Apoptosis In H9c2 Cardiomyoblast Cells Research Articles

Benomyl induced oxidative stress related DNA damage and apoptosis in H9c2 cardiomyoblast cells

Benomyl, benzimidazole group pesticide, has been prohibited in Europe and USA since 2003 due to its toxic effects and it has been still determined as food and environmental contaminant. In the present study, the toxic effect mechanisms of benomyl were evaluated in rat cardiomyoblast (H9c2) cells. Cytotoxicity was determined by MTT and NRU assay and, oxidative stress potential was evaluated by reactive oxygen species (ROS) production and glutathione levels. DNA damage was assessed by alkaline comet assay. Relative expressions of apoptosis related genes were evaluated; furthermore, NF-κB and JNK protein levels were determined. At 4 μM concentration (at which cell viability was >70%), benomyl increased 2-fold of ROS production level and 2-fold of apoptosis as well as DNA damage. Benomyl down-regulated miR21, TNF-α and Akt1 ≥ 48.75 and ≥ 97.90; respectively. PTEN, JNK and NF-κB expressions were upregulated. The dramatic changes in JNK and NF-κB expression levels were not observed in protein levels. These findings showed the oxidative stress related DNA damage and apoptosis in cardiomyoblast cells exposed to benomyl. However, further mechanistic and in vivo studies are needed to understand the cardiotoxic effects of benomyl and benzimidazol fungucides.

Protective effect of cholecystokinin octapeptide on angiotensin II-induced apoptosis in H9c2 cardiomyoblast cells.

Cholecystokinin (CCK) and its receptors are expressed in mammalian cardiomyocytes and are involved in cardiovascular system regulation; however, the exact effect and underlying mechanism of CCK in cardiomyocyte apoptosis remain to be elucidated. We examined whether sulfated CCK octapeptide (CCK-8) protects H9c2 cardiomyoblast cells against angiotensin II (Ang II)-induced apoptosis. The H9c2 cardiomyoblasts were subjected to Ang II with or without CCK-8 and the viability and apoptotic rate were detected using a Cell Counting Kit-8 assay, Hoechst 33342 staining, terminal deoxyribonucleotide transferase-mediated nick-end labeling assays, and flow cytometry. In addition, specific antiapoptotic mechanisms of CCK-8 were investigated using specific CCK1 (Devazepide) or CCK2 (L365260) receptor antagonists, or the PI3K inhibitor LY294002. The expression of CCK, CCK1 receptor, CCK2 receptor, Akt, p-Akt, Bad, p-Bad, Bax, Bcl-2, and caspase-3 were detected by Western blot analysis and real-time polymerase chain reaction. We found that CCK and its receptor messenger RNA (mRNA) and protein are expressed in H9c2 cardiomyoblasts. Ang II-induced increased levels of CCK mRNA and protein expression and decreased levels of CCK1 receptor protein and mRNA. Pretreatment of CCK-8 attenuated Ang II-induced cell toxicity and apoptosis. In addition, pretreatment of H9c2 cells with CCK-8 markedly induced expression of p-Akt, p-bad, and Bcl-2 and decreased the expression levels of Bax and caspase-3. The protective effects of CCK-8 were partly abolished by Devazepide or LY294002. Our results suggest that CCK-8 protects H9c2 cardiomyoblasts from Ang II-induced apoptosis partly via activation of the CCK1 receptor and the phosphatidyqinositol-3 kinase/protein kinase B (PI3K/Akt) signaling pathway.

Carthamus tinctorius L. extract activates insulin-like growth factor-I receptor signaling to inhibit FAS-death receptor pathway and suppress lipopolysaccharides-induced H9c2 cardiomyoblast cell apoptosis.

