Nicotine induces H9C2 cell apoptosis via Akt protein degradation

  • Authors:
    • Chong Huang
    • Xiong Guo
    • Huishou Zhao
    • Rui An
    • Kun Lian
    • Xiaomeng Zhang
    • Jinglong Zhang
    • Feng Yan
    • Huaning Xie
    • Shan Wang
    • Ling Tao
  • View Affiliations

  • Published online on: August 22, 2017     https://doi.org/10.3892/mmr.2017.7331
  • Pages: 6269-6275
Metrics: Total Views: 0 (Spandidos Publications: | PMC Statistics: )
Total PDF Downloads: 0 (Spandidos Publications: | PMC Statistics: )


Abstract

Smoking is highly associated with cardiovascular diseases. However, the effect of nicotine, a key ingredient in smoking products, on cardiomyocyte apoptosis remains controversial. The present study aims to clarify the role of nicotine on cardiomyocyte cell apoptosis and to investigate the underlying mechanism. In the present study, H9C2 cells were exposed to nicotine at various concentrations (0, 10 and 100 µM) for 48 h. Cell Counting Kit‑8 and TUNEL assays were performed to assess cell viability and apoptosis, respectively, and reverse transcription‑quantitative polymerase chain reaction and western blot analysis were used to investigate the mRNA and protein expression. PYR‑41, a ubiquitin E1 inhibitor, was employed to investigate whether the ubiquitin‑proteasome system was involved in the downregulation of Akt. An Akt1 overexpression plasmid was used to demonstrate the role of Akt in H9C2 cells apoptosis. Tetratricopeptide repeat domain 3 (TTC3) small interfering RNA (siRNA) was used to investigate the effect of TTC3 on Akt protein degradation. The results demonstrated that nicotine induced apoptosis in H9C2 cells compared with control cells (P<0.05). The protein level of Akt was downregulated by nicotine in a concentration‑dependent manner (P<0.05). PYR‑41 treatment restored the protein level of Akt. The cell viability was significantly improved by Akt overexpression when cells were exposed to nicotine at 10 µM, compared with control cells. Nicotine also upregulated the level of TTC3 mRNA (P<0.05) and the protein level of Akt, and cell viability was recovered by TTC3 siRNA. In conclusion, the current study demonstrated that nicotine induced H9C2 cell apoptosis via Akt protein degradation, which may be mediated by TTC3.

Introduction

It is well established that smoking is a serious health concern and is associated with cancer and various other diseases (13). Cardiovascular disease is a frequently-occurring and common disease, with a higher incidence and mortality among smokers (4). Nicotine is the key ingredient in smoking products and there are two distinctive arguments regarding nicotine and apoptosis. Certain reports have indicated that nicotine promotes proliferation and inhibits apoptosis by acting on neuronal nicotinic acetylcholine receptors (nAChRs) (5,6). However, another report indicated that nicotine induces human cell apoptosis by influencing Hsp90 α expression (7). Therefore, the effect of nicotine on apoptosis requires further investigation. Previously, it was demonstrated that nicotine has a harmful effect on cardiomyocytes via the promotion of apoptosis (8), which is associated with various cardiology diseases and high cardiovascular mortality, however, the underlying mechanism is not well established.

Akt, a serine/threonine kinase, has an important role in cell survival, proliferation, migration and apoptosis. Akt is phosphorylated and activated by phosphoinositide 3-kinase (PI3K), which is mediated by the insulin pathway, and subsequently regulates fatty acid β-oxidation and promotes survival (9,10). An et al (11) reported that melatonin attenuates sepsis-induced cardiac dysfunction via activation of Akt. Due to the critical role of Akt, the regulatory mechanism of Akt is important. Various methods of Akt regulation exist, one of which is Akt protein degradation (12). A previous study demonstrated that ubiquitin-mediated Akt protein degradation leads to normal human lung fibroblast apoptosis (13). Tetratricopeptide repeat domain 3 (TTC3) and mitochondrial E3 ubiquitin protein ligase 1 (MUL1), which are important regulating factors of E3 ligases for Akt, have been reported to cause Akt ubiquitination and proteasomal degradation (14,15).

