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AKT signaling pathway involvement in fluoride-induced apoptosis in C2C12 cells
Accepted Manuscript PI3K/AKT signaling pathway involvement in fluoride-induced apoptosis in C2C12 cells
Bian-hua Zhou, Pan-pan Tan, Liu-shu Jia, Wen-peng Zhao, Ji-cang Wang, Hongwei Wang PII:
S0045-6535(18)30259-5
DOI:
10.1016/j.chemosphere.2018.02.057
Reference:
CHEM 20815
To appear in:
Chemosphere
Received Date:
29 December 2017
Revised Date:
06 February 2018
Accepted Date:
08 February 2018
Please cite this article as: Bian-hua Zhou, Pan-pan Tan, Liu-shu Jia, Wen-peng Zhao, Ji-cang Wang, Hong-wei Wang, PI3K/AKT signaling pathway involvement in fluoride-induced apoptosis in C2C12 cells, Chemosphere (2018), doi: 10.1016/j.chemosphere.2018.02.057
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PI3K/AKT signaling pathway involvement in fluoride-induced apoptosis in C2C12 cells Bian-hua Zhou1*, Pan-pan Tan1, Liu-shu Jia1, Wen-peng Zhao1, Ji-cang Wang1, Hong-wei
2
Wang1*
3 4
1
5
Safety, College of Animal Science and Technology, Henan University of Science and
6
Technology, Kaiyuan Avenue 263, Luoyang, Henan, 471000, People’s Republic of China
Henan Provincial Open Laboratory of Key Disciplines, Environment and Animal Products
Corresponding author: Bian-hua Zhou (E-mail: [email protected]) and Hong-wei Wang (E-
*
mail: [email protected]). College of Animal Science and Technology, Henan University of Science and Technology, 263 Kaiyuan Avenue, Luoyang, Henan 471000, People’s Republic of China.
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Abstract
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To investigate the mechanisms of fluoride-induced apoptosis, a fluoride-induced C2C12
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skeletal muscle cell (C2C12 cell) model was established in this study, and the viability of the
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C2C12 cells was measured using an MTT assay. Cell morphological changes were observed via
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haematoxylin and eosin staining and transmission electron microscopy. Apoptosis was monitored
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through Hoechst staining. The mRNA and protein expression of PI3K, PDK1, AKT1, BAD, Bcl-
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2, Bax and caspase-9 were detected through real-time PCR and western blotting, respectively. The
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results showed that the survival rates of C2C12 cells decreased gradually with an increasing
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fluoride doses. The C2C12 cell structure was seriously damaged by fluoride, presenting with
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pyknosis, mitochondrial ridge disruption and swollen endoplasmic reticulum. Furthermore, the
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expression of mRNA in PI3K, BAD, Bcl-2, Bax and caspase-9 were significantly increased in the
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fluoride group (P < 0.01), while the expression of PDK1 was markedly decreased (P < 0.01). The
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expression of protein in BAD, Bcl-2 and Bax were significantly increased in the fluoride group (P
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< 0.01), while the expression of PDK1 and P-AKT1 was markedly decreased (P < 0.01). In
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conclusion, fluoride-induced apoptosis in C2C12 cells is related to the PI3K/AKT signaling
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pathway.
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Keywords: fluoride, C2C12 cells, apoptosis, PI3K, AKT
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1. Introduction
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The PI3K/AKT pathway is important for cell signaling pathway and mediates a wide range of
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cellular functions, such as survival, proliferation, migration and differentiation (Yang et al., 2012).
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It is well known that phosphatidylinositol 3-kinase (PI3K), a crucial regulatory factor in the
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PI3K/AKT pathway, can be activated by various environmental stimuli (Lee, 2009). Following the
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changes that occur upon PI3K activation, phosphoinositide-dependent protein kinase-1 (PDK1)
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recruits protein kinase B (AKT) to the plasma membrane and activates the kinase (Tang et al.,
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2015; Paez and Sellers, 2003). Apoptosis, a type of programmed cell death, is an important
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mechanism that regulates organism growth, cell senescence and tissue homeostasis. Moreover, the
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PI3K/AKT pathway, via activation of caspases and the Bcl-2 family, tightly regulates it (Numata
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et al., 2011; Guo et al., 2015). Excessive apoptosis may overwhelm cellular clearance mechanisms
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and contribute to a reduction in disease in several tumour cell types (Di Serio et al., 2008; Li et al.,
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2011; Wang et al., 2014). However, excessive apoptosis induced by noxious risk factors can,
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alternatively, cause disease (Guo et al., 2012). Recent studies indicated that many noxious
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environmental stimuli could cause serious damage, inducing apoptosis in cells via the PI3K/AKT
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pathway (Zhao and Zhang, 2017; Sarkar and Sil, 2014; Zhang et al, 2017).
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Fluoride is one of the essential trace elements for life, and is widely present in food and
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water. Fluoride, in low concentrations, contributes to the growth and development of skeletal
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tissues, such as bones and teeth (Gu et al., 2016; Zhou et al., 2015). However, excessive fluoride
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can damage the liver (Zhou et al., 2015; Zhan et al., 2006), thyroid (Wang et al., 2009; Ge et al.,
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2005), kidney (Wasana et al., 2015; Chen et al., 2015), brain (Dec et al., 2017; Ge et al., 2006) and
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reproductive tissues (Zhou et al., 2013; Sun et al., 2010), which seriously affects the normal
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structures and physiological functions of the body. Several studies reported that excessive fluoride
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could induce cell apoptosis in HL-60 cells (Anuradha et al., 2000), ameloblasts (Wang et al.,
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2016) and MC3T3-E1 cells (Gu et al., 2016). In the present study, to measure the toxicity of
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fluoride in C2C12 cells and investigate the involvement of the PI3K/AKT pathway, a model of
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fluoride-induced C2C12 cells was established. The C2C12 cell structures, ultrastructure and levels
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of apoptosis were measured by microscopy, cell activity was measured by MTT method, and the
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mRNA and protein expression levels were assayed by real-time PCR and western blotting.
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2. Materials and methods
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2.1 Cell culture
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C2C12 cells were obtained from the cell bank of the Chinese Academy of Sciences. The
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C2C12 cells were cultured in DMEM media (Gibco, USA) containing 10% fetal bovine serum (
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Gibco, USA) and 1% penicillin-streptomycin solution (Solarbio, Beijing), maintained at 37 °C in
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a 5% CO2 cell incubator (Thermo, USA).
