Role of thioredoxin-ASK1 in doxorubicin-induced apoptosis of neonatal rat cardiac myocytes

AIM: To investigate the role of thioredoxin(Trx)-apoptosis signal-regulating kinase 1(ASK1) in doxorubicin-induced apoptosis of neonatal rat cardiac myocytes (NRCMs). METHODS: Primary cardiomyocytes were isolated from newborn Sprague-Dawley rats with the purity of NRCMs >95%. NRCMs were pretreated with the indicated concentrations of ebselen 2 h prior to the addition of doxorubicin, then treated with doxorubicin at concentration of 1 μmol/L for another 24 h. The viability of the cells was examined by MTT assay.Reactive oxygen species(ROS) levels were measured by a ROS-specific probe 2',7'-dichlorodihydrofluorescein diacetate (H₂DCFDA). Apoptotic cardiomyocytes were determined by Hoechst 33258 nuclear staining. The activity of caspase-3 was detected with a caspase-3 colorimetric assay kit. The protein levels of poly(ADP-ribose) polymerase 1(PARP1), ASK1, p-ASK1, p38 and p-p38 were determined by Western blotting. Immunoprecipitation and immunoblotting were performed to detect whether the Trx-ASK1 was dissociated. RESULTS: Doxorubicin induced significant apoptosis of NRCMs. The levels of ROS were significantly increased. Ebselen significantly decreased the apoptosis. Compared with control group, increased activity of caspase-3 was showed in doxorubicin group (P<0.01). Increased protein levels of PARP1, ASK1 and p38 were observed (P<0.01). The increase in the dissociated Trx-ASK1 was also found. Compared with doxorubicin group, ebselen decreased the activity of caspase-3 (P<0.01), the levels of PARP1,ASK1 and p38 proteins (P<0.05), and the dissociated Trx-ASK1. CONCLUSION: Doxorubicin induces significant apoptosis of NRCMs. ASK1 is partly dissociated from Trx, and starts the ASK1-mediated apoptotic signaling. The process is significantly attenuated by pretreatment with ebselen. Trx-ASK1 plays an important role in doxorubicin-induced apoptosis of cardiomyocytes.     

TPX2 induces apoptosis of rectal cancer HR-8348 cells through p38 MAPK signaling pathway

AIM: To study the effect of targeting protein for Xenopus kinesin-like protein 2 (TPX2) expression knockdown on the apoptosis of rectal cancer HR-8348 cells.METHODS: The HR-8348 cells transfected with TPX2 small interfering RNA (siRNA) served as TPX2 siRNA group. The non-transfected cells were used as control group. The cells transfected with siRNA negative control (siRNA-NC) were used as siRNA-NC group. The TPX2 siRNA-transfected cells exposed to p38 MAPK inhibitor SB203580 served as TPX2 siRNA+SB203580 group. The expression of TPX2 at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell viability was measured by MTT assay, the apoptosis was analyzed by flow cytometry. The protein levels of p38 MAPK, p-p38 MAPK, cleaved caspase-3 and Bcl-2 in the HR-8348 cells were determined by Western blot.RESULTS: After transfection, the expression of TPX2 at mRNA and protein levels was decreased in TPX2 siRNA-transfected cells (P<0.05). Transfection with siRNA-NC had no effect on TPX2 mRNA and protein levels in the cells. After knockdown of TPX2 expression, the viability of rectal cancer HR-8348 cells and the expression of Bcl-2 were decreased, while the apoptotic rate and the protein levels of cleaved caspase-3 and p-p38 MAPK/p38 MAPK were increased significantly reduced (P<0.05). Compared with TPX2 siRNA group, the apopto-tic rate and the protein levels of cleaved caspase-3 and p-p38 MAPK/p38 MAPK in TPX2 siRNA+SB203580 group were significantly decreased, while the viability was significantly increased (P<0.05).CONCLUSION: Knockdown of TPX2 expression promotes apoptosis of rectal cancer HR-8348 cells by activating p38 MAPK signaling pathway.     

