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【求助】做Akt的Western blot为什么要做P－Akt和总Akt？
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向各位达人求教，做Akt的Western blot为什么要做P－Akt和总Akt？说明什么？
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Akt的持续活化是其发挥促细胞生存、抑制细胞凋亡功能的重要前提, 而Ser473和，或Thr308位点的磷酸化是Akt激活的必要条件。所以一般检测的时候是要检测P-Akt和总Akt的，这个基本是要看你做出来的结果来分析，比如说P-Akt不断减弱，但是总Akt有可能变化很小，也有可能不断减弱，这样处理对结果的解释就会不一样，所以两个都要做。
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谢谢baiy的指导，我正要做些这方面的工作～～～
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Akt有三个isoform,不知道你是做哪一个?Aktα (T308, S473), Aktβ(T309, S474), Aktβ(T305, S472).单单T位磷酸化的话活性只有10%,只有T和S都磷酸化的话,其活性才出来.
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关于丁香园Expression and clinical significance of TRB3 and p-AKT in oral tongue squamous cell carcinoma--《Chinese Archives of Otolaryngology-Head and Neck Surgery》2012年02期
Expression and clinical significance of TRB3 and p-AKT in oral tongue squamous cell carcinoma
ZHANG Jing1,SUN Chuanzheng2,WEN Haojie2,GUO Zhuming3,ZENG Musheng3,JIANG Yu'e2,HE Xiaoguang4 (1 Department of Orthopedics,2 Department of Head and Neck Surgery,the Third Affiliated Hospital of Kunming Medical University,Kunming,Yunnan,650118,C 3 State Key Laboratory of Oncology in South China and Department of Head Neck Surgery,Sun Yat-Sen University Cancer Center,Guangzhou,Guangdong,
Department of Head and Neck Surgery, the First Affiliated Hospital of Kunming Medical University, Kunming,Yunnan,650032,China)
OBJECTIVE To investigate the expression and clinical significance of TRB3 and p-AKT in oral tongue squamous cell carcinoma(OTSCC). METHODS Expression of TRB3 mRNA in 18 paired OTSCC tissue and adjacent noncancerous mucosa was assessed using reverse transcription polymerase chain reaction(RT-PCR).Western blot and immunohistochemistry assays were applied to detect expression of TRB3 and p-AKT protein.RESULTS In 18 paired tissues,expression of TRB3 mRNA in 15 (83.3%) OTSCC tissues were significantly higher than that in adjacent noncancerous mucosa.And expression of TRB3 protein and p-AKT protein were both significantly higher in OTSCC tissue than in adjacent noncancerous mucosa in 13/18(72.2%) Western blot assays. Immunohistochemical assays showed high expression of TRB3 protein in 63/128(49.2%) and p-AKT protein in 88/128(68.8%) paraffin-embedded OTSCC samples. Expression of TRB3 and p-AKT were significantly higher in OTSCC tissues than that in adjacent noncancerous tissues(both P0.001).The Expression of TRB3 and p-AKT were significantly correlated with tumor pathological T stage,N stage and tumor recurrence.The expression level of TRB3 protein showed a significant negative relation with the expression level of p-AKT (r=-0.27,P0.01).CONCLUSION TRB3 and p-AKT were overexpressed in OTSCC and were related to tumor prognosis.Expression of p-AKT was correlated negatively with expression of TRB3.
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(C)2006 Tsinghua Tongfang Knowledge Network Technology Co., Ltd.(Beijing)(TTKN) All rights reserved[Influence of polypeptide extract from scorpion venom on PI3K and p-Akt signaling protein expression and cell proliferation of K562 cells].
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):872-5.[Influence of polypeptide extract from scorpion venom on PI3K and p-Akt signaling protein expression and cell proliferation of K562 cells]. [Article in Chinese]1, , , , , , .1Tianjin University of Traditional Chinese Medicine, Tianjin, China.AbstractThis study was aimed to investigate the effect of polypeptide extract from scorpion venom (PESV) on PI3K, p-Akt signal protein regulating K562 cell apoptosis and its mechanism. The K562 cells were cultured with PESV for different time, the cell growth curve was determined by MTT method, the levels of PI3K and p-Akt proteins were detected by Western blot. The results showed that as compared with control group, the apoptosis rate of K562 cells treated with PESV increased, the levels of PI3K and p-Akt expression decreased. It is concluded that the PESV inhibits the proliferation and promotes the apoptosis of K562 cells probably through suppressing the expression of PI3K and p-Akt signal proteins.PMID:
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External link. Please review our .Effect of Neferine combined with Imatinib on Akt/p-Akt protein expression in primary cells of chronic myelogenous leukemia--《China Journal of Modern Medicine》2015年30期
Effect of Neferine combined with Imatinib on Akt/p-Akt protein expression in primary cells of chronic myelogenous leukemia
Si-run HE;Min HU;Qun QIN;Rui YANG;Xiangya Hospital,Central South U
【Objective】 To investigate the effect of a combination of Neferine and Imatinib on Akt/p-Akt protein expression in chronic myelogenous leukemia(CML) primary cells.【Methods】 Akt/p-Akt protein expression was detected by Western blot.【Results】 Neither Neferine(4 μmol),Imatinib(1 μmol) alone nor their combination had influence on the Akt protein expression in CML primary cells(P
0.05).On the contrary,pAkt expression was reduced in the three groups,and even lower in the combination group(P
0.05).【Conclusions】 p-Akt protein expression is reduced by the combination of Neferine and Imatinib,which may be one of the mechanisms of Neferine sensitizing CML primary cells to Imatinib.