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2.2 Determination of IC50 in C2C12 cells
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After the C2C12 cells were seeded at 3×103 cells per well in 96-well plates for 24 h, fluoride
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(0, 0.05, 0.1, 0.5, 1, 2.5, 5, 10, 20 and 50 mmol/L) was added to the medium for 24 h, and then 10
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µl of Cell Counting Kit-8 (CCK-8, Beyotime, China) was added for 4 h. The absorbance was read
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at 450 nm in a microplate reader (Multiskan FC, Thermo Scientific, USA), and the IC50 of the
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C2C12 cells was calculated using GraphPad Prism 5. The experiment for each dose was given in
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six times.
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2.3 MTT assay
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C2C12 cells were seeded at 3×103 cells per well in 96-well plates for 24 h, then incubated in
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a medium containing fluoride (0, 0.05, 0.5, 1, 2.5, 5 and 10 mmol/L) for 24, 48, 72 and 96 h. Next,
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10 µl of the CCK-8 reagent was added to each well for 4 h. The absorbance was recorded using a
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microplate reader at 450 nm. The growth of the C2C12 cells was recorded by inverted microscope
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after treatment with fluoride at 0, 1 and 2.5 mmol/L for 48 h.
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2.4 Haematoxylin and Eosin (HE) staining
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Based on the calculated concentration of IC50, the fluoride doses at 0 and 2.5 mmol/L were
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used in the subsequent experiments. Thus, the C2C12 cells were seeded onto coverslips at a
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density of 5×104 cells per well in 6-well plates for attachment. They were then incubated with 0
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and 2.5 mmol/L of fluoride for 48 h. The cells were washed three times with PBS before being
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fixed in paraformaldehyde, and the cells were then incubated with the haematoxylin and eosin
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before observation using a light microscope.
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2.5 Transmission electron microscope (TEM) observation
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To analyse the effect of fluoride on C2C12 cell ultrastructures, the cells were treated with 0
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and 2.5 mmol/L of fluoride for 48 h after culturing in T-25 flasks for 24 h. The cells were
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collected, fixed in 2.5% glutaraldehyde phosphate buffer (PB) (pH 7.4) at 4 ºC and washed by PB.
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Afterward, it was fixed in 1% osmic acid for 1.5 h. The cells were dehydrated using 50%, 70%,
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80%, 90% and 100% alcohol and were embedded in araldite resin. Ultra-thin sections were
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serially cut and stained with uranyl acetate and lead citrate before TEM examination (H-7500,
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Hitachi, Japan).
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2.6 Hoechst staining
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To assess the effect of fluoride on apoptosis in C2C12 cells, cell coverslips were prepared
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and treated with fluoride at 0 and 2.5 mmol/L for 48 h. The C2C12 cells were fixed in
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paraformaldehyde and incubated with Hoechst 33258 staining (Beyotime, China) for 10 min at 4
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°C in the dark before being washed three times with PBS. The cell coverslips were embedded in
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an anti-fade mounting medium before being observed and photographed using fluorescence
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microscopy (CKX41S, Olympus, Japan).
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2.7 Real-time PCR
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The cells were treated with 0 and 2.5 mmol/L of fluoride for 48 h after culturing in T-25
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flasks for 24 h. The total RNA was extracted from the C2C12 cells using the Trizol reagent
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(Invitrogen, Life Technologies) and purified using the RNeasy® Mini Kit (TaKaRa, QIAGEN),
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according to the manufacturer’s instructions. The cDNA was synthesised from 5 µl of RNA using
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SuperScriptTM II Reverse Transcriptase Kit (Invitrogen, Life Technologies). Real-time PCR was
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performed in a reaction mixture containing SYBR Green PCR master mix and 1 µl of first-stand
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cDNA as a template, with primers specific for PI3K, PDK1, AKT1, BAD, Bcl-2, Bax and
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caspase-9, which are shown in Table 1. The expression of the target genes relative to β-actin was
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determined.
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2.8 Western blotting analysis
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After 48 h of fluoride treatment, the C2C12 cells were collected and lysed in RIPA buffer
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(Google Biotechnology, Wuhan) containing protease inhibitors and then incubated on ice 30 min
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before centrifugation at 12, 000 g for 5 min. The total protein concentration was measured using
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the BCA protein assay (Google Biotechnology, China). Equal amounts of protein samples were
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run in 10% SDS-PAGE and transferred to PVDF membranes. They were then blocked with 5%
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non-fat milk for 60 min, and an overnight incubation at 4 °C with PI3K (A0265, Boster, Wuhan,
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China), PDK1 (5662, CST, USA), T-AKT1 (BM1612, Boster, Wuhan, China), P-AKT1 (AF0908,
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Affinity, USA), BAD (GB11198, Servicebio, Wuhan, China), Bcl-2 (GB12008, Servicebio,
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Wuhan, China), Bax (GB11007, Servicebio, Wuhan, China), caspase-9 (9502, CST, USA) and β-
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actin (GB13001-1, Servicebio, Wuhan, China) primary antibodies. After the membranes were
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washed three times for 10 min with 0.5% TBS-Tween 20 buffer (TBST), they were incubated with
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secondary antibodies (074-1506, KPL, USA) for 30 min at room temperature. Finally, the
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membranes were washed three times for 5 min with TBST. The bands were visualised using
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enhanced chemiluminescence (ECL) reagents.
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2.9 Statistical analysis
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The results are expressed as the mean ± standard deviation (SD). All data were analysed using
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the SPSS software version 13.0 and the student’s t test. A P value less than 0.05 was considered as
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statistically significant.
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3. Results
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3.1 Effects of fluoride on the viability of C2C12 cells
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The viability of the C2C12 cells was determined via an MTT assay. As shown in Fig. 1, the
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survival rates of the C2C12 cells decreased significantly with the doses of fluoride added, and an
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IC50 of 3.209 mmol/L was measured. In addition, the MTT results indicated that the absorbance
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of the C2C12 cells increased with all the doses of fluoride added, and the time for which it was
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added (Fig. 2). Under microscopic observation, the number of C2C12 cells was obviously
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decreased, the shapes of the C2C12 cells changed, and the intercellular spaces became larger
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under the influence of fluoride in doses of 1 mmol/L (Figs. 3b and 3b1) and 2.5 mmol/L for 48 h
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(Figs. 3c and 3c1). These results were consistent with the MTT assay.