The present study, to the best of our knowledge, demonstrated for the first time that nicotine induced H9C2 cell apoptosis via Akt protein degradation. In addition, the results indicated that nicotine upregulated the level of TTC3 mRNA, which may be responsible for Akt protein degradation.

Materials and methods

Cell culture

H9C2 embryonic rat myocardium-derived cells were obtained from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (Shanghai, China). The cells were cultured in Dulbecco's modified Eagle's medium (Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) containing 10% heat-inactivated fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 100 U/ml penicillin and 100 µg/ml streptomycin at 37°C in a humidified incubator with 5% CO2 until 70–80% confluence, and sub-cultured (1:3 split ratio) using trypsin (0.25%) containing EDTA (0.02%).

Cell treatments

Nicotine (Sigma Aldrich, Merck KGaA, Darmstadt, Germany) was dissolved in DMEM containing 10% FBS to concentration of 10 and 100 µM, then 80% confluent cells were incubated with different concentrations of nicotine (10 or 100 µM) or standard medium for 48 h at 37°C, or together with a PYR-41 pretreatment for 30 min. PYR-41 (Selleckchem, TX, USA) was dissolved in DMEM containing 10% FBS to concentration of 5 and 10 µM.

Cell viability assay

Cell Counting Kit-8 (CCK-8) assay was purchased from Dojindo Molecular Technologies, Inc. (Kumamoto, Japan) and was used to measure the cell viability in different nicotine treatment groups, according to the manufacturer's protocol. Briefly, cells were seeded in a 96-well plate at a density of 5×103 cells/well with four replicates for each group and cultured overnight at 37°C, cells were subsequently exposed to various concentrations of nicotine (10 or 100 µM) or standard medium as a control for 48 h at 37°C. Following treatment, 100 µl fresh medium containing 10 µl CCK-8 solution was added to each well in an incubator for 30 min at 37°C. The optical density of each well was determined at 450 nm by a microplate reader to calculate the cell viability.

Quantification of apoptotic cells

According to the manufacturer's protocol, the cell apoptosis at single-cell level was detected and quantified by TUNEL assay, using the in situ Cell Death Detection Fluorescein kit (Roche Molecular Systems, Inc., Pleasanton, CA, USA). Briefly, cells cultured on glass coverslips at 37°C at 70–80% confluence, were treated with various concentrations of nicotine (0, 10 or 100 µM) for 48 h at 37°C, washed with PBS, fixed with 4% paraformaldehyde in PBS for 1 h at 37°C and permeabilized with 0.5% Triton X-100 in 0.1% sodium citrate for 2 min on ice. Following incubation with the enzyme solution mixture and label solution for 1 h at 37°C in a dark, humid chamber, nuclei were counterstained with 100 ng/ml DAPI for 3–5 min at 37°C. The percentage of cells undergoing apoptosis was determined with a fluorescence microscope and 25 random fields were quantified by an investigator who was blind to the treatment.

mRNA expression analysis

Total RNA was prepared from 95% confluent cultured cells using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.), according to the manufacturer's protocol. RNase-free DNase I (cat. no. RR047A; Takara Biotechnology, Co., Ltd., Dalian, China) was used to remove DNA contamination and cDNA was generated from 2 µg total RNA using the PrimeScript™ RT reagent kit with gDNA Eraser (cat. no. RR047A; Takara Biotechnology, Co., Ltd.), according to the manufacturer's protocol. The 20 µl reaction mixture was incubated as 50°C of 15 min followed with enzyme inactivation by incubation at 85°C for 5 sec. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed using a CFX96 Real-Time PCR System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and the QuantiTect SYBR-Green PCR kit (Takara Biotechnology, Co., Ltd.), with the following An initial predenaturation step at 95°C for 30 sec, followed by 40 cycles of denaturation at 95°C for 5 sec and annealing at 60°C for 30 sec. Data processing and normalization was undertaken as previously described (16). All RT-qPCR experiments were performed in triplicate. Primers used are listed in Table I.

Table I.

Primer sequences used for reverse transcription-quantitative polymerase chain reaction.

Table I.

Primer sequences used for reverse transcription-quantitative polymerase chain reaction.