Carthamus tinctorius L. (Compositae) is used in Chinese medicine to treat heart disease and inflammation. In our previous study, we found that C. tinctorius L. inhibited lipopolysaccharides (LPS)-induced tumor necrosis factor-alpha (TNF-α) activation, JNK expression, and apoptosis in H9c2 cardiomyoblast cells. The present study was performed to investigate the protective effect of C. tinctorius extract (CTF) on LPS-challenged H9c2 myocardioblast cell and to explore the possible underlying mechanism. Cell viability assay showed that LPS treatment decreased the cell viability of H9c2 cell, whereas CTF treatment reversed LPS cytotoxicity in a dose-dependent manner, especially in the LPS + CTF 25 (μg/mL) group. LPS treatment-induced apoptosis was determined by transferase-mediated dUTP nick end labeling assay, and by Western blot. LPS-induced apoptotic bodies were decreased following CTF treatment. Expression of TNF-α, FAS-L, FAS, FADD, caspase-8, BID, and t-BID was significantly increased in LPS-treated H9c2 cells. In contrast, it was significantly suppressed by the administration of CTF extract. In addition, CTF treatment activates antiapoptotic proteins, Bcl-2 and p-Bad, and downregulates Bax, cytochrome-c, caspase-9, caspase-3, and apoptosis-inducing factor expression. Furthermore, CTF exerted cytoprotective effects by activating insulin-like growth factor-I (IGF-I) signaling pathway leading to downregulation of the apoptotic proteins involved in FAS death receptor pathway. In addition, AG1024 and IGF-I receptor (IGF-IR) inhibitor and siRNA silencing reverses the effect of CTF implying that CTF functions through the IGF-IR pathway to inhibit LPS-induced H9c2 apoptosis. These results suggest that treatment with CTF extract prevented the LPS-induced apoptotic response through IGF-I pathway.

Suppression of isoproterenol-induced apoptosis in H9c2 cardiomyoblast cells by daidzein through activation of Akt.

Increased serum norepinephrine level is one of pathological processes relating to heart disease (HD). Estrogens are considered as potential therapeutics for the treatment of HD; however, estrogen supplementation shows some side-effects, such as increasing the risk of developing breast, endometrial and ovarian cancers. This study investigated the cardio-protective effects of daidzein (Dai), a selective estrogen receptor modulator (SERM) from soy bean extract, in H9c2 cardiomyoblast cells treated with isoproterenol (ISO), a norepinephrine analog. In this in vitro model, H9c2 cells treated with Dai at different concentrations showed no statistical difference in cell viability. TdT-mediated digoxigenin-dUTP nick-end labeling (TUNEL) data and western blotting results indicated that Dai treated-H9c2 cells recovered from the damage induced by ISO. The recovery effects of Dai on ISO-induced damage were blocked by inhibition of Akt activation through adding Akt inhibitor. On the other hand, the fold changes of phosphorylated Akt (p-Akt)/Akt normalized with the control for con, 0.25, 0.5, 1, 3 and 24 h of treatment were 1, 2, 5, 13, 11 and 10, respectively. In conclusion, Dai ameliorates apoptosis of cardiomyoblasts induced by ISO through Akt signaling pathway.

CREB Negatively Regulates IGF2R Gene Expression and Downstream Pathways to Inhibit Hypoxia-Induced H9c2 Cardiomyoblast Cell Death.

During hypoxia, gene expression is altered by various transcription factors. Insulin-like growth factor-II (IGF2) is known to be induced by hypoxia, which binds to IGF2 receptor IGF2R that acts like a G protein-coupled receptor, might cause pathological hypertrophy or activation of the mitochondria-mediated apoptosis pathway. Cyclic adenosine monophosphate (cAMP) responsive element-binding protein (CREB) is central to second messenger-regulated transcription and plays a critical role in the cardiomyocyte survival pathway. In this study, we found that IGF2R level was enhanced in H9c2 cardiomyoblasts exposed to hypoxia in a time-dependent manner but was down-regulated by CREB expression. The over-expression of CREB in H9c2 cardiomyoblasts suppressed the induction of hypoxia-induced IGF2R expression levels and reduced cell apoptosis. Gel shift assay results further indicated that CREB binds to the promoter sequence of IGF2R. With a luciferase assay method, we further observed that CREB represses IGF2R promoter activity. These results suggest that CREB plays an important role in the inhibition of IGF2R expression by binding to the IGF2R promoter and further suppresses H9c2 cardiomyoblast cell apoptosis induced by IGF2R signaling under hypoxic conditions.