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3.2 Effects of fluoride on histopathological and ultrastructural changes in C2C12 cells
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As shown in Fig. 4, the C2C12 cells in the control group grew well, and the structure of the
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cells was normal (Fig. 4a). Conversely, with the increasing doses of fluoride, the number of cells
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decreased and the gap between the cells increased. Nuclear pyknosis and deformities were seen
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under the light microscope in the fluoride group (Fig. 4b).
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The ultrastructural changes in the C2C12 cells are shown in Fig. 4. In the control group, the
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structure of the C2C12 cells remained normal, and the outline of cells was easy to observe. The
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distribution of chromatin in the nucleus was uniform, the double nuclear membrane was clearly
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visible and the mitochondrion ridge was clearly seen (Figs. 4A, 4A1 and 4A2). However, in the
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fluoride group, the amount of chromatin in the nucleus was visibly decreased, nuclear
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condensation had commenced and apoptotic bodies began to form. The mitochondria were
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severely damaged, due to vacuolisation, and the ridge was indistinct. The endoplasmic reticulum
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also appeared swollen (Figs. 4B, 4B1 and 4B2).
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3.3 Effects of fluoride on apoptosis of C2C12 cells
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With Hoechst staining, shown in Figs. 4C and 4D, the number of positively stained cells
150
increased compared to the control group, and the colour of the cells became pale (Fig. 4D).
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3.4 Effects of fluoride on the mRNA expression of PI3K, PDK1, AKT1, BAD, Bcl-2, Bax and
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caspase-9
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The mRNA expression levels of PI3K, PDK1, AKT1, BAD, Bcl-2, Bax and caspase-9 in
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C2C12 cells were assessed (Fig. 5). In the fluoride group, the mRNA expressions levels of PI3K,
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BAD, Bcl-2, Bax and caspase-9 were significantly up-regulated (P < 0.01), respectively, while the
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mRNA expressions levels of PDK1 was significantly down-regulated compared with the control
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group (P < 0.01).
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3.5 Effects of fluoride on the protein expression of PI3K, PDK1, T-AKT1, P-AKT1, BAD,
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Bcl-2, Bax and caspase-9
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The protein expression levels of PI3K, PDK1, T-AKT1, P-AKT1, BAD, Bcl-2, Bax and
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caspase-9 in C2C12 cells were detected by western blotting (Fig. 6). After treatment with fluoride,
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the levels of BAD, Bcl-2 and Bax were significantly increased compared with control group (P <
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0.01), while the levels of PDK1 and P-AKT1 were significantly decreased (P < 0.01),
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respectively. The changes in the expression levels of these proteins matched the changes seen in
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the mRNA expression levels.
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4. Discussion
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Fluoride can readily penetrate cellular membranes and seriously damage the structure and
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physiological function of cells (Zhou et al., 2015). Several studies have shown that excessive
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fluoride can decrease the viability of cells and that the growth of cells is inhibited by the excess
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presence of fluoride ion (Chae et al., 2016; Yan et al., 2015; Yang et al., 2015). Our previous work
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confirmed this inhibition in HeLa cells (Wang et al., 2017a). In the present study, the viability of
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C2C12 cells was significantly decreased with increasing fluoride concentrations, and the IC50 was
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shown to be 3.209 mmol/L. The IC50 of fluoride exposure was 1.9 mmol/L in human bronchial
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epithelial cells (BEAS-2B) and was 5.8 mmol/L in human liver cells (HL-7702) (Ying et al.,
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2017). These results suggested that high concentrations of fluoride affect cell viability of all types
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and inhibit cellular growth, though the tolerance of different cells exposed to fluoride is different.
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This finding provides evidence for fluoride-induced toxicology in C2C12 cells.
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To explore the theory that fluoride would decrease the viability of C2C12 cells, the
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histopathological and ultrastructural changes were observed. The C2C12 cellular structures were
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obviously changed from the control group, with nuclear pyknosis and deformities seen in the
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fluoride group. The number of C2C12 cells was also reduced by the presences of excess fluoride.
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This result was consistent with the fluoride-induced decrease in viability of the C2C12 cells.
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Under TEM observation, the mitochondria were observed to be swollen and vacuolised, with
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indistinct ridges. Furthermore, the endoplasmic reticulum was dilated, and nuclear pyknosis was
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visible in the C2C12 cells in the fluoride group. The above histopathological characteristics were
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due to the beginnings of apoptosis. Several studies indicated that the fluoride-induced
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mitochondrial damage and endoplasmic reticulum swelling were highly associated with the
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apoptosis process (Wang et al., 2017b; Zhang et al., 2015; Deng et al., 2016). Moreover, the
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increasing number of positive cells observed in the Hoechst staining provided direct evidence in
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this case for fluoride-induced apoptosis in C2C12 cells.
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PI3K, PDK1 and AKT, proteins in the PI3K/AKT pathway, play an important role in the
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regulation of cell cycle progression (Chang et al., 2003). The phosphorylation of AKT is an
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important link in the cell apoptosis process (Gu et al., 2016). Phosphorylated AKT could facilitate
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cell survival and proliferation and regulate various signaling pathways (Sussman, 2007). The
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present study found that with the significant decrease of the expression level of PDK1, the P-
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AKT1 expression level significantly decreased in C2C12 cells exposed to fluoride. Due to the
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week suppression to BAD by P-AKT1 made it possible to induce apoptosis in C2C12 cells with
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the fluoride treatment.
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Bcl-2 is a widely studied modulator of programmed cell death. Moreover, Bax, as a member
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of the Bcl-2 family, can form heterologous dimeric complexes with Bcl-2 and accelerate
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programmed cell death (Lou et al., 2014). Recent studies reported that the Bcl-2 levels decreased
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and Bax levels increased in presence of excessive fluoride (Liu et al., 2013; Cao et al., 2013).