Primer sequence

GeneAccession no.ForwardReverse
GAPDHNM_017008.4 5′-GGCACAGTCAAGGCTGAGAATG-3′ 5′-ATGGTGGTGAAGACGCCAGTA-3′
Total-AktNM_033230.2 5′-ATGGACTTCCGGTCAGGTTCA-3′ 5′-GCCCTTGCCCAGTAGCTTCA-3′
NM_017093.1
NM_031575.1
MUL1NM_001106695.1 5′-GCAAGTTACAGCCACCACCTGA-3′ 5′-CCAGACTTTGTGTTGCTGCTGA-3′
TTC3NM_001108315 5′-GTGGGCACAAGTTTCACAAAGG-3′ 5′-AAGTGCATGGTGCATTAGTGAGG-3′

[i] MUL1, mitochondrial E3 ubiquitin protein ligase 1; TTC3, tetratricopeptide repeat domain 3.

Gene silencing with small interfering RNA (siRNA) and gene overexpression with plasmid vector

The siRNA against TTC3 mRNA (5′-UUGCAACUUGCUAGAAGAAUU-3′) and scrambled siRNA (5′-UUAACGUUGAACGAUCUUCUU-3′) were designed and purchased from Shanghai GenePharma Co., Ltd. (Shanghai, China). The Akt1 overexpression pCMV6-XL5 plasmid vector and control pCMV6-XL5 plasmid vector were designed by GeneCopoeia, Inc. (Rockville, MD, USA). H9C2 cells were plated onto 6-well plates at a density of 1.5×104 cells/well and transfected with TTC3 siRNA (75 pmol) or pCMV6-XL5-Akt1 (2 µg) at 80% confluence at 37°C using Lipofectamine 2000 according to the manufacturer's protocol (Invitrogen; Thermo Fisher Scientific, Inc.). At 48 h post-transfection, RT-qPCR was used to determine the expression levels of TTC3 and western blotting was performed to determine the expression levels of Akt1. At 36 h post-transfection, the cells were subjected to treatment with nicotine (10 µM) or standard medium for 48 h at 37°C. Subsequently, the cells were harvested for further analysis.

Western blot analysis

Cells were cultured in a 6-well plates until 95% confluence and were lysed in ice-cold radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) supplemented with 1% phenylmethylsulfonyl fluoride (Beyotime Institute of Biotechnology). Protein concentrations were detected with a bicinchoninic acid assay. Lysates (40 µg/lane) were separated on 12% SDS-PAGE gels, transferred to nitrocellulose filter membranes. Subsequently, the membrane was incubated with 10% skim milk for 1 h at 37°C to block nonspecific binding, and incubated overnight at 4°C with antibodies directed against Akt (cat. no. 9272; 1:1,000), phosphorylated (p)-Akt (ser473; cat. no. 4060; 1:1,000), caspase-3 (cat. no. 14220; 1:1,000), cleaved-caspase-3 (cat. no. 9664; 1:1,000; all from Cell Signaling Technology, Inc., Danvers, MA, USA), β-actin (cat. no. sc-130300; 1:500, Santa Cruz Biotechnology, Inc., Dallas, TX, USA). The blots were subsequently incubated with horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (cat. no. 7074; 1:2,000) or an anti-mouse IgG (cat. no. 7076; 1:2,000; Cell Signaling Technology, Inc.) for 1 h at 37°C. Bands were visualized with an enhanced chemiluminescence kit (EMD Millipore, Billerica, MA, USA) in a ChemiDoc XRS system (Bio-Rad Laboratories, Inc.). The protein level was quantified with Quantity One software version 4.6.2 (Bio-Rad Laboratories, Inc.).

Statistical analysis

Data are presented as the mean ± standard error of the mean. SPSS 15.0 software (SPSS, Inc., Chicago, IL, USA) was used for all statistical analyses. Statistical comparisons between two groups were performed with the unpaired Student's t-test. The differences between more than two groups were analyzed with one-way analysis of variance followed by Student-Newman-Keuls post hoc test. P≤0.05 was considered to indicate a statistically significant difference.