Effect of p38 MAPK on cisplatin-induced renal tubular cell apoptosis

AIM:To investigate the role of p38 MAPK in cisplatin-induced rat renal proximal tubular cell (RPTC) apoptosis. METHODS:To determine the optimal concentration of cisplatin to induce RPTC apoptosis, the cells were treated with 0, 5, 10 and 20 μmol/L cisplatin for 24 h, and then the cell lysates were collected for Western blot analysis of cleaved PARP, p38 and phosphor ylated p38 (p-p38). To determine the role of p38 MAPK in cisplatin-induced RPTC apoptosis, the cells were divided into control group, cisplatin group (the cells were treated with cisplatin for 24 h) and cisplatin+p38 MAPK inhibitor group (the cells were treated with p38 MAPK inhibitor SB203580 for 1 h, and then treated with cisplatin for another 24 h). The morphological changes of apoptotic cells were observed under phase-contrast fluorescence microscope. The apoptotic rate of the cells were analyzed by flow cytometry. The caspase activity of RPTC lysates was examined using Ac-DEVD-AFC kit. The protein levels of p-p38, p38, cleaved PARP and cleaved caspase-3 were determined by Western blot. The pH value of extracellular environment of the cells was measured by pH meter. RESULTS:Cisplatin at 20 μmol/L obviously induced apoptosis of RPTC. The p38 MAPK was phosphorylated and its phosphorylation peaked at 15 min after cisplatin treatment. The apoptotic rate of RPTC was 12.08% after cisplatin induction. Cisplatin treatment also enhanced caspase activity, and increased cleavage of PARP and caspase-3 proteins (P<0.05). The p38 MAPK inhibitor SB203580 effectively inhibited the phosphorylation of p38 MAPK, down-regulated the RPTC apoptosis rate and caspase activity, and reduced the cleavage of PARP and caspase-3 proteins. The pH value change in RPTC culture medium was also inverted by SB203580. CONCLUSION:The phosphorylation of p38 MAPK is involved in cisplatin-induced apoptosis of RPTC. The apoptosis induced by cisplatin results in the change of acidic extracellular environment, which is inhibited by p38 MAPK inhibitor SB203580.     

p38 MAPK involves in cepharanthine-induced apoptosis in cardiomyocytes

AIM: To investigate the apoptotic effect of cepharanthine (CEP) on neonatal rat cardiomyocytes(NRCMs) and the underlying mechanisms. METHODS: MTT assay was used to detect the viability of the cells. CEP-induced apoptosis in NRCMs was evaluated by Hoechst 33342 staining and the expression of activated caspase-3. The phosphorylation levels of mitogen-activated protein kinases (MAPKs),such as extracellular signal-regulated kinase (ERK), c-jun N-terminal kinase (JNK) and p38 MAPK,were examined by Western blotting. The specific inhibitors of ERK and p38 MAPK were applied for identifying the roles of the corresponding signal pathways in CEP-induced apoptosis of cardiomyocytes. RESULTS: CEP inhibited the viability of NRCMs in a dose-and time-dependent manners. Positive nuclear fragmentation and activated caspase-3 were found in CEP-treated NRCMs. The phosphorylation levels of ERK and p38 MAPK were significantly elevated in CEP-treated NRCMs, but the change of JNK was not obvious. SB203580, an inhibitor of p38 MAPK, significantly alleviated the apoptotic effect induced by CEP. However, PD98059, an inhibitor of ERK1/2, did not significantly reduce the apoptotic effect.CONCLUSION: p38 MAPK is involved in CEP-induced apoptosis in NRCMs.     

Erianin induces apoptosis of human lung cancer A549 cells via ROS/p38 MAPK pathway

AIM:To investigate the effect of erianin on the viability and apoptosis of human lung cancer A549 cells and its possible mechanism. METHODS:A549 cells and BEAS-2B cells were cultured in vitro and treated with erianin at 10, 20, 40, 80 and 160 nmol/L for 48 h. The cell viability was measured by CCK-8 assay. The apoptosis and reactive oxygen species (ROS) were analyzed by flow cytometry. The activity of superoxide dismutase (SOD) was detected by WST-8 method, and the content of malondialdehyde (MDA) was detected by barbituric acid method. The protein levels of nuclear factor E2-related factor 2 (Nrf2), NAD(P)H:quinone oxidoreductase-1 (NQO1), heme oxygenase-1 (HO-1), p38 MAPK, p-p38 MAPK, caspase-3 and cleaved caspase-3 were determined by Western blot.RESULTS:Erianin remarkably reduced the viability of A549 cells in a dose-dependent manner (P<0.05) with IC₅₀ at 52.64 nmol/L. Erianin also induced apoptosis (P<0.05), increased ROS level and MDA content (P<0.05), diminished SOD activity (P<0.05), and down-regulated the protein levels of Nrf2, NQO1 and HO-1 (P<0.05), in a dose-dependent manner. Meanwhile, erianin up-regulated the levels of p-p38 MAPK and cleaved caspase-3 (P<0.05), and these effects were inhibited by N-acetyl-L-cysteine and SB203580 (P<0.05).CONCLUSION:Erianin may induce apoptosis of human lung cancer A549 cells most likely via inhibiting SOD activity and down-regulating the protein levels of Nrf2, NQO1 and HO-1, thus resulting in an increase in ROS and activation of p38 MAPK.     