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However, the present study showed that the expression levels of Bcl-2 and Bax both increased in
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the C2C12 cells exposed to excessive fluoride. The increases in both Bcl-2 and Bax expression
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might be a feedback effect following the C2C12 cells apoptosis caused by fluoride (Lou et al.,
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2014). Furthermore, Bax, an opposition partner of Bcl-2, its over expression promotes the level of
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Bcl-2 increased. Our results indicated that Bax and Bcl-2 play a pivotal role in the PI3K/AKT
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pathway involving the fluoride-induced apoptosis of the C2C12 cells. Caspase-9 is the key
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initiating caspase for the intrinsic pathway to cell death (Yan et al., 2009). In the present study, the
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level of caspase-9 expression in C2C12 cells significantly increased in the fluoride group. It is
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reported that caspase-9 can be activated through Apaf1 binding to cytochrome c in the cytoplasm,
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thereby forming a multi-protein complex known as an apoptosome that leads to apoptosis (Wu et
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al., 2014). BAD, a pro-apoptotic protein, heterodimerises with anti-apoptotic proteins such as Bcl-
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2 and Bcl-xL to promote cell death (Martelli et al., 2006; Franke et al., 2003; Li et al., 2013). It
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was reported that the increased expression of BAD promoted the apoptosis induced by fluoride in
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human oral squamous cell carcinoma HSC-2 cells (Otsuki et al., 2011). In the present study, the
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expression levels of BAD significantly increased promotes the fluoride-induced apoptosis in
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C2C12 cells. Several studies suggested that a deficit of BAD can result in diminished
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mitochondria-based glucokinase activity and blunted mitochondria respiration in response to
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glucose, which may be associated with apoptosis (Danial et al., 2003; Ranger et al., 2003; Seo et
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al., 2004), thus this mechanism requires further study. These results, taken together, indicated that
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the PI3K/AKT pathway is involved in the fluoride-induced apoptosis of C2C12 cells.
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In conclusion, the viability of C2C12 cells seriously decreased in the presence of excess
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fluoride, and the IC50 of this was shown to be 3.209 mmol/L. The results provide powerful
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evidence for the fluoride-induced apoptosis of C2C12 cells via the PI3K/AKT signaling pathway.
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Acknowledgements
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This work is sponsored by the National Natural Science Foundation of China (grant no.
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31201963) and Youth Backbone Teachers Project in Henan Province Department of Education,
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China (grant no. 2016GGJS-061).
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Fig. 1 IC50 of C2C12 cells exposed to fluoride (n=6) After 24 h of fluoride exposure, the survival rate of C2C12 cells decreased in proportion to the dose of fluoride added, and the IC50 of this was shown to be 3.209 mmol/L.
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Fig. 2 MTT determination of C2C12 cells viability (n=6) The viability of C2C12 cells increased with lower concentration of fluoride over 24 h, and cell growth was inhibited by a high fluoride concentration. After fluoride exposure for 48, 72 and 96 h, the viability of the C2C12 cells significantly decreased with the doses of fluoride. *P < 0.05 and **P < 0.01 indicated statistical significance compared to the control group.
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Fig. 3 Morphological changes in C2C12 cells (a) and (a1), (b) and (b1), (c) and (c1) show the morphological changes in the C2C12 cells between the control group, the 1.0 mmol/L fluoride group, and the 2.5 mmol/L fluoride group, respectively. After fluoride exposure for 48 h, the number of C2C12 cells obviously decreased, the shapes of the C2C12 cells changed, and the intercellular spaces widened with the increasing doses of fluoride.
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Fig. 4 Histopathological and ultrastructural changes and Hoechst staining in C2C12 cells The morphological changes between C2C12 cells from the control group and the fluoride group are showed in (a) and (b), respectively, with a normal histopathological structure seen in (a) and nuclear pyknosis and deformation of the C2C12 cells exposed to excessive fluoride shown in (b) with an increasing gap between cells also obvious. (A) shows the normal ultrastructure of C2C12 cells in the control group. (A1) and (A2) show the normal mitochondria (Mi), endoplasmic reticulum (ER) and nuclear membrane (NM) of C2C12 cells. (B) shows the ultrastructure of C2C12 cells exposed to excessive fluoride, with the nuclear condensation clearly seen. (B1) and (B2) show the mitochondria vacuolisation and ER swelling. (C) and (D) show the Hoechst staining results for the C2C12 cells. (D) shows that the number of positive cells increased compared to the control group, and the colour of the cells became pale.
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Fig. 5 Real-time PCR analysis of mRNA expression levels of different genes in the C2C12 cells (n=3). **P < 0.01 (the fluoride group compared with the control group).
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Relative density
b
Control group 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1
Fluoride group
**
**
**
** ** B ax ca sp as e9
cl -2 B
A D B
K T1
PA
1
K T1
TA
PD K
PI 3K
0.0
Fig. 6 Western blotting analysis of the expression levels of PI3K, PDK1, T-AKT1, PAKT1, BAD, Bcl-2, Bax and caspase-9 in C2C12 cells (n=3). (a) Western blotting electrophoretic pattern and (b) relative expression levels. **P < 0.01 (the fluoride group compared with the control group).
ACCEPTED MANUSCRIPT Highlights Fluoride causes histopathological changes in C2C12 cells. Fluoride exposure damages ultrastructure in C2C12 cells. Fluoride exposure induces apoptosis in C2C12 cells. PI3K/AKT signaling pathway is involved in fluoride-induced apoptosis in C2C12 cells.
ACCEPTED MANUSCRIPT Table 1 Oligonucleotide Primer sets for real-time PCR
Gene
P13K
PDK1
AKT1
BAD Bcl-2 Bax caspase-9
β-actin
Primers Forward (5′→3′) Primers Reverse (5′→3′) AGCCGCCAGCTCTGATAATA TCTCCCCAGTACCATTCAGC CAAGAACTCCGACCAGAAGC TTTCTGCACCACTTGTGAGC ACTCATTCCAGACCCACGAC CCGGTACACCACGTTCTTCT AGGACTTATCAGCCGAAGCA GCTCAAACTCTGGGATCTGG CTGCAAATGCTGGACTGAAA TCAGGAGGGTTTCCAGATTG TGCAGAGGATGATTGCTGAC GATCAGCTCGGGCACTTTAG TTCCCAGGTTTTGTCTCCTG GGGACTGCAGGTCTTCAGAG TGTTACCAACTGGGACGACA GGGGTGTTGAAGGTCTCAAA
Accession Number
Tm (℃)
Bp
NM_001077495.2
60
183
AF079535.1
55
110
NM_009652.3
55
176
NM_007522.3
59
187
NM_009741.5
59
158
NM_007527.3
59
173
NM_015733.5
55
106
NM_007393.5
55
165
Bian-hua Zhou, Pan-pan Tan, Liu-shu Jia, Wen-peng Zhao, Ji-cang Wang, Hongwei Wang PII:
S0045-6535(18)30259-5
DOI:
10.1016/j.chemosphere.2018.02.057
Reference:
CHEM 20815
To appear in:
Chemosphere
Received Date:
29 December 2017
Revised Date:
06 February 2018
Accepted Date:
08 February 2018
Please cite this article as: Bian-hua Zhou, Pan-pan Tan, Liu-shu Jia, Wen-peng Zhao, Ji-cang Wang, Hong-wei Wang, PI3K/AKT signaling pathway involvement in fluoride-induced apoptosis in C2C12 cells, Chemosphere (2018), doi: 10.1016/j.chemosphere.2018.02.057
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 1
PI3K/AKT signaling pathway involvement in fluoride-induced apoptosis in C2C12 cells Bian-hua Zhou1*, Pan-pan Tan1, Liu-shu Jia1, Wen-peng Zhao1, Ji-cang Wang1, Hong-wei
2
Wang1*
3 4
1
5
Safety, College of Animal Science and Technology, Henan University of Science and
6
Technology, Kaiyuan Avenue 263, Luoyang, Henan, 471000, People’s Republic of China
Henan Provincial Open Laboratory of Key Disciplines, Environment and Animal Products
Corresponding author: Bian-hua Zhou (E-mail: [email protected]) and Hong-wei Wang (E-
*
mail: [email protected]). College of Animal Science and Technology, Henan University of Science and Technology, 263 Kaiyuan Avenue, Luoyang, Henan 471000, People’s Republic of China.