Results

Nicotine promotes H9C2 cell apoptosis in vitro

To investigate whether nicotine may promote cardiac myoblast cell apoptosis, the present study employed H9C2 cells to determine the role of nicotine on apoptosis in cultured cells. Cell viability was determined by CCK-8 assay. The viability of H9C2 cells exposed to nicotine at 10 and 100 µM was significantly reduced in a concentration-dependent manner after 48 h, compared with control cells (P<0.01; Fig. 1A). To further investigate the effect of the nicotine on H9C2 cells, the protein expression level of caspase-3, which is involved in apoptosis, was also investigated by western blotting. The results demonstrated that nicotine led to activation of caspase-3 (cleaved-caspase-3) in the 100 µM nicotine treatment, but not the 10 µM treatment group compared with the control (Fig. 1B and C). In addition, apoptosis was evaluated by TUNEL analysis. Compared with control cells, nicotine at 10 and 100 µM induced a significant increase in the number of TUNEL-positive cells after 48 h of treatment (Fig. 1D and E). These results indicate that nicotine may inhibit H9C2 cell viability and promote H9C2 cell apoptosis.

Nicotine induces Akt protein degradation through the ubiquitin-proteasome system in a concentration-dependent manner

To investigate the mechanism of nicotine-induced cells apoptosis, the present study investigated whether nicotine altered Akt and p-Akt protein levels. Similar to the reduction in cell viability, protein levels of p-Akt and total-Akt were reduced by a 48 h nicotine exposure in a concentration-dependent manner (Fig. 2A-C), compared with control cells. In order to further investigate the mechanism, the level of Akt mRNA expression was determined, and it was observed that Akt mRNA expression was unchanged following 48 h exposure to 10 and 100 µM nicotine (Fig. 2D). To investigate the involvement of the ubiquitin-proteasome system in the reduced protein expression of Akt following nicotine treatment, H9C2 cells were treated with different concentrations of PYR-41 (5 or 10 µM), which is a ubiquitin E1 inhibitor and also a valuable tool for investigating ubiquitination (17). After a 48 h period of nicotine exposure at 10 µM, which is the concentration that is closest to the levels observed in the blood of smokers, the reduction in the protein expression of Akt induced by 10 µM nicotine was significantly blocked by PYR-41 (Fig. 2E and F). Taken together, the results indicate that nicotine may cause Akt protein degradation via the ubiquitin-proteasome system in H9C2 cells.

Akt overexpression improves cell viability when exposed to nicotine

Due to the important role of Akt in cell survival and apoptosis, and as the results of the present study indicated that nicotine induced Akt protein degradation in a concentration-dependent manner, the present study further investigated the association between apoptosis and Akt. H9C2 cells were transfected with an Akt1 overexpression plasmid or vector-only. The results indicated that the level of Akt mRNA and protein were significantly upregulated in the vector-Akt group compared with the vector-only group when treated with 10 µM nicotine (Fig. 3A and B). Furthermore, the results demonstrated that Akt overexpression significantly inhibited nicotine-induced reductions in cell viability (Fig. 3C).

Nicotine induces Akt ubiquitination by TTC3

The results discussed in Fig. 2, where an inhibitor of ubiquitin E1 (PYR-41) was used, indicated that Akt protein degradation following exposure to nicotine occurred via ubiquitination and the ubiquitin-proteasome system. TTC3 and MUL1 are both E3 ligases that facilitate Akt ubiquitination. Therefore, to determine the type of E3 ligase against Akt that was involved in nicotine-induced Akt ubiquitination, the mRNA levels of TTC3 and MUL1 were determined using RT-qPCR in nicotine-exposed H9C2 cells. Nicotine augmented TTC3 mRNA expression in a concentration-dependent manner after 48 h of treatment (Fig. 4A). By contrast, MUL1 mRNA was unchanged under the same conditions (Fig. 4B). To further demonstrate the role of TTC3 in nicotine-induced Akt ubiquitination and degradation, TTC3 siRNA was used to inhibit TTC3 mRNA expression. Knockdown of TTC3 expression by TTC3 siRNAs led to a significant reduction in Akt ubiquitination and degradation induced by nicotine, compared with nicotine-treated cells transfected with scrambled siRNA (Fig. 4C and D). Furthermore, the current study determined whether knockdown of TTC3 expression may affect nicotine-induced reductions in cell viability. Compared with scrambled siRNA, cell viability was significantly increased by TTC3 siRNA (Fig. 4E), indicating that apoptosis levels may have been reduced.