The Protective Effect of INH2BP, a Novel PARP Inhibitor 5-Iodo-6-Amino-1,2-Benzopyrone, Against Hydrogen Peroxide-Induced Apoptosis Through ERK and p38 MAPK in H9c2 Cells

INH₂BP (5-iodo-6-amino-1,2-benzopyrone), a poly-ADP ribose polymerase inhibitor, has been shown to possess anti-cancer, anti-viral, and anti-inflammation properties. The aim of this study was to investigate the protective effect of INH₂BP against oxidative stress-induced apoptosis in H9c2 cardiomyoblast cells. While the treatment of H9c2 cardiomyoblasts cells with hydrogen peroxide (H₂O₂) caused a loss of cell viability and an increase in the number of apoptotic cells, INH₂BP significantly protected the cells against H₂O₂-induced cell death without any cytotoxicity. Our data also shows that INH₂BP significantly scavenged intracellular reactive oxygen species (ROS), and markedly enhanced the expression of antioxidant enzymes such as Mn-SOD (superoxide) and Cu/Zn-SOD, and heme oxygenase-1, which was accompanied by the concomitant activation of extracellular regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) phosphorylation in H9c2 cells. The effects of INH₂BP on ERK1/2 and p38 MAPK phosphorylation were abrogated by PD98059, an ERK1/2 inhibitor, and SB203580, a p38 inhibitor. In addition, inhibition of ERK1/2 and p38 MAPK by these inhibitors significantly attenuated INH₂BP-mediated H9c2 viability as well as cleaved caspases-3, Bax, and Bcl-2 activation. Taken together, these results demonstrate that INH₂BP prevents H₂O₂-induced apoptosis in H9c2 cells by reducing the production of intracellular ROS, regulating apoptotic-related proteins, and the activation of the ERK1/2 and p38 MAPK.

Cardioprotective effects of fucoidan against hypoxia-induced apoptosis in H9c2 cardiomyoblast cells

Context: Cardiomyocyte apoptosis plays a critical role in the progress of heart diseases. Fucoidan, a complex-sulfated polysaccharide, has been reported to possess potential cardioprotective efficacy in vivo.Objective: The present study determines whether fucoidan could provide cardioprotection on hypoxia-induced cardiomyocyte apoptosis.Materials and methods: H9c2 cardiomyoblast cells were incubated with various concentrations (15, 30, and 60 μg/ml) of fucoidan in a humidified incubator at 37 °C with 95% O2 and 5% CO2. After 6 h, hypoxia was processed and the cardioprotective effects of fucoidan were evaluated by applying MTT, ELISA, Hoechst 33258 nucleus staining, and western blot.Results: Following a 6 h exposure of H9c2 to hypoxic condition, significant reduction was found in cell survival (0.57-fold) and superoxide dismutase (SOD) activity (0.56-fold), which were associated with the increase of malondialdehyde (MDA) level (2.58-fold), creatine phosphokinase (CK, 3.57-fold), and lactate dehydrogenase (LDH) activities (2.39-fold). Moreover, hypoxia-induced apoptosis was confirmed by Hoechst 33258 nuclear staining, and these changes were accompanied by the increase of Bcl-2 (1.27-fold) and Bax expression (2.6-fold). However, preincubation of the cells with fucoidan prior to hypoxia exposure elevated the cell viability (30 μg/ml, 1.18-fold; 60 μg/ml, 1.32-fold) and SOD activity (30 μg/ml, 1.12-fold; 60 μg/ml, 1.25-fold), but decreased the MDA level (30 μg/ml, 0.70-fold; 60 μg/ml, 0.80-fold), CK (30 μg/ml, 0.69-fold; 60 μg/ml, 0.76-fold), and LDH (30 μg/ml, 0.67-fold; 60 μg/ml, 0.86-fold) leakages. Hoechst 33258 nuclear staining observations demonstrated the same protective effect of fucoidan on hypoxia-induced myocardial injury. Also, cardioprotective effects of fucoidan were reflected by increasing Bcl-2 (60 μg/ml, 1.84-fold), as well as decreasing Bax (60 μg/ml, 0.6-fold).Conclusion: Fucoidan had protective effect against hypoxia-induced cardiomyocytes apoptosis, and the mechanism might involve protections of the cell from oxidative injury.

Protective effect of Danggui (Radix Angelicae Sinensis) on angiotensin II-induced apoptosis in H9c2 cardiomyoblast cells.