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TAN Yi,HE Wen(Department of Pharmacy,Renmin Hospital of Wuhan University,Wuhan,Hubei 430060,China);[J];Journal of Guangdong Pharmaceutical C2010-04
ZHOU Nenga,b LIANG Yi-Zengb LIU Shaoca(Institute of Natural Products,Department of Chemistry-Biology,Yulin Normal University,Yulin,Guangxi 537000,P.R.China)b(Research Center of Modernization of Chinese Medicines,College of Chemistry and Chemical Engineering,Central South University,Changsha 410083,P.R.China)c(Department of Pharmacy of Xiangya Hospital,Central South University,Changsha 410008,P.R.China);[J];Chinese Journal of Spectroscopy L2010-01
ZHENC Wei, ZHOU Jie, CHEN Juan, CONG Rong, FENG Youmei Department of Biochemistry and Molecular Biology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, C[J];Foreign Medical S2005-03
1,YUAN Li-hua
2,TIAN Mao
(1.Women and Children's Health Care Hospital of Heshan District,Hunan Yiyang 413002,C
2.Tumor Hospital of Taojiang County,Hunan Taojiang 413400,C
3.Enviromental Monitoring Station of Xiangxi Autonomous Perfecture,Hunan Jishou 416000,China);[J];Trace Elements S2005-01
LIN Wen-jin,XU Rong-qing,ZHANG Ya-min,XU Zheng-wei(Fujian Medical Test Key Laboratory Room,Fujian Institute of Medical Sciences,Fuzhou Fujian 350001);[J];World Journal of Integrated Traditional and Western M2009-06
【Secondary Citations】
Chinese Journal Full-text Database
yong, ZHANG Sheng
hua, ZHEN Yong
su*Department of Oncology, Institute of Medicinal Biotechnology, Chinese Academyof Medical Sciences &Peking Union Medical College, Beijing 100050, P
C[J];CHINESE JOURNAL OF CANCER;2000-08
qing1, CAO Jian
guo1*, LIAO Duan
fang21.Institute of Oncology, Nanhua University, Hengyang 421001, P.R.China2.Department of Pharmacology, Nanhua University, Hengyang 421001, P.R.C[J];Chinese Journal of C2001-08
?Li Dacheng?1
Liu Bailin?1
Tang Xingyao?2
Zhang Taihuai?3?Ouyang Xuesong?1
Liu Shuqiu?1?1?Lab of Molecular Biology, School of Basic Medical Sciences (Chengdu 610041);
2.Lab of Herb?Medicine, School of P
3. D[J];JOURNAL OF WEST CHINA UNIVERSITY OF MEDICAL SCIENCES;1998-01
Li, FENG You
Mei, CONG Rong, ZONG Yi
Qiang, WANG Chun
Ben and FENG Zong
(Department of Biochemistry, Tongji Medical University, Wuhan 430030, China);[J];CHINESE JOURNAL OF ARTERIOSC LEROSIS;1999-01
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(C)2006 Tsinghua Tongfang Knowledge Network Technology Co., Ltd.(Beijing)(TTKN) All rights reservedHydrogen sulfide protects H9c2 cardiac cells against doxorubicin-induced cytotoxicity through the PI3K/Akt/FoxO3a pathway
International Journal of Molecular Medicine
Hydrogen sulfide protects H9c2 cardiac cells against doxorubicin-induced cytotoxicity through the PI3K/Akt/FoxO3a pathway
Published online on: April 14, 2016
https://doi.org/10.3892/ijmm.
Doxorubicin (DOX) is an efficient drug used in cancer therapy that also produces reactive oxygen species (ROS) that induces severe cytotoxicity, which limits its clinical application. Hydrogen sulfide (H2S), a novel gasotransmitter, has been shown to exert cardioprotective effects. The present study aimed to determine whether exogenous H2S protects H9c2 cardiac cells against DOX-induced cytotoxicity and whether these protective effects are mediated through the PI3K/Akt/FoxO3a pathway. The H9c2 cardiac cells were exposed to 5 uM DOX for 24 h to establish a model of DOX-induced cardiotoxicity. The results showed that the treatment of H9c2 cardiac cells with sodium hydrosulfide (NaHS) for 30 min prior to DOX exposure markedly attenuated the phosphorylation of Akt and FoxO3a. Notably, pre-treatment of the H9c2 cells with NaHS significantly attenuated the nuclear localization of FoxO3a as well as the apoptosis of H9c2 cells induced by DOX. The treatment of H9c2 cells with N-acetyl-L-cysteine (NAC), a scavenger of ROS, prior to DOX exposure, also markedly increased the phosphorylation of Akt and FoxO3a which was inhibited by DOX alone. Furthermore, pre-treatment with LY294002, a selective inhibitor of PI3K/Akt, reversed the protective effect of H2S against DOX-induced injury of cardiomyocytes, as demonstrated by an increased number of apoptotic cells, a decrease in cell viability and the reduced phosphorylation of Akt and FoxO3a. These findings suggested that exogenous H2S attenuates DOX-induced cytotoxic effects in H9c2 cardiac cells through the PI3K/Akt/FoxO3a pathway.