Discussion

The present study demonstrated that nicotine reduced the viability and induced H9C2 cells apoptosis, Akt protein expression was downregulated when exposed to nicotine at various concentrations for 48 h, PYR-41, a ubiquitin E1 inhibitor, restored the protein expression of Akt, Akt overexpression inhibited H9C2 cell apoptosis induced by nicotine, nicotine upregulated the expression of TTC3 mRNA, nicotine-induced reductions in the protein expression of Akt protein level were reversed when TTC3 was silenced by siRNA and nicotine-induced reductions in cell viability were inhibited when TTC3 was silenced by siRNA.

It is clear that smoking is associated with numerous diseases, particularly cardiovascular diseases. Recent study reported by Baber et al (18) reported that smoking was an independent predictor of major bleeding following percutaneous coronary intervention with drug-eluting stents. In addition, Stone et al (19) discovered that smoking was associated with hospitalization for heart failure. Furthermore, Das et al (20), using pigs as experimental animals, revealed that smoking induced myocardial injury by release of cardiac troponin-T and -I in the serum, oxidative stress, inflammation, apoptosis, thrombosis and collagen deposition in the myocardium. Sumanasekera et al (21) also reported that smoking induced reduced cardiac stem cell viability, migration reduction and led to exacerbation of the damage.

Nicotine, which is a key ingredient in smoking products, may be responsible for various risks associated with smoking (22). Marrs and Maynard (23) reported that nicotine is the classic nAChR agonist and it has also been used as an insecticide. Li et al (24) discovered that nicotine induced cardiomyocyte hypertrophy through the transient receptor potential cation channel subfamily C member 3-mediated Ca2+/nuclear factor of activated T-cells signaling pathway. Furthermore, Zhou et al (8) reported that nicotine promoted cardiomyocyte apoptosis via oxidative stress and altered expression of apoptosis-associated genes. In the present study, the reduction of cell viability, the increased percentage of TUNEL-positive cells and the activation of caspase-3 indicated that nicotine also promoted H9C2 cell apoptosis.

Akt, a serine/threonine kinase, is a crucial molecule that is involved in various biological actions, including apoptosis, survival, proliferation and cell migration. Activated Akt, which is phosphorylated by PI3K, suppresses apoptosis by inactivating proapoptotic factors, maintaining mitochondrial integrity and stabilizing anti-apoptotic factors (25). In addition, Akt prevents apoptosis by phosphorylating apoptosis signal-regulating kinase 1 (26), which causes the activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase (27). Furthermore, Akt leads to p53 ubiquitination and degradation by phosphorylating the ubiquitin ligase mouse double minute homolog 2, which leads to apoptosis inhibition (28). Stulpinas et al (29) previously demonstrated that inhibition of Akt kinase signaling pathways leads to muscle-derived stem cell death. Regarding the cardiovascular system, Kerr et al (30) discovered that endothelial Akt deletion induced retinal vascular smooth muscle cell loss and basement membrane deterioration, and resulted in vascular regression and retinal atrophy. In addition, Fujio et al (31) reported that Akt protected cardiomyocytes against ischemia-reperfusion injury. The results of the present study indicated that the protein level of Akt was downregulated when exposed to nicotine in a concentration-dependent manner, while the cell viability in H9C2 cells was increased following nicotine treatment when Akt expression was upregulated.

There are various methods of Akt regulation, and one mechanism is Akt degradation. Kim et al (32) reported that non-thermal plasma induced Akt degradation via the ubiquitin-proteasome system, subsequently leading to head and neck cancer cell death. They also demonstrated that cigarette smoke induced normal human lung fibroblast cell apoptosis by Akt protein degradation via the ubiquitin-proteasome system (13). The present study demonstrated that PYR-41, a ubiquitin E1 inhibitor, restored the protein expression of Akt following nicotine treatment. Therefore, it was hypothesized that nicotine induced Akt protein degradation via the ubiquitin-proteasome system. The results of the current study also indicated that the cell viability was restored when Akt was overexpressed.