BackgroundDanggui (Radix Angelicae Sinensis) is an herb often used in Traditional Chinese medicine. It is used to promote blood flow and has been used in the treatment of myocardial ischemia-reperfusion injury in animal models. Angiotensin II (Ang II) has been shown to play important roles in mediating cardiovascular diseases, and may cause cardiac hypertrophy and apoptosis. This study aimed to investigate whether Danggui has protective effects on Ang II-induced apoptosis in H9c2 cardiomyoblast cells and study the mechanisms involved.MethodsWe evaluated the effect of Danggui on Ang II-induced apoptosis in an in vitro model. H9c2 cardiomyoblast cells were cultured in serum-free medium for 4 hr, then treated with Danggui (50, 100 μg/ml) 1 hr pre- or post-Ang II treatment. After a further 23 hr of culture, cells were harvested for analyses with assays for apoptosis markers and cell signaling pathways.ResultsOur results showed that Ang II induced upregulation of pro-apoptotic Bad, instability of the mitochondria membrane potential, cytochrome c release, caspase-9 and caspase-3 activation and cardiomyocyte apoptosis. Pre- or post-treatment with Danggui reversed all of the above Ang II-induced apoptotic effects in H9c2 cells. Furthermore, the JNK (SP600125) inhibitor completely blocked Danggui inhibition of caspase-3 activation in Ang II-treated H9c2 cells.ConclusionsOur results showed that Danggui either pre-treatment or post-treatment highly attenuated the Ang II-induced apoptosis in cardiomyoblast cells. The findings demonstrated that the anti-apoptosis effect of Danggui is mediated by JNK and PI3k inhibitors.Electronic supplementary materialThe online version of this article (doi:10.1186/1472-6882-14-358) contains supplementary material, which is available to authorized users.

Impact of LPS-induced cardiomyoblast cell apoptosis inhibited by earthworm extracts.

Dilong is an earthworm extract with a dense nutritional content, widely used in Chinese herbal medicine to remove stasis and stimulate wound healing. Earthworm extracts are traditionally used by indigenous people throughout the world. How this Dilong inhibits Lipopolysaccharide (LPS)-induced cardiomyoblast cell apoptosis is still unclear. This study investigates the Dilong extract effect on LPS-induced apoptosis in H9c2 cardiomyoblast cells. LPS (1 μg/ml) administration for 24 h induced apoptosis in H9c2 cells. Cell apoptosis was detected using MTT, LDH, TUNEL assay and JC-1 staining. Western blot analysis was used to detect pro-apoptotic and anti-apoptotic proteins. Dilong extract totally blocked the LPS impact, leading to the activation of anti-apoptotic proteins, Bcl-2 and Bcl-xL, stabilized the mitochondria membrane and down-regulated the extrinsic and intrinsic pro-apoptotic proteins, TNF-α, active caspase-8, t-Bid, Bax, active caspase-9 and active caspase-3. Dilong could potentially serve as a cardio protective agent against LPS-induced H9c2 cardiomyoblast cell apoptosis.

The protective effect of hispidin against hydrogen peroxide-induced apoptosis in H9c2 cardiomyoblast cells through Akt/GSK-3β and ERK1/2 signaling pathway

Hispidin, a phenolic compound from Phellinus linteus (a medicinal mushroom), has been shown to possess strong anti-oxidant, anti-cancer, anti-diabetic, and anti-dementia properties. However, the cardioprotective efficacy of hispidin has not yet been investigated. In the present study, we investigated the protective effect of hispidin against oxidative stress-induced apoptosis in H9c2 cardiomyoblast cells and neonatal rat ventricular myocytes. While the treatment of H9c2 cardiomyoblast cells with hydrogen peroxide caused a loss of cell viability and an increase in the number of apoptotic cells, hispidin significantly protected the cells against hydrogen peroxide-induced cell death without any cytotoxicity as determined by XTT assay, LDH release assay, Hoechst 33342 assay, and Western blotting of apoptosis proteins such as caspase-3, Bax, and Bcl-2. Our data also shows that hispidin significantly scavenged intracellular ROS, and markedly enhanced the expression of antioxidant enzymes such as heme oxygenase-1 and catalase, which was accompanied by the concomitant activation of Akt/GSK-3β and ERK1/2 phosphorylation in H9c2 cardiomyoblast cells. The effects of hispidin on Akt and ERK phosphorylation were abrogated by LY294002 (a PI3K/Akt inhibitor) and U0126 (an ERK1/2 inhibitor). The effect of hispidin on GSK-3b activities was also blocked by LY294002. Furthermore, inhibiting the Akt/GSK-3β and ERK1/2 pathway by these inhibitors significantly reversed the hispidin-induced Bax and Bcl-2 expression, apoptosis induction, and ROS production. These findings indicate that hispidin protects against apoptosis in H9c2 cardiomyoblast cells exposed to hydrogen peroxide through reducing intracellular ROS production, regulating apoptosis-related proteins, and the activation of the Akt/GSK-3β and ERK1/2 signaling pathways.