Introduction
Doxorubicin (DOX) is one of the most widely used
anticancer drugs because of its potent therapeutic effects on
various types of cancer, including leukemia, lymphoma and breast
cancer. However, the clinical use of DOX is limited by severe
cardiotoxicity, which may lead to dilated cardiomyopathy and
congestive heart failure (). The
production of reactive oxygen species (ROS) is involved in the
toxic effect elicited by DOX on cardiomyocytes and endothelial
cells, and in the promotion of endothelial dysfunction () and apoptosis (). A number of pharmacological
interventions have been suggested as therapies to protect against
DOX-induced cardiotoxicity.
Hydrogen sulfide (H2S) is considered a
toxic gas, and has been classified as the third gasotransmitter,
together with nitric oxide and carbon monoxide, and exerts various
effects on the cardiovascular system (). Previous findings have shown that
H2S protects the heart from myocardial
ischemia-reperfusion (IR) injury (). In a previous study, it was
demonstrated that the increased generation of endogenous
H2S in the early reperfusion phase has an important
function in ischemic pre-conditioning-elicited protection in
isolated hearts ().
The forkhead box class O (FoxO) subfamily of
forkhead transcription factors comprises the members FoxO1, FoxO3a
and FoxO4, which are downstream targets of Akt (). FoxO3a is involved in the regulation
of various cell processes, including proliferation and apoptosis,
as well as protection against oxidative stress and metabolism
(). A model of β-amyloid-induced
neuron death was used to demonstrate that FoxO3a is activated,
translocates to the nucleus, and subsequently mediates neuron death
through Bim (). In neonatal rat
ventricular myocytes (NRVMs), hyperglycemia was demonstrated to
markedly enhance the apoptosis of NRVMs through the trans-location
of FoxO3a to the nucleus, and the resultant enhanced
transcriptional activity of FoxO3a (). Li et al found that the
PI3K/Akt/FoxO3a pathway is involved in neuronal apoptosis in the
brain of a developing rat ().
In a previous study, it was demonstrated that resveratrol protects
PC12 cells against high glucose-induced oxidative stress and
apoptosis through the activation of the PI3K/Akt/FoxO3a signaling
pathway (). In addition,
sodium tanshinone IIA sulfonate () and bromelain () have been found to protect rat
hearts against IR injury through the activation of the
PI3K/Akt/FoxO3a pathway. Therefore, we hypothesized that the
PI3K/Akt/FoxO3a pathway may be involved in the protective effect of
exogenous H2S against DOX-induced cardiotoxicity in H9c2
cardiac cells.
To examine this hypothesis, H9c2 cells were treated
with 5 uM DOX in the present study to establish a model of
chemotherapy-induced cardiotoxicity as previously described
(). Subsequently, we examined:
i) the effect of DOX on the phosphorylation of Akt and FoxO3a; ii)
the effect of exogenous H2S on the DOX-induced
translocation of FoxO3a to the nucleus where it subsequently
mediates H9c2 cardiac cell death through B and iii) whether
exogenous H2S protects H9c2 cells against DOX-induced
cardiotoxicity through the PI3K/Akt/FoxO3a pathway.
Materials and methods
Methyl thiazolyl tetrazolium (MTT), Hoechst 33258,
2′,7′-dichlorofluorescein diacetate (DCFH-DA), DOX, sodium
hydrosulfide (NaHS), and N-acetyl-L-cysteine (NAC) were purchased
from Sigma-Aldrich (St. Louis, MO, USA). LY294002 was purchased
from Calbiochem (Billerica, MA, USA). Cell culture medium
components were purchased from Thermo Fisher Scientific (Waltham,
MA, USA) unless otherwise noted. The H9c2 cardiac myocytes were
obtained from the Shanghai Cell Library of China [originally from
the American Type Culture Collection (ATCC); Manassas, VA,
Cell culture
The H9c2 cardiac cells were cultured in Dulbecco's
modified Eagle's medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), 100 ug/ml streptomycin (Life Technologies,
Carlsbad, CA, USA) and 100 U/ml penicillin-streptomycin (Life
Technologies) in a humidified 5% CO2 atmosphere at 37°C.
The H9c2 cardiac myocytes were passaged every 2 days. Subsequently,
they were seeded at a density of 2×106 cells/dish in
100-mm dishes with 10% calf serum, incubated for 24 h and then, the
medium was replaced with 0.5% FBS DMEM for 24-h serum starvation.
To determine the degree of apoptosis, the H9c2 cardiac myocytes
were treated with NaHS (100 uM) for 30 min or NAC (1,000
uM) for 60 min, followed by exposure to DOX for 24 h. In
some experiments, the H9c2 cells were treated with LY
uM) prior to NaHS stimulation.
The MTT assay is a standard method used to assess
cell viability. Prior to each experiment, the H9c2 cardiac myocytes
(5×102 cells/well) were seeded in 96-well microtiter
plates. After incubation with the phosphatidylinositol-3-kinase
(PI3K) inhibitor LY uM) and/or NaHS for 30 min,
the cells were exposed to 5 uM DOX for a further 24 h.
Subsequently, 10 ul MTT solution was added to each well, and
the plates were incubated for 4 h at 37°C. The absorbance was
measured at 470 nm using a SpectraMax 190 Absorbance Microplate
Reader (Molecular Devices LLC, Sunnyvale, CA, USA) and used to
calculate the relative ratio of cell viability. Three independent
experiments were performed for each experimental condition.