TTC3 is an Akt-specific E3 ligase that binds to Akt and facilitates its ubiquitination and degradation within the nucleus (14,33,34). The present study demonstrated that nicotine upregulated the level of TTC3 mRNA, whereas the level of Akt mRNA was not changed. Furthermore, when TTC3 was silenced by siRNA, the nicotine-induced reduction in the protein expression of Akt was significantly reversed, and the cell viability was also improved. Taken together, these results indicate that nicotine may induce Akt protein degradation by the ubiquitin-proteasome system via TTC3 upregulation.

In conclusion, the current study demonstrated that nicotine induced H9C2 cell apoptosis by facilitating Akt protein degradation, it was also demonstrated that inhibition of the degradation of Akt protein protected H9C2 cells from reduction of cell viability induced by nicotine. These results may contribute to the investigation of the mechanism of diseases caused by smoking and provide a novel therapeutic target for smoking-associated diseases. Due to the effect of nicotine on the apoptosis of H9C2 cells, and the role of nicotine on Akt protein degradation, we hypothesize that nicotine may aggravate apoptosis following ischemia/reperfusion, which requires further investigation.

Acknowledgements

The present study was supported by the Program for National Science Fund for Distinguished Young Scholars of China (grant no. 81225001), the National Key Basic Research Program of China (973 Program; grant no. 2013CB531204), the Key Science and Technology Innovation Team in Shaanxi Province (grant no. 2014KCT-19), the Program for Changjiang Scholars and Innovative Research Team in University (grant no. PCSIRT-14R08), the National Science Funds of China (grant nos. 81170186, 81470478 and 81400201), the Major Science and Technology Project of China ‘Significant New Drug Development’ (grant no. 2012ZX09J12108-06B) and the Fourth Military Medical University's Young Talent Project (the first level).

References

1 

Glickman MS and Schluger N: Adding insult to injury: Exacer-bating TB risk with smoking. Cell Host Microbe. 19:432–433. 2016. View Article : Google Scholar : PubMed/NCBI

2 

Purdue MP and Silverman DT: Clearing the air: Summarizing the smoking-related relative risks of bladder and kidney cancer. Eur Urol. 70:467–468. 2016. View Article : Google Scholar : PubMed/NCBI

3 

Sharma R, Harlev A, Agarwal A and Esteves SC: Cigarette smoking and semen quality: A new meta-analysis examining the effect of the 2010 World Health Organization laboratory methods for the examination of human semen. Eur Urol. 70:635–645. 2016. View Article : Google Scholar : PubMed/NCBI

4 

Office of the Surgeon General (US); Office on Smoking and Health (US), . The Health Consequences of Smoking: A Report of the Surgeon General. Atlanta (GA): Centers for Disease Control and Prevention (US); 2004

5 

Singh S, Pillai S and Chellappan S: Nicotinic acetylcholine receptor signaling in tumor growth and metastasis. J Oncol. 2011:4567432011. View Article : Google Scholar : PubMed/NCBI

6 

Lau JK, Brown KC, Thornhill BA, Crabtree CM, Dom AM, Witte TR, Hardman WE, McNees CA, Stover CA, Carpenter AB, et al: Inhibition of cholinergic signaling causes apoptosis in human bronchioalveolar carcinoma. Cancer Res. 73:1328–1339. 2013. View Article : Google Scholar : PubMed/NCBI

7 

Wu YP, Kita K and Suzuki N: Involvement of human heat shock protein 90 alpha in nicotine-induced apoptosis. Int J Cancer. 100:37–42. 2002. View Article : Google Scholar : PubMed/NCBI

8 

Zhou X, Sheng Y, Yang R and Kong X: Nicotine promotes cardiomyocyte apoptosis via oxidative stress and altered apoptosis-related gene expression. Cardiology. 115:243–250. 2010. View Article : Google Scholar : PubMed/NCBI

9 

Franke TF: PI3K/Akt: Getting it right matters. Oncogene. 27:6473–6488. 2008. View Article : Google Scholar : PubMed/NCBI

10 

Manning BD and Cantley LC: AKT/PKB signaling: Navigating downstream. Cell. 129:1261–1274. 2007. View Article : Google Scholar : PubMed/NCBI

11 

An R, Zhao L, Xi C, Li H, Shen G, Liu H, Zhang S and Sun L: Melatonin attenuates sepsis-induced cardiac dysfunction via a PI3K/Akt-dependent mechanism. Basic Res Cardiol. 111:82016. View Article : Google Scholar : PubMed/NCBI