Dilong prevents the high-KCl cardioplegic solution administration-induced apoptosis in H9c2 cardiomyoblast cells mediated by MEK.

Infusion of high-KCl cardioplegic solution (High-KCS) is the most common method used to induce asystole before cardiac surgery. However, our previous study showed the High-KCS can cause the apoptosis of cardiomyocytes in patients who were administered High-KCS prior to undergoing coronary artery bypass graft (CABG) to treat coronary artery disease (CAD). Therefore, it is urgent today to find a complementary medicine to reduce this damage. Dilong (earthworm) has been used as a traditional medicine in China for several thousand years, and extract from the dilong has been empirically used in Asia for the treatment of vascular disorders. In this study, we applied dilong extract to reduce myocardial cell damage from High-KCS infusion and further investigated the mechanisms. H9c2 cardiomyoblast cells were cultured in serum-free medium for 4 h and then treated with dilong at 31.25, 62.5, 125, and 250 mg/mL for 24 h, which was then followed by High-KCS treatment for 3 h to detect the protective mechanisms of dilong behind cardiomyocyte apoptosis and cardiac fibrosis. Cells were harvested for MTT assay, TUNEL assay, and western blot analysis. We found that High-KCS-induced cardiomyocyte apoptosis, enhanced the protein level of pro-apoptotic Bad, released cytochrome c, and activated caspase-3 in H9c2 cells. The IGF-I/IGF-IR/ERK pathway involved in non-cardiomyocyte proliferation, and the expression/activation of uPA, Sp-1 and CTGF, which are implicated in the development of cardiac fibrosis were up-regulated, but the Akt for cardiomyocyte survival was greatly deactivated in postcardioplegic H9c2 cardiomyoblast cells. However, dilong was highly protective and totally reversed the apoptosis and cardiac fibrosis effects induced by High-KCS. Chemical inhibitors P38 (SB203580), JNK (SP600125), MEK (U0126), IGF-1 (AG1024), and PI3K (LY294002) were applied to investigate which is the mediator for dilong attenuated High-KCS stimulated caspase 3 activation. MEK (U0126) inhibitor completely blocked dilong inhibited caspase 3 activation in High-KCS treated H9c2 cells. The MEK siRNA was further applied to knockdown MEK to confirm our finding. We found dilong worked through MEK to inhibit caspase 3 activity induced by High-KCS in H9c2 cells. Furthermore, we used the pure component of dilong, Lumbrokinase, to block the High-KCS effect. Using the microscope to observe the cell viability, we found Lumbrokinase could reverse the High-KCS effect. Lumbrokinase could also reduce the protein levels of caspase 8, caspase 9, and caspase 3, and enhance the survival related proteins PI3K/Akt and Bcl2. These results demonstrate that dilong could be used as a potential agent to block the side effects caused by High-KCS in CABG surgery patients.

SIRT4 Prevents Hypoxia-Induced Apoptosis in H9c2 Cardiomyoblast Cells

Aims: Apoptosis plays a critical role in cardiomyocyte loss during ischaemic heart injury. A detailed understanding of the mechanism involved has a substantial impact on the optimization and development of treatment strategies. Here, we report that the expression of SIRT4, a mitochondrial sirtuin, is markedly down-regulated in hypoxia-induced apoptosis of H9c2 cardiomyoblast cells. Methods and Results: SIRT4 interference significantly alters H9c2 cell viability, apoptotic cell number and caspase-3/7 activity. Furthermore, SIRT4 expression can affect the ratio of pro-caspase 9/caspase 9 or pro-caspase 3/caspase 3, an affect Bax translocation, which in turn alters the development of H9c2 cell apoptosis. Conclusion: These results suggest that SIRT4 is a key player in hypoxia-induced cardiomyocyte apoptosis, and that strategies based on its enhancement might be of benefit in the treatment of ischaemic heart disease.