Assessment of H9c2 cell apoptosis
The analysis of apoptosis was performed by
fluorescence microscopy with the chromatin dye Hoechst 33258. After
various treatments, the cells were fixed in ice-cold 4%
paraformaldehyde dissolved in phosphate-buffered saline (PBS) at
room temperature for 20 min. Non-specific binding was blocked using
5% normal goat serum in 0.01 M PBS containing 0.3% Triton X-100
(PBS+T). The cells were washed twice with PBS and incubated with 10
ug/ml Hoechst 33258 for 15 min at room temperature in the
dark. The cells were visualized under a fluorescence microscope
(BX50-FLA; Olympus, Tokyo, Japan). Apoptotic cells exhibited
condensed, fractured or distorted nuclei, whereas viable cells
exhibited normal nuclear size and uniform fluorescence.
Measurement of intracellular ROS
The determination of intracellular ROS levels was
performed by measuring the level of a fluorescent product formed by
the oxidation of DCFH-DA. Briefly, the culture medium was plated
into 96-well microtiter plates. After various treatments, the cells
were washed with PBS 3 times. Following the addition of fresh
culture medium, the cells were incubated with DCFH-DA at a final
concentration of 10 umol/l for 30 min at 37°C. The cells
were washed again with PBS 3 times and then lysed with 500
ul 90% DMSO and 10% PBS for 10 min at room temperature in
the dark. The supernatant (200 ul) was transferred to
another 96-well microtiter plate. The fluorescence intensity of the
oxidized product, 2′,7′-dichlorofluorescein (DCF), was measured
using a Model F-4500 fluorescence spectrophotometer (Hitachi,
Tokyo, Japan) at 495 nm (excitation) and 520 nm (emission). The
values were expressed as the percentage of fluorescence intensity
relative to the control wells.
Western blot analysis
H9c2 cells were treated with 0.1, 1, 5 and 10
uM DOX for 24 h ()
and exposed to 5 uM DOX for 3, 6, 12 and 24 h (). The cells were homogenized
directly in cell lysis buffer (Cell Signaling Technology, Danvers,
MA, USA) and phosphatase inhibitor cocktail (Sigma-Aldrich). The
lysates were centrifuged at 12000 × g for 10 min at 4°C. The
protein concentration was determined with the use of a BCA protein
assay kit according to the manufacturer's instructions. For
nuclear/cytoplasmic fractionation, the cultured H9c2 cells were
fractionated into nuclear and cytoplasmic lysates using a Nuclear
and Cytoplasmic Protein Extraction kit (Beyotime Institute of
Biotechnology, Shanghai, China) according to the manufacturer's
instructions. The extracted proteins were mixed with 5% sodium
dodecyl sulfate (SDS)-PAGE sample buffer, boiled at 100°C for 7 min
and then separated by electrophoresis on a 10% SDS-polyacrylamide
gel. Following electrophoresis, the proteins were transferred to
polyvinylidene difluoride membranes (Beyotime Institute of
Biotechnology). The membranes were blocked in Tris-buffered saline
(TBS) containing 0.1% Tween-20 (TBS-T) with 5% non-fat dry milk,
for 2 h at room temperature, with rotation. After blocking, the
membranes were incubated with the following antibodies: rabbit
anti-Akt polyclonal antibody (cat. no. ,000), rabbit
anti-phosphorylated (p-) Akt (Ser 473) monoclonal antibody (cat.
no. ,000), rabbit anti-FoxO3a polyclonal antibody (cat.
no. 1,000), rabbit anti-p-FoxO3a (ser 253) polyclonal
antibody (cat. no. ,000) (all from Cell Signaling
Technology) and rabbit anti-Bim poly-clonal antibody (ab32158,
1:200; Abcam, Cambridge, MA, USA). The membranes were then
incubated in 5% milk or bovine serum albumin overnight at 4°C. The
primary antibody was removed by washing the membranes 3 times in
TBS-T, and subsequently incubating the membranes for 2 h with the
appropriate horseradish peroxidase-conjugated secondary antibodies.
After washing the membranes 3 times in TBS-T, the antigen-antibody
bands were detected using an enhanced chemiluminescence reagent kit
and quantified using a densitometry program. The data from the
western blot analysis of p-Akt and p-FoxO3a were presented as a
ratio of the p-forms to their total forms, respectively. The
immunoblot of Bim was corrected to the bands of GAPDH.
Statistical analysis
The results are presented as the means ± SEM.
Statistical analysis was performed using the Student's t-test or
analysis of variance (ANOVA) with SPSS 13.0 software (SPSS, Inc.,
Chicago, IL, USA). In all cases, P&0.05 was considered to
indicate a statistically significant difference.
DOX decreases the phosphorylation of Akt
and FoxO3a in H9c2 cells
To investigate the role of the PI3K/Akt/FoxO3a
pathway in DOX-induced cytotoxicity, we investigated the
phosphorylation of Akt and FoxO3a in the H9c2 cells following
exposure to DOX. The H9c2 cells were treated with DOX at different
concentrations for different time periods, and the effect on the
phosphorylation of Akt and FoxO3a was determined using western blot
show that DOX decreased the
phosphorylation of Akt and FoxO3a in the H9c2 cells in a
concentration- and time-dependent manner. DOX inhibited the
phosphorylation of Akt and FoxO3a in the H9c2 cells at a
concentration of 1 uM and the maximal effect was reached at
a concentration of 10 uM ().
shows that 5
uM DOX induced a significant decrease in the levels of p-Akt
and p-FoxO3a at 6 and 12 h, and it almost completely abolished the
phosphorylation of Akt and FoxO3a at 24 h in the H9c2 cells.