12 

Liao Y and Hung MC: Physiological regulation of Akt activity and stability. Am J Transl Res. 2:19–42. 2010.PubMed/NCBI

13 

Kim SY, Lee JH, Huh JW, Ro JY, Oh YM, Lee SD, An S and Lee YS: Cigarette smoke induces Akt protein degradation by the ubiquitin-proteasome system. J Biol Chem. 286:31932–31943. 2011. View Article : Google Scholar : PubMed/NCBI

14 

Suizu F, Hiramuki Y, Okumura F, Matsuda M, Okumura AJ, Hirata N, Narita M, Kohno T, Yokota J, Bohgaki M, et al: The E3 ligase TTC3 facilitates ubiquitination and degradation of phosphorylated Akt. Dev Cell. 17:800–810. 2009. View Article : Google Scholar : PubMed/NCBI

15 

Bae S, Kim SY, Jung JH, Yoon Y, Cha HJ, Lee H, Kim K, Kim J, An IS, Kim J, et al: Akt is negatively regulated by the MULAN E3 ligase. Cell Res. 22:873–885. 2012. View Article : Google Scholar : PubMed/NCBI

16 

Livak KJ and Schmittgen TD: Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 25:402–408. 2001. View Article : Google Scholar : PubMed/NCBI

17 

Yang Y, Kitagaki J, Dai RM, Tsai YC, Lorick KL, Ludwig RL, Pierre SA, Jensen JP, Davydov IV, Oberoi P, et al: Inhibitors of ubiquitin-activating enzyme (E1), a new class of potential cancer therapeutics. Cancer Res. 67:9472–9481. 2007. View Article : Google Scholar : PubMed/NCBI

18 

Baber U, Mehran R, Giustino G, Cohen DJ, Henry TD, Sartori S, Ariti C, Litherland C, Dangas G, Gibson CM, et al: Coronary thrombosis and major bleeding after PCI with drug-eluting stents: Risk scores from PARIS. J Am Coll Cardiol. 67:2224–2234. 2016. View Article : Google Scholar : PubMed/NCBI

19 

Stone GW, Selker HP, Thiele H, Patel MR, Udelson JE, Ohman EM, Maehara A, Eitel I, Granger CB, Jenkins PL, et al: Relationship between infarct size and outcomes following primary PCI: Patient-level analysis from 10 randomized trials. J Am Coll Cardiol. 67:1674–1683. 2016. View Article : Google Scholar : PubMed/NCBI

20 

Das A, Dey N, Ghosh A, Das S, Chattopadhyay DJ and Chatterjee IB: Molecular and cellular mechanisms of cigarette smoke-induced myocardial injury: Prevention by vitamin C. PLoS One. 7:e441512012. View Article : Google Scholar : PubMed/NCBI

21 

Sumanasekera WK, Tran DM, Sumanasekera TU, Le N, Dao HT and Rokosh GD: Cigarette smoke adversely affects functions and cell membrane integrity in c-kit+ cardiac stem cells. Cell Biol Toxicol. 30:113–125. 2014. View Article : Google Scholar : PubMed/NCBI

22 

Lee J and Cooke JP: Nicotine and pathological angiogenesis. Life Sci. 91:1058–1064. 2012. View Article : Google Scholar : PubMed/NCBI

23 

Marrs TC and Maynard RL: Neurotranmission systems as targets for toxicants: A review. Cell Biol Toxicol. 29:381–396. 2013. View Article : Google Scholar : PubMed/NCBI

24 

Li N, Si B, Ju JF, Zhu M, You F, Wang D, Ren J, Ning YS, Zhang FQ, Dong K, et al: Nicotine induces cardiomyocyte hypertrophy through TRPC3-mediated Ca2+/NFAT signalling pathway. Can J Cardiol. 32:1260.e1–1260.e10. 2016. View Article : Google Scholar

25 

Zhou BP, Liao Y, Xia W, Spohn B, Lee MH and Hung MC: Cytoplasmic localization of p21Cip1/WAF1 by Akt-induced phosphorylation in HER-2/neu-overexpressing cells. Nat Cell Biol. 3:245–252. 2001. View Article : Google Scholar : PubMed/NCBI

