers were significantly
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upregulated compared with those in control livers, but serum irisin levels remained unchanged.
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Additionally, we evaluated the effects of different concentrations of human recombinant modified and
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active (glycosylated) irisin (IM) or human recombinant nonmodified irisin (INM) on cell migration,
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proliferation, viability, and invasiveness. CCK8, transwell, and scratching assays demonstrated that
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irisin significantly increased cell proliferation, invasion, and migration through activation of the
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PI3K/AKT pathway. Irisin-induced cell proliferation, migration, and invasion were blocked by a PI3K
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inhibitor (LY294002). Irisin also decreased the cytotoxicity of doxorubicin in HepG2 cells. These data
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indicate that increased irisin levels may have protective roles in liver cancer cells through partial
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activation of the PI3K/AKT pathway, which may facilitate liver cancer progression and decrease the
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sensitivity to chemotherapy.
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Keywords: Irisin, proliferation, doxorubicin, phosphorylated AKT
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ACCEPTED MANUSCRIPT 1. Introduction
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Irisin (FNDC5) is a recently identified adipokine/myokine [1] that has been widely studied worldwide.
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After intense exercise, PGC1-α, a transcriptional coactivator of the PPARc nuclear receptor,
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stimulates the expression of FNDC5, FNDC5 N-terminal domain (1-31 amino-acids (aa)) is
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proteolytically cleaved, and irisin (32-143 aa) is formed and released into the blood. Irisin was
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initially identified in skeletal muscles and adipose tissues [2], but its expression has been observed in
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the brain, pancreas, liver, stomach [3], heart [4], spleen, and skin [5]. Additionally, the expression of
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irisin was determined to increase in patients with metabolic disorder [6], cervical cancer, endometrial
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cancer [7],cancer cachexia [8], and prior gestational diabetes mellitus [9], and determined to decrease
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in type 2 diabetes patients [10, 11] and non-alcoholic fatty liver disease [12]. Irisin serum levels have
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been shown to be decreased in breast cancer [13], and irisin was shown to have considerable
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suppressive effects on malignant breast cancer cells [14]. Primary liver cancer was the sixth most
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frequent cancer and the second leading cause of cancer-related death worldwide in 2015, accounting
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for 810,500 deaths [15]. However, the detailed molecular mechanisms underlying liver cancer
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development and progression remain unclear. Although a significant increase in irisin levels has been
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detected in hepatocellular carcinoma (HCC) [16], no previous in vitro or in vivo experiments have
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confirmed the potential role of irisin in HCC.
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Here, we aimed to determine the expression levels of irisin in HCC tissues and serum samples,
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and, for the first time, to characterize the effects of irisin on liver cancer and molecular mechanisms
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underlying these effects.
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2. Materials and Methods 3
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This study has been approved by the Ethics Committee of Qing Dao municipal hospital. Twenty
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patients with HCC were recruited at the Qing Dao Municipal Hospital in 2016 and 2017.
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Corresponding non-tumor liver specimens were used as the controls. The study was conducted in
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collaboration with the Liver Center of the Qingdao Municipal Hospital. Only adult patients were
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enrolled, and informed consent was obtained from all patients. Exclusion criteria were as follows: (1)
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acute liver failure; (2) non-HCC liver neoplasms (i.e., cholangiocarcinoma, adenomatosis, hepatic
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hemangioma); (3) acute and/or chronic kidney disease (defined as serum creatinine 1.5 mg/dL at
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transplantation); (4) body-mass index (BMI) > 30 kg/m2; (5) autoimmune liver disease on active
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steroid treatment; (6) hepatitis C virus (HCV)-related disease treated with interferon; (7) a history of
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major cardiovascular events, such as myocardial infarction, unstable angina, or stroke; (8) diabetes; (9)
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history of chemotherapy and/or radiotherapy; (10) occurrence of transfer; (11) polycystic ovary
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syndrome; or (12) mental impairment.
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2.2. Determination of human serum FNDC5/irisin level
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The serum FNDC5/irisin level was determined using an enzyme-linked immunosorbent assay (ELISA)
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kit (USCN Life Science, Wuhan, China) according to the manufacturer’s instructions. The absorbance
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of the samples was measured at 450 nm (Bio-Tek ELX800, Winooski, USA).