Exogenous H2S ameliorates the
DOX-induced decrease in the levels of p-Akt and p-FoxO3a in H9c2
To determine whether the cytoprotective effect of
H2S against DOX-induced toxicity was associated with the
PI3K/Akt/FoxO3a pathway in H9c2 cells, we examined the effect of
NaHS on the expression of p-Akt and p-FoxO3a. The results showed
that treating the H9c2 cells with 100 uM NaHS (a donor of
H2S) for 30 min prior to exposure to 5 uM DOX for
24 h significantly increased the phosphorylation of Akt and FoxO3a
(). Furthermore, NaHS
treatment alone also significantly increased the levels of p-Akt
and p-FoxO3a compared with the DOX-treated groups. The total Akt
and FoxO3a levels remained unchanged among the four groups. These
results suggested that the PI3K/Akt/FoxO3a pathway was involved in
the protective effect of H2S.
NAC ameliorates the DOX-induced decrease
in the levels of p-Akt and p-FoxO3a in H9c2 cells
The H9c2 cells were treated with 1,000 uM NAC
(ROS scavenger) for 60 min prior to exposure to 5 uM DOX for
24 h, to confirm whether the protective effect of H2S on
the DOX-induced decrease in p-Akt and p-FoxO3a is associated with
antioxidation. As shown in , the pretreatment of the H9c2 cells with NAC markedly
increased the expression of p-Akt and p-FoxO3a and this was similar
to the protective effect observed with NaHS pre-treatment. The
total Akt and FoxO3a levels remained unchanged in the four groups.
The results revealed that an antioxidant effect contributed to the
protective effect of H2S against DOX-induced
Exogenous H2S induces Akt and
FoxO3a phosphorylation through the PI3K/Akt pathway in H9c2
To investigate the role of the PI3K/Akt pathway in
the protective effects of H2S, the H9c2 cells were
treated with the PI3K inhibitor LY294002 prior to exposure to NaHS
plus DOX. The activation of Akt and FoxO3a was determined as
described above. LY294002 abolished the stimulation of p-Akt and
p-FoxO3a in the presence of NaHS (), but elicited no effect on the
expression levels of total Akt and FoxO3a. These results suggested
that the PI3K/Akt/FoxO3a pathway was involved in the protective
effect of H2S.
DOX enhances the nuclear localization of
FoxO3a in H9c2 cells whereas H2S blocks the effect of
The transcription factor FoxO3a functions through
its phosphorylation and subcellular localization. The
phosphorylation of FoxO3a by Akt causes it to localize in the
cytoplasm and inhibit the functions of FoxO3a, including
pro-apoptotic effects (). By
contrast, dephosphorylation of this protein promotes the
translocation of FoxO3a to the nucleus and triggers apoptosis. To
investigate the effects of H2S and DOX on FoxO3a, we
studied the subcellular localization of FoxO3a following exposure
to these reagents. Nuclear and cytosolic proteins from H9c2 cells
were extracted, and the subcellular localization of FoxO3a was
determined.
shows that DOX
enhanced the nuclear localization of FoxO3a in the H9c2 cells,
whereas NaHS blocked the effect of DOX. Co-treatment with the PI3K
inhibitor, LY294002, abolished the protective effect of NaHS.
Exogenous H2S downregulates
Bim expression through the PI3K/Akt-dependent signaling
A significant down-regulation of Bim protein was
observed in the NaHS + DOX group compared with the DOX-treated
group. Furthermore, co-treatment with LY294002 increased Bim
protein expression compared with the control group (). These results indicated that
pre-treatment with NaHS downregulated the expression of Bim through
a PI3K/Akt-dependent signaling pathway.
Exogenous H2S inhibits
DOX-induced cytotoxicity
shows that
the exposure of the H9c2 cells to DOX for 24 h induced marked
cytotoxicity, leading to a decrease in cell viability. However,
cell pre-treatment with 100 uM NaHS for 30 min prior to DOX
exposure significantly attenuated the degree of DOX-induced
cytotoxicity, as demonstrated by an increase in cell viability. The
preceding results ( and
) showed that H2S
attenuated the DOX-induced decrease in p-Akt in the H9c2 cells.
Thus, we aimed to confirm whether the PI3K/Akt signaling pathway is
involved in the protective effect of H2S. The treatment
of the H9c2 cells with LY294002 and NaHS for 30 min prior to DOX
exposure for 24 h abolished the protective effect of
H2S, leading to an decrease in cell viability (). NaHS alone did not alter the
viability of the H9c2 cells. These findings suggested that
H2S exerts a protective effect against DOX-induced
cytotoxicity, which may occur through the PI3K/Akt signaling
Exogenous H2S reduces
DOX-induced apoptosis in H9c2 cells
The effects of H2S on DOX-induced
apoptosis were observed.
shows that the apoptotic rate of the H9c2 cells exposed to 5
uM of DOX for 24 h increased significantly. However, cell
pre-treatment with 100 uM NaHS for 30 min prior to DOX
exposure markedly decreased the DOX-induced increase in the
apoptotic rate. To ascertain whether the PI3K/Akt signaling pathway
is involved in apoptosis induced by DOX, the H9c2 cells were
treated with LY294002 prior to exposure to NaHS plus DOX. The
results revealed that pretreatment with LY294002 abolished the
protective effect of H2S. NaHS alone did not markedly
alter the percentage of apoptotic H9c2 cells. These data strongly
suggest that H2S protects DOX-exposed H9c2 cells against
apoptosis and this may occur through the PI3K/Akt signaling
Exogenous H2S reduces
DOX-induced oxidative stress in H9c2 cells
The effect of H2S on the DOX-induced
production of ROS was investigated to elucidate whether the
antioxidant activity of H2S affords a cytoprotective
effect against DOX-induced cardiotoxicity.
shows that the exposure of H9c2
cells to 5 uM DOX evidently enhanced the generation of
intracellular ROS. However, NaHS pre-conditioning for 30 min
markedly attenuated the DOX-elicited generation of ROS. Notably,
pre-treatment with LY294002 abolished the protective effect of
H2S. However, NaHS alone did not alter the basal levels
of intracellular ROS.