26 

Kim AH, Khursigara G, Sun X, Franke TF and Chao MV: Akt phosphorylates and negatively regulates apoptosis signal-regulating kinase 1. Mol Cell Biol. 21:893–901. 2001. View Article : Google Scholar : PubMed/NCBI

27 

Tobiume K, Matsuzawa A, Takahashi T, Nishitoh H, Morita K, Takeda K, Minowa O, Miyazono K, Noda T and Ichijo H: ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep. 2:222–228. 2001. View Article : Google Scholar : PubMed/NCBI

28 

Zhou BP, Liao Y, Xia W, Zou Y, Spohn B and Hung MC: HER-2/neu induces p53 ubiquitination via Akt-mediated MDM2 phosphorylation. Nat Cell Biol. 3:973–982. 2001. View Article : Google Scholar : PubMed/NCBI

29 

Stulpinas A, Imbrasaitė A and Kalvelytė AV: Daunorubicin induces cell death via activation of apoptotic signalling pathway and inactivation of survival pathway in muscle-derived stem cells. Cell Biol Toxicol. 28:103–114. 2012. View Article : Google Scholar : PubMed/NCBI

30 

Kerr BA, West XZ, Kim YW, Zhao Y, Tischenko M, Cull RM, Phares TW, Peng XD, Bernier-Latmani J, Petrova TV, et al: Stability and function of adult vasculature is sustained by Akt/Jagged1 signalling axis in endothelium. Nat Commun. 7:109602016. View Article : Google Scholar : PubMed/NCBI

31 

Fujio Y, Nguyen T, Wencker D, Kitsis RN and Walsh K: Akt promotes survival of cardiomyocytes in vitro and protects against ischemia-reperfusion injury in mouse heart. Circulation. 101:660–667. 2000. View Article : Google Scholar : PubMed/NCBI

32 

Kim SY, Kim HJ, Kang SU, Kim YE, Park JK, Shin YS, Kim YS, Lee K and Kim CH: Non-thermal plasma induces AKT degradation through turn-on the MUL1 E3 ligase in head and neck cancer. Oncotarget. 6:33382–33396. 2015. View Article : Google Scholar : PubMed/NCBI

33 

Dey-Guha I, Alves CP, Yeh AC, Salony, Sole X, Darp R and Ramaswamy S: A mechanism for asymmetric cell division resulting in proliferative asynchronicity. Mol Cancer Res. 13:223–230. 2015. View Article : Google Scholar : PubMed/NCBI

34 

Toker A: TTC3 ubiquitination terminates Akt-ivation. Dev Cell. 17:752–754. 2009. View Article : Google Scholar : PubMed/NCBI

Related Articles

Journal Cover

November-2017
Volume 16 Issue 5

Print ISSN: 1791-2997
Online ISSN:1791-3004

Sign up for eToc alerts

Recommend to Library

Copy and paste a formatted citation
x
Spandidos Publications style
Huang C, Guo X, Zhao H, An R, Lian K, Zhang X, Zhang J, Yan F, Xie H, Wang S, Wang S, et al: Nicotine induces H9C2 cell apoptosis via Akt protein degradation. Mol Med Rep 16: 6269-6275, 2017
APA
Huang, C., Guo, X., Zhao, H., An, R., Lian, K., Zhang, X. ... Tao, L. (2017). Nicotine induces H9C2 cell apoptosis via Akt protein degradation. Molecular Medicine Reports, 16, 6269-6275. https://doi.org/10.3892/mmr.2017.7331
MLA
Huang, C., Guo, X., Zhao, H., An, R., Lian, K., Zhang, X., Zhang, J., Yan, F., Xie, H., Wang, S., Tao, L."Nicotine induces H9C2 cell apoptosis via Akt protein degradation". Molecular Medicine Reports 16.5 (2017): 6269-6275.
Chicago
Huang, C., Guo, X., Zhao, H., An, R., Lian, K., Zhang, X., Zhang, J., Yan, F., Xie, H., Wang, S., Tao, L."Nicotine induces H9C2 cell apoptosis via Akt protein degradation". Molecular Medicine Reports 16, no. 5 (2017): 6269-6275. https://doi.org/10.3892/mmr.2017.7331