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2.3. Real-time quantitative polymerase chain reaction (qPCR)
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Total RNA was extracted from tissues using Trizol (Invitrogen, California, USA). RNA was
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reverse-transcribed using reverse transcriptase (Takara, Dalian, China). To determine irisin mRNA
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levels, real-time qPCR was performed using an ABI 7500 Fast Real-Time PCR system (Applied
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ACCEPTED MANUSCRIPT Biosystems, Foster City, CA, USA). The results were normalized to the levels of the internal reference,
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glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
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2.4. Cell culture and treatments
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HepG2 and SMMC7721 cells were cultured in Dulbecco's Modified Eagle Medium (DMEM,
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Hyclone Laboratories, Utah, USA) supplemented with 10% fetal bovine serum (FBS; Excell Bio,
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Shanghai, China) and penicillin (100 U/mL) and streptomycin (100 U/mL). The cells were transferred
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into culture flasks and cultured at 37°C in a humidified atmosphere with 5% CO2. The cells were
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treated for 24 h with either human recombinant modified (glycosylated) irisin (IM) from PlexBio (San
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Francisco, CA, USA) or human recombinant nonmodified irisin (INM; Cayman Chemical, Ann Arbor,
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MI, USA) at concentrations of 0.625 nM, 1.25 nM, 2.5 nM, 5 nM, 10 nM, or 20 nM, dissolved in
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culture media.
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2.5. Cell proliferation
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Cell proliferation was assessed using the Cell Counting Kit 8 (CCK 8; 7-sea, Haimen, China). HepG2
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and SMCC7721 cells were seeded into 96-well plates at 2000 cells/well in 200 µL of medium, and
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treated with different concentrations of IM or INM (0.625, 1.25, 2.5, 5, 10, and 20 nM) or without
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these molecules (N=8) for 24 h in a humidified atmosphere with 5% CO2. Subsequently, the cells
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were then incubated with 10 µL of CCK8 per well at 37°C for 1 h. An ELISA reader was then used to
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measure the optical density at 450 nm.
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Furthermore, HepG2 and SMCC7721 cells were treated with IM (2.5 nM) for 0, 12, 24, 48, and
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72 h (N=8), 10 µL of CCK8 solution was added to each well, and the cultures were incubated at 37°C
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for 1 h. The optical density of each well was determined at 450 nm.
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Cell migration was assessed by scratching monolayers of cells cultured in six-well plates. HepG2 cells
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were treated with or without 2.5 nM IM for 24 h prior to the introduction of the scratch. Subsequently,
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the cells were washed three times with phosphate-buffered saline (PBS), and the medium was
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removed from the wells and replaced with serum-free medium (N=5). Migration was observed after
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24 h and imaged under a light microscope (Olympus, Tokyo, Japan). Cell invasion was evaluated
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using Transwell insert chambers with an 8-µm pore size (Corning, Maine, USA) coated with Matrigel
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(BD Biosciences, Bedford, MA), and 5×104 cells were seeded in the serum-free medium in the upper
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chamber. The lower chamber was filled with DMEM with 10% FBS. After 24 h, the cells that had
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traversed the membrane were fixed, stained in 0.1% crystal violet solution for 30 min, and counted
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(N=5).
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2.7. Western blot
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Total protein was extracted from HepG2 cells. The protein concentration was determined using the
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BCA method, as recommended by the manufacturer (Beyotime Biotechnology, China). The collected
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proteins were boiled for 5 min and separated using SDS-PAGE (10%), after which they were
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transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The
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PVDF membranes were blocked with 5% bovine serum albumin (BSA) for 2 h at 25°C and then
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incubated with primary antibodies against AKT (1:1000) and phospho (p)-AKT (1:2000) (CST, Santa
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Cruz Biotechnology, CA, USA), at 4°C overnight. After washing with phosphate-buffered saline with
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Tween 20 (PBST) three times, the membranes were incubated with the secondary rabbit polyclonal
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antibody (1:2000) (Abgent, Suzhou, China) for 1 h at 25°C. Labeled protein bands were revealed by
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ECL detection reagent (Millipore, MA, USA). The obtained bands were quantified by determining the
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2.8. Annexin V/propidium iodide (PI) staining
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HepG2 cells were treated with medium or Dox (10 µM) with and without IM (2.5 nM) for 24 h. The
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efficiency of Dox against HepG2 cells was determined by using annexin V/PI staining. HepG2 cells
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were trypsinized, harvested, and washed with PBS two times, after which they were harvested by
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centrifugation at 1000 rpm for 3 min. Apoptotic cells were detected using an Annexin V-FITC
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apoptosis detection kit (Beyotime Biotechnology, Shanghai, China), according to the manufacturer’s
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instructions. Briefly, 1×105 treated cells were collected and incubated with Annexin V-FITC and PI,
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and analyzed using flow cytometry (BD FACS Calibur, BD, USA).
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2.9. Statistical analyses
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Data are presented as the mean ± standard deviation. Comparisons between the groups were
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performed using one–way analysis of variance (ANOVA). Student’s t-test was performed to determine
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the differences between two groups. P<0.05 was considered statistically significant.