Discussion
DOX is one of the most widely used and successful
antitumor drugs, although the clinical use of DOX is limited by
cumulative and dose-dependent cardiotoxic effects. With an
increasing population of cancer survivors, there is a growing need
to develop preventive strategies and effective therapies against
DOX-induced cardiotoxicity, and particularly, late-onset
cardio-myopathy. Although the cardiotoxic effects of DOX have been
previously examined, the underlying mechanisms responsible for
these effects remain to be elucidated. Mounting evidence supports
the hypothesis that free radical-induced oxidative stress, which
leads to cardiomyocyte death by apoptosis and necrosis, is a key
contributor to DOX-induced cardiotoxicity ().
Previous findings have demonstrated that exogenous
H2S offers protection against DOX-induced cardiotoxicity
through antoixidant effects and the downregulation of inflammatory
responses (,,). Guo et al have demonstrated
that exogenous H2S attenuates DOX-induced inflammation
and cytotoxicity through the inhibition of the p38 MAPK/NF-κB
pathway in H9c2 cells ().
H2S also attenuates DOX-induced cardiotoxicity through
the inhibition of endoplasmic reticulum stress in H9c2 cells
(). Su et al have
demonstrated that the downregulation of endogenously generated
H2S is probably involved in the pathogenesis of
DOX-induced cardiomyopathy, as H2S reduces lipid
peroxidation, increases the activity of antioxidant enzyme systems
and inhibits oxidative stress-induced injury ().
Erythropoietin has been found to protect the
myocardium against DOX-induced impairment of heart function and
inhibits the apoptosis of cardiomyocytes by activating the PI3K/Akt
cell-survival pathway (). In
addition, neureg-ulin-1 (),
tanshinone IIA (), and
urotensin II () have been
demonstrated to prevent the apoptosis of cardiomyocytes exposed to
DOX, partly through the Akt signaling pathways. Thus, these agents
may promote cell survival and exert cardioprotective effects.
FoxO3a is regulated by the PI3K/Akt pathway and plays an important
role in mediating the cytotoxic effects of DOX (). It has also been demonstrated that
in the presence of serum and growth factors, the survival kinase
Akt is phosphorylated, which in turn phosphorylates FoxO
transcription factors, thereby leading to nuclear exclusion,
cytoplasmic retention and the inactivation of FoxO transcription
factors (). Conversely,
oxidative stress has been shown to induce the re-localization of
FoxO transcription factors from the cytoplasm to the nucleus and
activate the target genes of FoxO transcription factors, including
the pro-apoptotic gene Bim, with subsequent cell apoptosis
The roles of FoxO3a in oxidative stress-induced
cardio-toxicity have received attention. Cardiac microvascular
endothelial cells (CMECs) are some of the predominant cells that
are immediately damaged after myocardial I/R injury. High glucose
() and hypoxia () have been observed to reduce the
phosphorylation of Akt and FoxO3a, induce FoxO3a activation, and
lead to ROS production and apoptosis in CMECs. Wang et al
reported that venlafaxine protects PC12 cells against
corticosterone-induced cell death by modulating the activity of the
PI3K/Akt/FoxO3a pathway ().
Simvastatin inhibits rapamycin-induced dysfunction and apoptosis of
CMECs, probably through the activation of the PI3K/Akt/FoxO3a
signaling pathway ().
Furthermore, erythropoietin activates the PI3K/Akt/FoxO3a signaling
pathway and protects neurons from 6-hydroxydopamine
(6-OHDA)-induced apoptosis ().
In addition, sodium tanshinone IIA sulfonate () and bromelain () have been demonstrated to inhibit
the FoxO3a pathway and apoptosis of cardiomyocytes. In the present
study, we have demonstrated that a statistically significant
reduction in the phosphorylation of Akt and FoxO3a protein was
observed in the DOX-treated H9c2 cells. The data suggest that the
PI3K/Akt/FoxO3a signaling pathway is important in DOX-induced
cytoxicity in cardiomyocytes.
To elucidate the potential protective effects of
H2S against DOX-induced cardiotoxicity as well as the
mechanisms responsible for these effects, we observed the effect of
NaHS on the phosphorylation of Akt and FoxO3a protein induced by
DOX exposure. The findings of the present study show that the
treatment of H9c2 cells with NaHS significantly prevented the
DOX-induced reduction in the levels of p-Akt and p-FoxO3a, and this
was accompanied by an increase in cell viability, indicating that
the PI3K/Akt/FoxO3a pathway may be involved in the protective
effects of exogenous H2S against DOX-induced
cardiotoxicity.
Notably, the results of the present study provide
novel evidence that an interaction between ROS and FoxO3a exists in
DOX-exposed H9c2 cells, because H2S attenuated the
DOX-induced reduction in p-Akt and p-FoxO3a levels. Similar to
exogenous H2S, the treatment of H9c2 cells with NAC (ROS
scavenger) prior to DOX exposure attenuated the phosphorylation of
Akt and FoxO3a. These results indicated that targeting the
interaction that occurs between ROS and FoxO3a in DOX-induced
cardiotoxicity may aid in the treatment and prevention of cardiac
injury. Another important novel finding of this study was that
exogenous H2S protects against DOX-induced
cardiotoxicity by activating the PI3K/Akt/FoxO3a pathway in H9c2
cells. The findings of the present study support this hypothesis.