Knockdown of dishevelled-1 attenuates cyclosporine A-induced apoptosis in H9c2 cardiomyoblast cells

Cyclosporine (CsA) has become a mainstay for immune suppression of organ transplants. It is known that patients receiving CsA manifest increased growth of aggressive cardiotoxicity. We have demonstrated that CsA induces myocardium cell apoptosis in vivo and vitro. Recently, dishevelled-1 (Dvl-1) protein, which is a cytoplasmic mediator of Wnt/β-catenin signaling, was explored in cardiac diseases. However, whether Dvl-1 is involved in CsA-induced apoptosis remains to be determined. The aim of this study was to explore the role of Dvl-1 in CsA-induced apoptosis in H9c2 cardiomyoblast cells and to investigate the role of the Wnt/β-catenin signaling cascade in this progress. H9c2 cells were treated with CsA in dose and time-dependent manners. We found that the appropriate concentrations and time-points of CsA-induced the expression of Dvl-1 and subsequent up-regulation of β-catenin and c-Myc, which is consistent with previously demonstrated concentrations and time-points when H9c2 cells apoptosis occurred. Then, cells were transfected with small interfering RNA (siRNA) against Dvl-1 and stimulated with previously demonstrated concentration of CsA. Dvl-1 down-regulation decreased the apoptotic rate, caspase-3 activity, and the Bax/Bcl-2 ratio in H9c2 cells treated with CsA. Furthermore, knocking down the expression of Dvl-1 partially suppressed the activity of the Wnt/β-catenin pathway. Moreover, we further deleted the downstream member β-catenin by specific siRNA, and found that CsA-induced the Bax/Bcl-2 ratio and the expression of c-Myc, which were attenuated. Our results are the first to unveil this novel aspect of Dvl-1 signaling. In addition, these data provide insight into the pathogenesis and the therapeutic strategies of CsA-induced myocardial injury.

Cyclosporin A induces apoptosis in H9c2 cardiomyoblast cells through calcium-sensing receptor-mediated activation of the ERK MAPK and p38 MAPK pathways

The cardiotoxicity of cyclosporine A (CsA) limits its clinical application in extensive and long-term therapies. Our group has shown that CsA induces myocardium cell apoptosis in vivo and increases calcium-sensing receptor (CaSR) expression. However, its molecular mechanism remains unknown. The purpose of this study was to determine whether CaSR plays an essential role in CsA-induced apoptosis in H9c2 cells and to investigate the role of the mitogen-activated protein kinase (MAPK) signaling cascade in this process. H9c2 cells were treated with CsA in a dose-dependent manner, and decreased Bcl-2 expression, increased Bax expression, and caspase-3 activation were observed. In a time-dependent manner, CsA increased CaSR expression, activated the extracellularly regulated kinase (ERK) and p38 MAPK pathways, and inactivated the c-Jun N-terminal kinase (JNK) MAPK signaling pathway. When H9c2 cardiomyoblast cells pretreated with gadolinium chloride (GdCl(3)), a CaSR activator, were treated with CsA, decreased phosphorylation of ERK1/2, increased phosphorylation of p38, decreased Bcl-2 expression, increased Bax expression, and activated caspase-3 were observed. Cells pretreated with the CaSR inhibitor NPS2390 inhibited this process. Furthermore, the MEK1/2 inhibitor U0126 and the p38 MAPK inhibitor SB203580 markedly blocked the effect of CsA on cell apoptosis, apoptotic-related protein expression, and caspase-3 activation. These findings showed that CsA induced apoptosis in H9c2 cells in vitro, and CaSR mediated the degradation of ERK MAPK and the upregulation of the p38 MAPK pathway involved in CsA-induced H9c2 cardiomyoblast cell apoptosis.

Carthamus tinctorius L. prevents LPS-induced TNFα signaling activation and cell apoptosis through JNK1/2–NFκB pathway inhibition in H9c2 cardiomyoblast cells