ACCEPTED MANUSCRIPT 8
Abstract
9
To investigate the mechanisms of fluoride-induced apoptosis, a fluoride-induced C2C12
10
skeletal muscle cell (C2C12 cell) model was established in this study, and the viability of the
11
C2C12 cells was measured using an MTT assay. Cell morphological changes were observed via
12
haematoxylin and eosin staining and transmission electron microscopy. Apoptosis was monitored
13
through Hoechst staining. The mRNA and protein expression of PI3K, PDK1, AKT1, BAD, Bcl-
14
2, Bax and caspase-9 were detected through real-time PCR and western blotting, respectively. The
15
results showed that the survival rates of C2C12 cells decreased gradually with an increasing
16
fluoride doses. The C2C12 cell structure was seriously damaged by fluoride, presenting with
17
pyknosis, mitochondrial ridge disruption and swollen endoplasmic reticulum. Furthermore, the
18
expression of mRNA in PI3K, BAD, Bcl-2, Bax and caspase-9 were significantly increased in the
19
fluoride group (P < 0.01), while the expression of PDK1 was markedly decreased (P < 0.01). The
20
expression of protein in BAD, Bcl-2 and Bax were significantly increased in the fluoride group (P
21
< 0.01), while the expression of PDK1 and P-AKT1 was markedly decreased (P < 0.01). In
22
conclusion, fluoride-induced apoptosis in C2C12 cells is related to the PI3K/AKT signaling
23
pathway.
24
Keywords: fluoride, C2C12 cells, apoptosis, PI3K, AKT
ACCEPTED MANUSCRIPT 26
1. Introduction
27
The PI3K/AKT pathway is important for cell signaling pathway and mediates a wide range of
28
cellular functions, such as survival, proliferation, migration and differentiation (Yang et al., 2012).
29
It is well known that phosphatidylinositol 3-kinase (PI3K), a crucial regulatory factor in the
30
PI3K/AKT pathway, can be activated by various environmental stimuli (Lee, 2009). Following the
31
changes that occur upon PI3K activation, phosphoinositide-dependent protein kinase-1 (PDK1)
32
recruits protein kinase B (AKT) to the plasma membrane and activates the kinase (Tang et al.,
33
2015; Paez and Sellers, 2003). Apoptosis, a type of programmed cell death, is an important
34
mechanism that regulates organism growth, cell senescence and tissue homeostasis. Moreover, the
35
PI3K/AKT pathway, via activation of caspases and the Bcl-2 family, tightly regulates it (Numata
36
et al., 2011; Guo et al., 2015). Excessive apoptosis may overwhelm cellular clearance mechanisms
37
and contribute to a reduction in disease in several tumour cell types (Di Serio et al., 2008; Li et al.,
38
2011; Wang et al., 2014). However, excessive apoptosis induced by noxious risk factors can,
39
alternatively, cause disease (Guo et al., 2012). Recent studies indicated that many noxious
40
environmental stimuli could cause serious damage, inducing apoptosis in cells via the PI3K/AKT
41
pathway (Zhao and Zhang, 2017; Sarkar and Sil, 2014; Zhang et al, 2017).
42
Fluoride is one of the essential trace elements for life, and is widely present in food and
43
water. Fluoride, in low concentrations, contributes to the growth and development of skeletal
44
tissues, such as bones and teeth (Gu et al., 2016; Zhou et al., 2015). However, excessive fluoride
45
can damage the liver (Zhou et al., 2015; Zhan et al., 2006), thyroid (Wang et al., 2009; Ge et al.,
46
2005), kidney (Wasana et al., 2015; Chen et al., 2015), brain (Dec et al., 2017; Ge et al., 2006) and
47
reproductive tissues (Zhou et al., 2013; Sun et al., 2010), which seriously affects the normal
ACCEPTED MANUSCRIPT 48
structures and physiological functions of the body. Several studies reported that excessive fluoride
49
could induce cell apoptosis in HL-60 cells (Anuradha et al., 2000), ameloblasts (Wang et al.,
50
2016) and MC3T3-E1 cells (Gu et al., 2016). In the present study, to measure the toxicity of
51
fluoride in C2C12 cells and investigate the involvement of the PI3K/AKT pathway, a model of
52
fluoride-induced C2C12 cells was established. The C2C12 cell structures, ultrastructure and levels
53
of apoptosis were measured by microscopy, cell activity was measured by MTT method, and the
54
mRNA and protein expression levels were assayed by real-time PCR and western blotting.
55
2. Materials and methods
56
2.1 Cell culture
57
C2C12 cells were obtained from the cell bank of the Chinese Academy of Sciences. The
58
C2C12 cells were cultured in DMEM media (Gibco, USA) containing 10% fetal bovine serum (
59
Gibco, USA) and 1% penicillin-streptomycin solution (Solarbio, Beijing), maintained at 37 °C in
60
a 5% CO2 cell incubator (Thermo, USA).