The treatment of H9c2 cells with NaHS (a donor of H2S)
prior to DOX exposure significantly ameliorated the reduction in
p-Akt and p-FoxO3a, attenuated the nuclear localization of FoxO3a
and the DOX-induced apoptosis of H9c2 cells, and exerted an
inhibitory effect on Bim expression. In addition, pre-treatment
with LY294002, a selective inhibitor of PI3K/Akt, reversed the
protective effect of H2S on DOX-induced cardiotoxicity,
as demonstrated by an increase in the number of apoptotic cells, a
reduction in cell viability and the phosphorylation of Akt and
FoxO3a, as well as a simultaneous increase in Bim expression.
Therefore, our results suggest that the protective effects of
H2S on FoxO3a nuclear translocation and Bim expression
are mediated by the PI3K/Akt pathway.
In conclusion, to the best of our knowledge, the
present study has demonstrated for the first time, that FoxO3a
plays a central role in the DOX-induced apoptosis of H9c2 cells.
Furthermore, data from the present study have revealed that
H2S protects H9c2 cardiomyocytes against DOX-induced
cytotoxicity through the activation of the PI3K/Akt/FoxO3a pathway.
To the best of our knowledge, this is the first study to show that
H2S is capable of acting on the PI3K/Akt/FoxO3a pathway
to enhance the survival of cardiomyocytes, thereby suggesting that
the FoxO3a pathway may be a novel therapeutic target in
cardiovascular disease.
Acknowledgments
The present study was supported by grants from the
National Natural Science Foundation of China (, Z.-S.J.)
and the Graduate student research innovation project of Hunan
province (CX, M.-H.L.).
References
Magnano LC, Martínez Cibrian N, Andrade
González X and Bosch X: Cardiac complications of chemotherapy: role
of prevention. Curr Treat Options Cardiovasc Med. 16:3122014.
Truong J, Yan AT, Cramarossa G and Chan
KK: Chemotherapy-induced cardiotoxicity: detection, prevention, and
management. Can J Cardiol. 30:869–878. 2014.
Spagnuolo RD, Recalcati S, Tacchini L and
Cairo G: Role of hypoxia-inducible factors in the
dexrazoxane-mediated protection of cardiomyocytes from
doxorubicin-induced toxicity. Br J Pharmacol. 163:299–312. 2011.
Zhang Y, Tang ZH, Ren Z, Qu SL, Liu MH,
Liu LS and Jiang ZS: Hydrogen sulfide, the next potent preventive
and therapeutic agent in aging and age-associated diseases. Mol
Cell Biol. 33:. 2013.
Osipov RM, Robich MP, Feng J, Liu Y,
Clements RT, Glazer HP, Sodha NR, Szabo C, Bianchi C and Sellke FW:
Effect of hydrogen sulfide in a porcine model of myocardial
ischemia-reperfusion: comparison of different administration
regimens and characterization of the cellular mechanisms of
protection. J Cardiovasc Pharmacol. 54:287–297. 2009.
Huang YE, Tang ZH, Xie W, Shen XT, Liu MH,
Peng XP, Zhao ZZ, Nie DB, Liu LS and Jiang ZS: Endogenous hydrogen
sulfide mediates the cardioprotection induced by ischemic
post-conditioning in the early reperfusion phase. Exp Ther Med.
Wang H, Zhou X, Huang J, Mu N, Guo Z, Wen
Q, Wang R, Chen S, Feng ZP and Zheng W: The role of Akt/FoxO3a in
the protective effect of venlafaxine against corticosterone-induced
cell death in PC12 cells. Psychopharmacology (Berl). 228:129–141.
van der Vos KE and Coffer PJ: The
extending network of FOXO transcriptional target genes. Antioxid
Redox Signal. 14:579–592. 2011.
Sanphui P and Biswas SC: FoxO3a is
activated and executes neuron death via Bim in response to
β-amyloid. Cell Death Dis. 4:e6252013.
Bao W, Pan F, Chen L, Su G, Gao X, Li Y,
Sun Q, Sun J, He K and Song H: The PI3K/AKT pathway and FOXO3a
transcription factor mediate high glucose-induced apoptosis in
neonatal rat ventricular myocytes. Iran Red Crescent Med J.
Li D, Qu Y, Mao M, Zhang X, Li J, Ferriero
D and Mu D: Involvement of the PTEN-AKT-FOXO3a pathway in neuronal
apoptosis in developing rat brain after hypoxia-ischemia. J Cereb
Blood Flow Metab. 29:. 2009.
Liu MH, Yuan C, He J, Tan TP, Wu SJ, Fu
HY, Liu J, Yu S, Chen YD, Le QF, et al: Resveratrol protects PC12
cells from high glucose-induced neurotoxicity via PI3K/Akt/FoxO3a
pathway. Cell Mol Neurobiol. 35:513–522. 2014.
Zhang MQ, Zheng YL, Chen H, Tu JF, Shen Y,
Guo JP, Yang XH, Yuan SR, Chen LZ, Chai JJ, et al: Sodium
tanshinone IIA sulfonate protects rat myocardium against
ischemia-reperfusion injury via activation of PI3K/Akt/FOXO3A/Bim
pathway. Acta Pharmacol Sin. 34:. 2013.