Expression of p-p38 MAPK in partial cerebral tissues after hypoxic-ischemic brain damage in neonatal A2AR knockout mice

AIM： To study the expression of p-p38 MAPK in partial cerebral tissues after hypoxic-ischemic brain damage (HIBD) in the neonatal adenosine A2A receptor knockout (A2AR-/-) mice. METHODS：Base on the modified Rice method, the model of HIBD was established. The total 64 C57/BL6 neonatal mice (7 days old) of A2AR-/-(KO) and corresponding wild type (A2AR+/+, WT) were randomized into sham-operated group and model group. The mice in model group were divided into 3 subgroups： 1 d after HIBD, 3 d after HIBD and 7 d after HIBD (n=8 for each group). The cortex and hippocampal CA1 region were used as the study areas. The neuronal apoptosis was detected using TUNEL assay combined with Nissl staining. The expression of p-p38 MAPK and activated caspase-3 was determined by the method of immunohistochemistry. The KO mice and WT mice were also taken from sham-operated group (SKO and SWT, n=10) and model group (MKO and MWT, n=30) 1 d after HIBD to assess the early neurological behavior. RESULTS：The apoptotic neurons, activated caspase-3 and p-p38 MAPK increased after HIBD and peaked at 1 d after HIBD in the cortex and the hippocampal CA1 region. The apoptotic neurons and the expression of activated caspase-3 in KO mice were significantly higher than those in WT mice at the same time point after HIBD. The expression level of p-p38 MAPK in KO mice were significantly higher than that in WT mice at 1 d and 3 d after HIBD. The expression of activated caspase-3 was positively correlated with the expression of p-p38 MAPK in neonatal mice after HIBD (in the cortex：r=0.957, P<0.01; in the hippocampal CA1 region： r=0.939, P<0.01). CONCLUSION：p-p38 MAPK might be involved in the aggravated neuron apoptosis and brain damage induced by A2AR knockout after neonatal HIBO.     

Effect of PARP-1 on cisplatin resistance in breast cancer MCF-7 cells

AIM: To study the effect of poly(ADP-ribose) polymerase-1 (PARP-1) on cisplatin resistance of human breast cancer MCF-7 cells and its possible mechanisms.METHODS: The expression of PARP-1 at mRNA and protein levels in MCF-7 cells and MCF-7/DDP cells was determined by real-time PCR and Western blot. The expression of PARP-1 in the MCF-7/DDP cells was blocked by PARP-1 siRNA. The cell viability and apoptosis were detected by the CCK-8 assay and flow cytometry analysis, respectively. Furthermore, the protein levels of PARP-1, Bcl-2, Bax, cleaved caspase-3, caspase-3, cytochrome C (Cyto-C), extracellular signal-regulated kinase (ERK) and phosphorylated ERK (p-ERK) were detected by Western blot.RESULTS: The expression of PARP-1 at both mRNA and protein levels was significantly up-regulated in the MCF-7/DDP cells. The expression of PARP-1 was increased in the MCF-7 cells treated with cisplatin. Knockdown of PARP-1 induced the apoptosis of MCF-7/DDP cells with an increased sensitivity to cisplatin. Meanwhile, knockdown of PARP-1 down-regulated the protein levels of Bcl-2/Bax and p-ERK, but up-regulated the protein levels of cleaved caspase-3 and Cyto-C. After incubated with a specific ERK inhibitor U0126, the cell viability in PARP-1 siRNA group was down-regulated significantly.CONCLUSION: Knockdown of PARP-1 increases the sensitivity of MCF-7/DDP cells to cisplatin, and promotes the cell apoptosis via mitochondrial apoptosis pathway. The mechanism may be related to the attenuation of ERK signaling pathway by inhibiting phosphorylation of ERK.     

Dexmedetomidine reduced isoflurane-induced neuroapoptosis by inhibiting activation of p38 and JNK proteins in hippocampus of neonatal rats

AIM：To investigate the effect of dexmedetomidine (Dex) on neuronal apoptosis induced by isoflurane (Iso) and its relationship with the expression of p38 mitogen-activated protein kinase (p38) and c-Jun N-terminal kinase (JNK) proteins in the hippocampus of neonatal rats. METHODS：Forty-eight neonatal SD rats at postnatal day 7 were randomly divided into control group (Con), Dex group, Iso group and Iso combined with Dex (Iso+Dex) group. Rats in Iso and Iso+Dex groups were exposed to 0.75% Iso for 6 h, while rats in Con and Dex groups were exposed to air for 6 h. Rats were intraperitoneally injected with 25 μg·kg-1 Dex (Dex and Iso+Dex groups) or 150 μL saline (Con and Iso groups) 20 min before exposure and 2 and 4 h after exposure. After the termination of anesthesia, the neuronal apoptosis in hippocampal CA1 region was detected by TUNEL staining, and the protein expression of cleaved caspase-3, phospho-p38 (p-p38), p38, phospho-JNK (p-JNK) and JNK in hippocampal tissues was detected by Western blotting. RESULTS：The number of TUNEL positive cells in hippocampal CA1 region of the rats in Iso group was increased by 447.57% (P<0.01) compared with Con group, while Dex significantly inhibited the increased TUNEL positive cells in Iso group by 75.18% (P<0.01). The expression of cleaved caspase-3 protein in Iso group was increased by 126.29% (P<0.01) compared with Con group, while Dex reversed the increased cleaved caspase-3 protein expression (P<0.01). Iso significantly increased the phosphorylation of p38 and JNK proteins (P<0.01), while Dex reversed the increased p-p38 and p-JNK proteins (P<0.01). CONCLUSION：Dex attenuates Iso-induced neuroapoptosis in the hippocampus of neonatal rats through inhibiting the phosphorylation of p38 and JNK proteins.     