61
2.2 Determination of IC50 in C2C12 cells
62
After the C2C12 cells were seeded at 3×103 cells per well in 96-well plates for 24 h, fluoride
63
(0, 0.05, 0.1, 0.5, 1, 2.5, 5, 10, 20 and 50 mmol/L) was added to the medium for 24 h, and then 10
64
µl of Cell Counting Kit-8 (CCK-8, Beyotime, China) was added for 4 h. The absorbance was read
65
at 450 nm in a microplate reader (Multiskan FC, Thermo Scientific, USA), and the IC50 of the
66
C2C12 cells was calculated using GraphPad Prism 5. The experiment for each dose was given in
67
six times.
68
2.3 MTT assay
69
C2C12 cells were seeded at 3×103 cells per well in 96-well plates for 24 h, then incubated in
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a medium containing fluoride (0, 0.05, 0.5, 1, 2.5, 5 and 10 mmol/L) for 24, 48, 72 and 96 h. Next,
71
10 µl of the CCK-8 reagent was added to each well for 4 h. The absorbance was recorded using a
72
microplate reader at 450 nm. The growth of the C2C12 cells was recorded by inverted microscope
73
after treatment with fluoride at 0, 1 and 2.5 mmol/L for 48 h.
74
2.4 Haematoxylin and Eosin (HE) staining
75
Based on the calculated concentration of IC50, the fluoride doses at 0 and 2.5 mmol/L were
76
used in the subsequent experiments. Thus, the C2C12 cells were seeded onto coverslips at a
77
density of 5×104 cells per well in 6-well plates for attachment. They were then incubated with 0
78
and 2.5 mmol/L of fluoride for 48 h. The cells were washed three times with PBS before being
79
fixed in paraformaldehyde, and the cells were then incubated with the haematoxylin and eosin
80
before observation using a light microscope.
81
2.5 Transmission electron microscope (TEM) observation
82
To analyse the effect of fluoride on C2C12 cell ultrastructures, the cells were treated with 0
83
and 2.5 mmol/L of fluoride for 48 h after culturing in T-25 flasks for 24 h. The cells were
84
collected, fixed in 2.5% glutaraldehyde phosphate buffer (PB) (pH 7.4) at 4 ºC and washed by PB.
85
Afterward, it was fixed in 1% osmic acid for 1.5 h. The cells were dehydrated using 50%, 70%,
86
80%, 90% and 100% alcohol and were embedded in araldite resin. Ultra-thin sections were
87
serially cut and stained with uranyl acetate and lead citrate before TEM examination (H-7500,
88
Hitachi, Japan).
89
2.6 Hoechst staining
90
To assess the effect of fluoride on apoptosis in C2C12 cells, cell coverslips were prepared
91
and treated with fluoride at 0 and 2.5 mmol/L for 48 h. The C2C12 cells were fixed in
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paraformaldehyde and incubated with Hoechst 33258 staining (Beyotime, China) for 10 min at 4
93
°C in the dark before being washed three times with PBS. The cell coverslips were embedded in
94
an anti-fade mounting medium before being observed and photographed using fluorescence
95
microscopy (CKX41S, Olympus, Japan).
96
2.7 Real-time PCR
97
The cells were treated with 0 and 2.5 mmol/L of fluoride for 48 h after culturing in T-25
98
flasks for 24 h. The total RNA was extracted from the C2C12 cells using the Trizol reagent
99
(Invitrogen, Life Technologies) and purified using the RNeasy® Mini Kit (TaKaRa, QIAGEN),
100
according to the manufacturer’s instructions. The cDNA was synthesised from 5 µl of RNA using
101
SuperScriptTM II Reverse Transcriptase Kit (Invitrogen, Life Technologies). Real-time PCR was
102
performed in a reaction mixture containing SYBR Green PCR master mix and 1 µl of first-stand
103
cDNA as a template, with primers specific for PI3K, PDK1, AKT1, BAD, Bcl-2, Bax and
104
caspase-9, which are shown in Table 1. The expression of the target genes relative to β-actin was
105
determined.
106
2.8 Western blotting analysis
107
After 48 h of fluoride treatment, the C2C12 cells were collected and lysed in RIPA buffer
108
(Google Biotechnology, Wuhan) containing protease inhibitors and then incubated on ice 30 min
109
before centrifugation at 12, 000 g for 5 min. The total protein concentration was measured using
110
the BCA protein assay (Google Biotechnology, China). Equal amounts of protein samples were
111
run in 10% SDS-PAGE and transferred to PVDF membranes. They were then blocked with 5%
112
non-fat milk for 60 min, and an overnight incubation at 4 °C with PI3K (A0265, Boster, Wuhan,
113
China), PDK1 (5662, CST, USA), T-AKT1 (BM1612, Boster, Wuhan, China), P-AKT1 (AF0908,
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Affinity, USA), BAD (GB11198, Servicebio, Wuhan, China), Bcl-2 (GB12008, Servicebio,
115
Wuhan, China), Bax (GB11007, Servicebio, Wuhan, China), caspase-9 (9502, CST, USA) and β-
116
actin (GB13001-1, Servicebio, Wuhan, China) primary antibodies. After the membranes were
117
washed three times for 10 min with 0.5% TBS-Tween 20 buffer (TBST), they were incubated with
118
secondary antibodies (074-1506, KPL, USA) for 30 min at room temperature. Finally, the
119
membranes were washed three times for 5 min with TBST. The bands were visualised using
120
enhanced chemiluminescence (ECL) reagents.
121
2.9 Statistical analysis
122
The results are expressed as the mean ± standard deviation (SD). All data were analysed using
123
the SPSS software version 13.0 and the student’s t test. A P value less than 0.05 was considered as
124
statistically significant.
125
3. Results
126
3.1 Effects of fluoride on the viability of C2C12 cells
127
The viability of the C2C12 cells was determined via an MTT assay. As shown in Fig. 1, the
128
survival rates of the C2C12 cells decreased significantly with the doses of fluoride added, and an
129
IC50 of 3.209 mmol/L was measured. In addition, the MTT results indicated that the absorbance
130
of the C2C12 cells increased with all the doses of fluoride added, and the time for which it was
131
added (Fig. 2). Under microscopic observation, the number of C2C12 cells was obviously
132
decreased, the shapes of the C2C12 cells changed, and the intercellular spaces became larger
133
under the influence of fluoride in doses of 1 mmol/L (Figs. 3b and 3b1) and 2.5 mmol/L for 48 h
134
(Figs. 3c and 3c1). These results were consistent with the MTT assay.