Juhasz B, Thirunavukkarasu M, Pant R, Zhan
L, Penumathsa SV, Secor ER Jr, Srivastava S, Raychaudhuri U, Menon
VP, Otani H, et al: Bromelain induces cardioprotection against
ischemia-reperfusion injury through Akt/FOXO pathway in rat
myocardium. Am J Physiol Heart Circ Physiol. 294:H1365–H.
Guo R, Wu K, Chen J, Mo L, Hua X, Zheng D,
Chen P, Chen G, Xu W and Feng J: Exogenous hydrogen sulfide
protects against doxorubicin-induced inflammation and cytotoxicity
by inhibiting p38MAPK/NFκB pathway in H9c2 cardiac cells. Cell
Physiol Biochem. 32:. 2013.
Tocchetti CG, Carpi A, Coppola C,
Quintavalle C, Rea D, Campesan M, Arcari A, Piscopo G, Cipresso C,
Monti MG, et al: Ranolazine protects from doxorubicin-induced
oxidative stress and cardiac dysfunction. Eur J Heart Fail.
16:358–366. 2014.
Wang XY, Yang CT, Zheng DD, Mo LQ, Lan AP,
Yang ZL, Hu F, Chen PX, Liao XX and Feng JQ: Hydrogen sulfide
protects H9c2 cells against doxorubicin-induced cardiotoxicity
through inhibition of endoplasmic reticulum stress. Mol Cell
Biochem. 363:419–426. 2012.
Su YW, Liang C, Jin HF, Tang XY, Han W,
Chai LJ, Zhang CY, Geng B, Tang CS and Du JB: Hydrogen sulfide
regulates cardiac function and structure in adriamycin-induced
cardiomyopathy. Circ J. 73:741–749. 2009.
Kim KH, Oudit GY and Backx PH:
Erythropoietin protects against doxorubicin-induced cardiomyopathy
via a phosphatidylinositol 3-kinase-dependent pathway. J Pharmacol
Exp Ther. 324:160–169. 2008.
An T, Zhang Y, Huang Y, Zhang R, Yin S,
Guo X, Wang Y, Zou C, Wei B, Lv R, et al: Neuregulin-1 protects
against doxorubicin-induced apoptosis in cardiomyocytes through an
Akt-dependent pathway. Physiol Res. 62:379–385. 2013.
Hong HJ, Liu JC, Chen PY, Chen JJ, Chan P
and Cheng TH: Tanshinone IIA prevents doxorubicin-induced
cardiomyocyte apoptosis through Akt-dependent pathway. Int J
Cardiol. 157:174–179. 2012.
Chen YL, Loh SH, Chen JJ and Tsai CS:
Urotensin II prevents cardiomyocyte apoptosis induced by
doxorubicin via Akt and ERK. Eur J Pharmacol. 680:88–94. 2012.
Ho KK, McGuire VA, Koo CY, Muir KW, de
Olano N, Maifoshie E, Kelly DJ, McGovern UB, Monteiro LJ, Gomes AR,
et al: Phosphorylation of FOXO3a on Ser-7 by p38 promotes its
nuclear localization in response to doxorubicin. J Biol Chem.
287:. 2012.
Wang Y, Zhou Y and Graves DT: FOXO
transcription factors: their clinical significance and regulation.
Biomed Res Int. 014.
Storz P: Forkhead homeobox type O
transcription factors in the responses to oxidative stress.
Antioxid Redox Signal. 14:593–605. 2011.
Peng C, Ma J, Gao X, Tian P, Li W and
Zhang L: High glucose induced oxidative stress and apoptosis in
cardiac microvascular endothelial cells are regulated by FoxO3a.
PLoS One. 8:e.
Zhang S, Zhao Y, Xu M, Yu L, Zhao Y, Chen
J, Yuan Y, Zheng Q and Niu X: FoxO3a modulates hypoxia stress
induced oxidative stress and apoptosis in cardiac microvascular
endothelial cells. PLoS One. 8:e.
Pan Q, Xie X, Guo Y and Wang H:
Simvastatin promotes cardiac microvascular endothelial cells
proliferation, migration and survival by phosphorylation of p70 S6K
and FoxO3a. Cell Biol Int. 38:599–609. 2014.
Jia Y, Mo SJ, Feng QQ, Zhan ML, OuYang LS,
Chen JC, Ma YX, Wu JJ and Lei WL: EPO-dependent activation of
PI3K/Akt/FoxO3a signalling mediates neuroprotection in in vitro and
in vivo models of Parkinson's disease. J Mol Neurosci. 53:117–124.
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Liu, M., Zhang, Y., He, J., Tan, T., Wu, S., Guo, D. ... Jiang, Z. (2016). Hydrogen sulfide protects H9c2 cardiac cells against doxorubicin-induced cytotoxicity through the PI3K/Akt/FoxO3a pathway. International Journal of Molecular Medicine, 37, . https://doi.org/10.3892/ijmm.
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Jiang, Z.&Hydrogen sulfide protects H9c2 cardiac cells against doxorubicin-induced cytotoxicity through the PI3K/Akt/FoxO3a pathway&. International Journal of Molecular Medicine 37.6 (2016): .
Zhang, Y.,
Jiang, Z.&Hydrogen sulfide protects H9c2 cardiac cells against doxorubicin-induced cytotoxicity through the PI3K/Akt/FoxO3a pathway&. International Journal of Molecular Medicine 37, no. 6 (2016): . https://doi.org/10.3892/ijmm.