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3. Results
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3.1. Irisin expression in HCC tissues
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Serum irisin levels did not differ between HCC patients and controls (Fig. 1A). Real-time qPCR
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analysis showed a significant increase in FNDC5 expression (over seven fold) in HCC tissues
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compared with that in the controls (Fig. 1B).
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3.2. Effects of irisin on cell proliferation, migration, and invasion
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The viability of HepG2 and SMCC7721 cells was shown to increase following the application of all
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Furthermore, the CCK8 assay demonstrated that IM treatment (2.5 nM) led to a significant increase in
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cell proliferation, compared with that of the control cells (Fig. 2B). Additionally, HepG2 cells treated
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with irisin (2.5 nM) displayed significantly increased cell migration (Fig. 2C) and invasiveness (Fig.
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2D), compared with those of the control cells.
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3.3. Irisin-mediated activation of PI3K/AKT signaling
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As shown in Figure 3A, compared with that in the control cells, p-AKT levels were shown to be
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significantly increased at 5, 15, and 30 min following irisin treatment. AKT phosphorylation induced
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by irisin was shown to be gradually suppressed by the PI3K inhibitor (40 µM) (Fig. 3B), whereas total
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AKT levels did not decrease. To further examine this effect, HepG2 cells were treated with LY294002
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(PI3K inhibitor) or without it in the presence of irisin. Irisin-induced HepG2 cell proliferation,
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migration, and invasiveness were shown to be significantly reduced following LY294002 treatment
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(Fig. 3C-3E).
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3.4. Effect of irisin on cellular sensitivity to Dox
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Dox treatment (10 µM) alone induced cell apoptosis, whereas addition of 2.5 nM IM significantly
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inhibited Dox cytotoxicity. To investigate the potential mechanism underlying this effect, we treated
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cells with LY294002 in the presence of irisin and Dox. Irisin significantly decreased the cytotoxicity
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of Dox, whereas the PI3K inhibitor inhibited the effects of irisin (Fig. 4).
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4. Discussion
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Recent studies found that overexpression of irisin in the hepatic tissue of HCC patients is associated
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proliferation and apoptosis have not been investigated. Consistent with the previously obtained results,
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we found that FNDC5 levels increased in the liver of patients with HCC. Irisin has been reported to
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significantly decrease the proliferation, viability, and migration of MDA-MB-231 and MCF-7 breast
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cancer cells [14]. However, in endometrial, colon, thyroid, and esophageal cancer cells in vitro, irisin
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was shown to have no effect on cell proliferation, adhesion, or colony formation relative to the
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findings in the control samples [17]. In contrast to the previous findings [14, 17], the findings
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obtained in this study suggest that post-translational modification of irisin and INM promote liver
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cancer cell proliferation, migration, and invasion. The discrepancy between our findings and those
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obtained previously may be due to differences in the cell lines used, and suggest that the effects of
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irisin may be tissue- and cell-specific.
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Many studies have shown that the PI3K/AKT pathway is involved in the development and
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progression of several cancers, including glioblastoma and ovarian, pancreatic, and liver cancers.
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PI3K/AKT has also been shown to regulate the expression of downstream target proteins, including
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mTOR [18, 19], NF-κB [20, 21], P70S6K [22], and Bax to regulate cell proliferation and migration.
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Further, AKT expression was found to be increased in liver cancer tissue and liver cancer cells in vitro
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[23], and was shown to be essential in the development and progression of liver cancer. Irisin has been
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shown to bind to as-yet unidentified receptors on the cell surface, activating p38 mitogen-activated
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protein kinase (p38MAPK), ERK mitogen-activated protein kinase (ERK-MAPK), and AKT to
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promote the browning of white adipocytes [24, 25], cell survival [26, 27], and pancreatic β cell
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proliferation [28]. In this study, we demonstrated that irisin significantly increases PI3K/AKT
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pathway activity in HepG2 cells. After 5 min, 15 min, or 30 min of treatment with 2.5 nM irisin, the
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addition of the PI3K inhibitor LY294002, the levels of AKT protein phosphorylation declined, as did
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cell proliferation, migration, and invasion. This indicates that the PI3K/AKT pathway plays an
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important role in irisin-induced cell proliferation, migration, and invasion.