135
3.2 Effects of fluoride on histopathological and ultrastructural changes in C2C12 cells
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As shown in Fig. 4, the C2C12 cells in the control group grew well, and the structure of the
137
cells was normal (Fig. 4a). Conversely, with the increasing doses of fluoride, the number of cells
138
decreased and the gap between the cells increased. Nuclear pyknosis and deformities were seen
139
under the light microscope in the fluoride group (Fig. 4b).
140
The ultrastructural changes in the C2C12 cells are shown in Fig. 4. In the control group, the
141
structure of the C2C12 cells remained normal, and the outline of cells was easy to observe. The
142
distribution of chromatin in the nucleus was uniform, the double nuclear membrane was clearly
143
visible and the mitochondrion ridge was clearly seen (Figs. 4A, 4A1 and 4A2). However, in the
144
fluoride group, the amount of chromatin in the nucleus was visibly decreased, nuclear
145
condensation had commenced and apoptotic bodies began to form. The mitochondria were
146
severely damaged, due to vacuolisation, and the ridge was indistinct. The endoplasmic reticulum
147
also appeared swollen (Figs. 4B, 4B1 and 4B2).
148
3.3 Effects of fluoride on apoptosis of C2C12 cells
149
With Hoechst staining, shown in Figs. 4C and 4D, the number of positively stained cells
150
increased compared to the control group, and the colour of the cells became pale (Fig. 4D).
151
3.4 Effects of fluoride on the mRNA expression of PI3K, PDK1, AKT1, BAD, Bcl-2, Bax and
152
caspase-9
153
The mRNA expression levels of PI3K, PDK1, AKT1, BAD, Bcl-2, Bax and caspase-9 in
154
C2C12 cells were assessed (Fig. 5). In the fluoride group, the mRNA expressions levels of PI3K,
155
BAD, Bcl-2, Bax and caspase-9 were significantly up-regulated (P < 0.01), respectively, while the
156
mRNA expressions levels of PDK1 was significantly down-regulated compared with the control
157
group (P < 0.01).
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3.5 Effects of fluoride on the protein expression of PI3K, PDK1, T-AKT1, P-AKT1, BAD,
159
Bcl-2, Bax and caspase-9
160
The protein expression levels of PI3K, PDK1, T-AKT1, P-AKT1, BAD, Bcl-2, Bax and
161
caspase-9 in C2C12 cells were detected by western blotting (Fig. 6). After treatment with fluoride,
162
the levels of BAD, Bcl-2 and Bax were significantly increased compared with control group (P <
163
0.01), while the levels of PDK1 and P-AKT1 were significantly decreased (P < 0.01),
164
respectively. The changes in the expression levels of these proteins matched the changes seen in
165
the mRNA expression levels.
166
4. Discussion
167
Fluoride can readily penetrate cellular membranes and seriously damage the structure and
168
physiological function of cells (Zhou et al., 2015). Several studies have shown that excessive
169
fluoride can decrease the viability of cells and that the growth of cells is inhibited by the excess
170
presence of fluoride ion (Chae et al., 2016; Yan et al., 2015; Yang et al., 2015). Our previous work
171
confirmed this inhibition in HeLa cells (Wang et al., 2017a). In the present study, the viability of
172
C2C12 cells was significantly decreased with increasing fluoride concentrations, and the IC50 was
173
shown to be 3.209 mmol/L. The IC50 of fluoride exposure was 1.9 mmol/L in human bronchial
174
epithelial cells (BEAS-2B) and was 5.8 mmol/L in human liver cells (HL-7702) (Ying et al.,
175
2017). These results suggested that high concentrations of fluoride affect cell viability of all types
176
and inhibit cellular growth, though the tolerance of different cells exposed to fluoride is different.
177
This finding provides evidence for fluoride-induced toxicology in C2C12 cells.
178
To explore the theory that fluoride would decrease the viability of C2C12 cells, the
179
histopathological and ultrastructural changes were observed. The C2C12 cellular structures were
ACCEPTED MANUSCRIPT 180
obviously changed from the control group, with nuclear pyknosis and deformities seen in the
181
fluoride group. The number of C2C12 cells was also reduced by the presences of excess fluoride.
182
This result was consistent with the fluoride-induced decrease in viability of the C2C12 cells.
183
Under TEM observation, the mitochondria were observed to be swollen and vacuolised, with
184
indistinct ridges. Furthermore, the endoplasmic reticulum was dilated, and nuclear pyknosis was
185
visible in the C2C12 cells in the fluoride group. The above histopathological characteristics were
186
due to the beginnings of apoptosis. Several studies indicated that the fluoride-induced
187
mitochondrial damage and endoplasmic reticulum swelling were highly associated with the
188
apoptosis process (Wang et al., 2017b; Zhang et al., 2015; Deng et al., 2016). Moreover, the
189
increasing number of positive cells observed in the Hoechst staining provided direct evidence in
190
this case for fluoride-induced apoptosis in C2C12 cells.
191
PI3K, PDK1 and AKT, proteins in the PI3K/AKT pathway, play an important role in the
192
regulation of cell cycle progression (Chang et al., 2003). The phosphorylation of AKT is an
193
important link in the cell apoptosis process (Gu et al., 2016). Phosphorylated AKT could facilitate
194
cell survival and proliferation and regulate various signaling pathways (Sussman, 2007). The
195
present study found that with the significant decrease of the expression level of PDK1, the P-
196
AKT1 expression level significantly decreased in C2C12 cells exposed to fluoride. Due to the
197
week suppression to BAD by P-AKT1 made it possible to induce apoptosis in C2C12 cells with
198
the fluoride treatment.
199
Bcl-2 is a widely studied modulator of programmed cell death. Moreover, Bax, as a member
200
of the Bcl-2 family, can form heterologous dimeric complexes with Bcl-2 and accelerate
201
programmed cell death (Lou et al., 2014). Recent studies reported that the Bcl-2 levels decreased
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and Bax levels increased in presence of excessive fluoride (Liu et al., 2013; Cao et al., 2013).