Aim of the study Severe and potentially fatal hypotension and cardiac contractile dysfunction are common symptoms in patients with sepsis. In our previous study, we found that estradiol and estrogen-receptor α have cardio-protective effects in myocardial cells exposed to LPS. Estradiol supplementation has been shown to induce breast and cervical cancers. Flos Carthami, the flower of Carthamus tinctorius L. (Compositae) is an important traditional Chinese medicine used for the treatment of heart disease and inflammation, and therefore might be a potential alternative to Estradiol in the prevention of heart damage. This study investigated the effect of Flos Carthami (FC EtOH) ethanolic extract on LPS-induced apoptosis in H9c2 cardiomyoblast cells. Materials and methods H9c2 cells induced apoptosis with LPS administration (1 μg/mL). H9c2 cells were divided into five groups: Control, LPS (1 μg/mL), and three FC EtOH (31.25, 62.5,and 125 μg/mL). We detected apoptosis using MTT, LDH, TUNEL assay. JC-1 staining and Western blot were used to detect pro-apoptosis proteins, anti-apoptosis proteins, MAPK proteins (JNK, ERK, and P38), and NFκB expression. Results FC EtOH (62.5 μg/mL) inhibited LPS-induced apoptosis by suppressing JNK1/2 activity, which resulted in the reduction of both IκB degradation and NFκB activation. In addition, FC EtOH led to the activation of anti-apoptotic proteins, Bcl-2 and Bcl-xL, the stabilization of the mitochondria membrane and the down-regulation of extrinsic and intrinsic pro-apoptotic proteins, such as TNFα, active caspase-8, t-Bid, Bax, active caspases-9, and -3. Conclusions Carthamus tinctorius L. possesses the ability to suppress JNK activity and inhibit LPS-induced TNFα activation and apoptosis in H9c2 cardiomyoblast cells. Carthamus tinctorius L could potentially serve as a cardio-protective agent against LPS-induced apoptosis.

Leu27IGF2 plays an opposite role to IGF1 to induce H9c2 cardiomyoblast cell apoptosis via Gαq signaling

This study examines the role of IGF2/mannose 6-phosphate receptor (IGF2R) signaling in the signaling transduction regulation and cell apoptosis in H9c2 cardiomyoblast cells. However, it is difficult to recognize the distinct activation of IGF2 signaling without interfacing with IGFI receptor (IGF1R) after exposure to IGF2. Leu27IGF2, an analog of IGF2 that interacts selectively with the IGF2R, was used to specifically activate IGF2R signaling in this study. DNA fragmentation and TUNEL assay revealed that in contrast to IGF1 treatment preventing angiotensin II and AG1024-induced cell apoptosis, Leu27IGF2 appears to synergistically increase apoptosis in those cells. We further found cell apoptosis induction and an increase in the active form of caspase 3 in the treatment of cells with Leu27IGF2, but not IGF1. To detect the interaction between IGF2R and Galphaq using the immunoprecipitation assay, we found that IGF2R could directly interact with Galphaq and after treatment with Leu27IGF2 the binding ability of Galphaq to IGF2R had increased. This sequentially resulted in the phosphorylation of phospholipase C-beta, a key downstream modulator of Galphaq, on serine 537. Moreover, disruption of the Galphaq protein by small interferon RNA reduced the cell apoptosis induced by Leu27IGF2. Our findings demonstrate that IGF2R activation appears to induce cell apoptosis via Galphaq-deriving signaling cascades and its effect is completely different from IGF1R survival signaling.

Enhancement of AG1024-Induced H9c2 Cardiomyoblast Cell Apoptosis via the Interaction of IGF2R with Gα Proteins and Its Downstream PKA and PLC-β Modulators by IGF-Ⅱ

Our previous studies found that insulin-like growth factor-I receptor (IGF1R) signaling blockade caused cardiac hypertrophy, and that apoptosis is required for upregulating the IGF-II and the IGF-II/ mannose 6-phosphate receptor (IGF2R) gene. However, the role of IGF-II in the regulation of cell apoptosis through IGF2R is little known. In this study, we hypothesized that IGF-II may induce cell apoptosis through IGF2R but is dependent on IGF1R activity. Western blots and TUNEL assay revealed that in the presence of IGF1R, exogenous IGF-II acts, like IGF-I, would increase phospho-Akt through IGF1R, but does not affect the caspase 3 activation and apoptotic induction in H9c2 cardiomyoblast cells. Conversely, AG1024, an inhibitor of IGF1R activity, causes cell apoptosis, and the treatment with IGF-II further enhances this process, implying that it occurs through IGF2R. Moreover, immunoprecipitation assay revealed that treatment with IGF-II could enhance the interaction of IGF2R with Galphai and Galphaq but reduce its binding with Galphas, resulting in the reduction of phospho-PKA and the activation of PLC-beta. Taken together, these data provide new insight into the dual role of IGF-II in the control of IGF1R dependent cell apoptosis and involved activation of IGF2R signaling. Improving IGF1R activity and suppressing IGF2R may be a good strategy to prevent the progression of heart disease with cardiomyocyte apoptosis.