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The PI3K/AKT pathway was previously shown to inhibit the initiation of apoptosis through the inhibition of caspase-9 phosphorylation [29], phosphorylation of Bad protein [30], inhibition of
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glycogen synthase kinase-3 β (p-GSK-3 β) activity [31], and regulation of the expression of
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transcription factors such as Forkhead [32] and NF-κB [33]. In addition, the PI3K/Akt signaling
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pathway participates in the multidrug resistance of human hepatocellular carcinoma [34]. Previous
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studies have demonstrated that irisin inhibits high glucose-induced apoptosis of endothelial cells [26]
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and pancreatic β cells [28]. Therefore, the present study examined whether irisin inhibited the
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apoptosis of HepG2 cells induced by Dox and whether the PI3K/AKT pathway was involved in the
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process. We demonstrated that the number of apoptotic cells increased significantly after intervention
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with Dox compared with that in the control group. HepG2 cell apoptosis significantly reduced in the
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irisin-treated group compared with that in the Dox group, and treatment with a PI3K inhibitor reduced
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this effect. Our results show that irisin can decrease the cytotoxicity of Dox in HepG2 cells through
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the PI3K/AKT pathway.
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In conclusion, the data presented in this study show that irisin regulates cell proliferation and
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migration of human liver cancer cells in vitro. We demonstrated that irisin activates the PI3K/AKT
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signaling pathway, promoting cell proliferation and decreasing the sensitivity to Dox. This indicates
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that the PI3K/AKT signaling pathway plays a central role in the irisin-induced proliferation of HepG2
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cells. Therefore, irisin may represent a novel potential target for future cancer therapies and its roles 10
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should be further investigated.
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Acknowledgments
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We would like to thank Editage [www.editage.cn] for English language editing.
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Funding
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This study was supported by research grants from the National Natural Science Fund of China (NO.
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81601617); Fundamental Research on Application of Qingdao Science and Technology Bureau (NO.
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12-1-4-16-（5）-jch).
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Figure legends
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Figure 1. Hepatic FNDC5 levels in hepatocellular carcinoma patients and controls. A: Plasma irisin
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levels did not different between HCC patients and controls. B: Heptic FNDC5/Irisin mRNA is
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over-expressed in HCC patients respect to the controls. *** P<0.001, compared with the controls.
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Figure 2. Effects of irisin on proliferation and migration of HepG2 and SMCC7721 cells. A: Viability
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of HepG2 and SMCC7721 cells treated with medium, or with different concentrations of modified
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irisin (IM) or nonmodified irisin (INM) ranging from 0.625 to 20 nM (N=8 in each group). B: Optical
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density at 450 nm, measured in the CCK8 assay, following treatment with IM (2.5 nM) at 24, 48, and
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72 h (N=8 in each group). C and D: Migration and invasiveness of the HepG2 cells treated as
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indicated (N=5 in each group). * P<0.05, ** P<0.01 and *** P<0.001, compared with the control. #
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P<0.05, compared with the control.
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Figure 3. Effects of irisin on adipocyte proliferation, migration, and invasion through the PI3K/AKT
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pathway. A: Phosphorylated and total AKT levels in HepG2 cells treated with phosphate-buffered
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saline (PBS; control) or r-irisin (2.5 nmol/L) at the indicated time points (N=3). B: Phosphorylated
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and total AKT levels in HepG2 cells pretreated with LY294002 at the indicated concentrations,
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followed by irisin treatment (N=3). C-E: Inhibition of the PI3K/AKT pathway reduces cell
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proliferation (N=8), migration (N=5), and invasion (N=5). * P<0.05, ** P<0.01, and *** P<0.001,
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compared with the control. # #P<0.01, and ### compared with the irisin group.
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Figure 4. Effects of irisin on HepG2 cell apoptosis induced by doxorubicin. HepG2 cells were treated
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with medium or doxorubicin (10 µM) with or without modified irisin (IM; 2.5 nM) or LY294002 for
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24 h (N=3). ** P<0.01, compared with the control. # P<0.05, compared with the Dox group. + P<0.05,
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compared with the Dox + irisin group.
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Highlights:
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Irisin levels are upgraded in patients with hepatocellular carcinoma. Irisin induces HEPG2 and SMCC7721 cell proliferation, migration, invasiveness, and inhibit appotosis of HEPG2 cell induced by Dox. Irisin induces the activation of PI3K/AKT pathway. Irisin has protective roles in liver cancer cells, inducing disease progression.
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Conflict of Interest Guangjun Shi declares that he has no conflict of interest. Nan Tang Shi declares that he has no conflict of interest. Jiantao Qiu declares that he has no conflict of interest. Deguo Zhang declares that he has no conflict of interest. Fei Huang declares that he has no conflict of interest. Kun Ding declares that she has no conflict of interest. Weisheng Li declares that he has no conflict of interest. Ping Zhang declares that she has no conflict of interest. Xueying Tan declares that he has no conflict of interest.