203
However, the present study showed that the expression levels of Bcl-2 and Bax both increased in
204
the C2C12 cells exposed to excessive fluoride. The increases in both Bcl-2 and Bax expression
205
might be a feedback effect following the C2C12 cells apoptosis caused by fluoride (Lou et al.,
206
2014). Furthermore, Bax, an opposition partner of Bcl-2, its over expression promotes the level of
207
Bcl-2 increased. Our results indicated that Bax and Bcl-2 play a pivotal role in the PI3K/AKT
208
pathway involving the fluoride-induced apoptosis of the C2C12 cells. Caspase-9 is the key
209
initiating caspase for the intrinsic pathway to cell death (Yan et al., 2009). In the present study, the
210
level of caspase-9 expression in C2C12 cells significantly increased in the fluoride group. It is
211
reported that caspase-9 can be activated through Apaf1 binding to cytochrome c in the cytoplasm,
212
thereby forming a multi-protein complex known as an apoptosome that leads to apoptosis (Wu et
213
al., 2014). BAD, a pro-apoptotic protein, heterodimerises with anti-apoptotic proteins such as Bcl-
214
2 and Bcl-xL to promote cell death (Martelli et al., 2006; Franke et al., 2003; Li et al., 2013). It
215
was reported that the increased expression of BAD promoted the apoptosis induced by fluoride in
216
human oral squamous cell carcinoma HSC-2 cells (Otsuki et al., 2011). In the present study, the
217
expression levels of BAD significantly increased promotes the fluoride-induced apoptosis in
218
C2C12 cells. Several studies suggested that a deficit of BAD can result in diminished
219
mitochondria-based glucokinase activity and blunted mitochondria respiration in response to
220
glucose, which may be associated with apoptosis (Danial et al., 2003; Ranger et al., 2003; Seo et
221
al., 2004), thus this mechanism requires further study. These results, taken together, indicated that
222
the PI3K/AKT pathway is involved in the fluoride-induced apoptosis of C2C12 cells.
223
In conclusion, the viability of C2C12 cells seriously decreased in the presence of excess
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fluoride, and the IC50 of this was shown to be 3.209 mmol/L. The results provide powerful
225
evidence for the fluoride-induced apoptosis of C2C12 cells via the PI3K/AKT signaling pathway.
226
Acknowledgements
227
This work is sponsored by the National Natural Science Foundation of China (grant no.
228
31201963) and Youth Backbone Teachers Project in Henan Province Department of Education,
229
China (grant no. 2016GGJS-061).
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Fig. 1 IC50 of C2C12 cells exposed to fluoride (n=6) After 24 h of fluoride exposure, the survival rate of C2C12 cells decreased in proportion to the dose of fluoride added, and the IC50 of this was shown to be 3.209 mmol/L.
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Fig. 2 MTT determination of C2C12 cells viability (n=6) The viability of C2C12 cells increased with lower concentration of fluoride over 24 h, and cell growth was inhibited by a high fluoride concentration. After fluoride exposure for 48, 72 and 96 h, the viability of the C2C12 cells significantly decreased with the doses of fluoride. *P < 0.05 and **P < 0.01 indicated statistical significance compared to the control group.
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Fig. 3 Morphological changes in C2C12 cells (a) and (a1), (b) and (b1), (c) and (c1) show the morphological changes in the C2C12 cells between the control group, the 1.0 mmol/L fluoride group, and the 2.5 mmol/L fluoride group, respectively. After fluoride exposure for 48 h, the number of C2C12 cells obviously decreased, the shapes of the C2C12 cells changed, and the intercellular spaces widened with the increasing doses of fluoride.
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Fig. 4 Histopathological and ultrastructural changes and Hoechst staining in C2C12 cells The morphological changes between C2C12 cells from the control group and the fluoride group are showed in (a) and (b), respectively, with a normal histopathological structure seen in (a) and nuclear pyknosis and deformation of the C2C12 cells exposed to excessive fluoride shown in (b) with an increasing gap between cells also obvious. (A) shows the normal ultrastructure of C2C12 cells in the control group. (A1) and (A2) show the normal mitochondria (Mi), endoplasmic reticulum (ER) and nuclear membrane (NM) of C2C12 cells. (B) shows the ultrastructure of C2C12 cells exposed to excessive fluoride, with the nuclear condensation clearly seen. (B1) and (B2) show the mitochondria vacuolisation and ER swelling. (C) and (D) show the Hoechst staining results for the C2C12 cells. (D) shows that the number of positive cells increased compared to the control group, and the colour of the cells became pale.
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Fig. 5 Real-time PCR analysis of mRNA expression levels of different genes in the C2C12 cells (n=3). **P < 0.01 (the fluoride group compared with the control group).
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Relative density
b
Control group 1.0 0.9 0.8 0.7 0.6 0.5 0.4 0.3 0.2 0.1
Fluoride group
**
**
**
** ** B ax ca sp as e9
cl -2 B
A D B
K T1
PA
1
K T1
TA
PD K
PI 3K
0.0
Fig. 6 Western blotting analysis of the expression levels of PI3K, PDK1, T-AKT1, PAKT1, BAD, Bcl-2, Bax and caspase-9 in C2C12 cells (n=3). (a) Western blotting electrophoretic pattern and (b) relative expression levels. **P < 0.01 (the fluoride group compared with the control group).
ACCEPTED MANUSCRIPT Highlights Fluoride causes histopathological changes in C2C12 cells. Fluoride exposure damages ultrastructure in C2C12 cells. Fluoride exposure induces apoptosis in C2C12 cells. PI3K/AKT signaling pathway is involved in fluoride-induced apoptosis in C2C12 cells.
ACCEPTED MANUSCRIPT Table 1 Oligonucleotide Primer sets for real-time PCR
Gene
P13K
PDK1
AKT1
BAD Bcl-2 Bax caspase-9
β-actin
Primers Forward (5′→3′) Primers Reverse (5′→3′) AGCCGCCAGCTCTGATAATA TCTCCCCAGTACCATTCAGC CAAGAACTCCGACCAGAAGC TTTCTGCACCACTTGTGAGC ACTCATTCCAGACCCACGAC CCGGTACACCACGTTCTTCT AGGACTTATCAGCCGAAGCA GCTCAAACTCTGGGATCTGG CTGCAAATGCTGGACTGAAA TCAGGAGGGTTTCCAGATTG TGCAGAGGATGATTGCTGAC GATCAGCTCGGGCACTTTAG TTCCCAGGTTTTGTCTCCTG GGGACTGCAGGTCTTCAGAG TGTTACCAACTGGGACGACA GGGGTGTTGAAGGTCTCAAA
Accession Number
Tm (℃)
Bp
NM_001077495.2
60
183
AF079535.1
55
110
NM_009652.3
55
176
NM_007522.3
59
187
NM_009741.5
59
158
NM_007527.3
59
173
NM_015733.5
55
106
NM_007393.5
55
165