p38MAPK expression and apoptosis in the rat unilateral ureteral obstruction model

AIM: To investigate the expression of p38 mitogen-activated protein kinase (p38MAPK) in the kidney after unilateral ureteral obstruction (UUO) in rats and the functional role of it on apoptosis and fibrosis.METHODS: Eighteen Wistar rats underwent UUO were killed at 3, 7, 14 days. Additional 7 rats were sham operated. Histological changes were observed by HE and Masson staining. Immunohistochemistry study was performed on renal tissue for proliferating cell nuclear antigen (PCNA). Apoptotic cells were determined by terminal deoxynucleotidyl transferase- mediated dUTP nick end labeling (TUNEL) and the electrophoresis analysis of genomic DNA. Western blotting of cysteinyl aspartate specific proteinase-3 (caspase-3), p38MAPK and p-p38MAPK were measured.RESULTS: UUO induced a significant increase in renal tubular and interstitial cell apoptosis, immunohistochemistry of PCNA and Western blotting of caspase-3, p-p38MAPK as well as severe morphology changes. However, there was no significant difference between UUO and the control in Western blotting of p38MAPK.CONCLUSION: An in vivo model of renal fibrosis after UUO demonstrates that activated or phosphorylated p38MAPK plays a role in apoptosis of renal tubulointerstitial cells.     

Effect of bim silencing by siRNA on hypoxia-induced apoptosis of rat cardiomyocytes

AIM：To investigate the effect of BH3-only protein Bim (Bcl-2 interacting mediator of cell death) on apoptosis of rat cardiomyocytes induced by hypoxia. METHODS：Rat cardiomyocytes were isolated from infant rats aged 1~3 days and then primarily cultured. The antibody targeting α-actin of striated muscle was used to identify the cardiomyocytes. The siRNAs of bim were transfected into the cardiomyocytes with liposome, and the expression of Bim was determined by Western blotting. The cardiomyocytes were divided into blank control group, hypoxia group, hypoxia+liposome group, hypoxia+negative control siRNA group and hypoxia+bim-siRNA group.The frequency and rhythm of cardiomyocyte beating were observed and recorded under inverted microscope. The activity of lactate dehydrogenase (LDH) in the culture medium was assessed by automatic biochemical analyzer. The viability of the cells was analyzed by MTT assay. The cell apoptotic rate was measured by flow cytometry. The protein expression of Bim, Bax, Bcl-2, p-p38 MAPK and p38 MAPK was detected by Western blotting. RESULTS：Immunohistochemical identification confirmed that the rat cardiomyocytes were successfully cultured. The expression of Bim was obviously inhibited after transfected with bim-siRNAs and the silencing efficiency of bim-siRNA-2 was the highest (86.73%). The frequency of cardiomyocyte beating was slowed down after hypoxia and the rhythm was disordered, while the frequency of beating was obviously increased after silencting the expression of bim. Compared with control group, the LDH in the culture medium was increased (P<0.01), and the viability of the cardiomyocytes was reduced in hypoxia group (P<0.05). The apoptotic rate was increased (P<0.01). After transfection with bim-siRNA, the release of LDH was decreased, and the viability of the cardiomyocytes was increased. The apoptotic rate was decreased. The results of Western blotting showed that hypoxia increased the expression of Bax and p-p38 MAPK (P<0.05), and decreased the expression of Bcl-2 (P<0.01), while transfection with bim-siRNA reduced the effects caused by hypoxia (P<005). These were greatly related to the decrease of apoptosis. However, the expression of p38 MAPK was not changed. CONCLUSION：The apoptosis of cardiomyocytes induced by hypoxia can be inhibited by silencing the expression of bim gene by down-regulation of p-p38 MAPK and Bax expression and up-regulation of Bcl-2 expression.     

Role of p38 MAPK signaling pathway in inflammatory effects on cerulein-induced pancreatic cells

AIM: To investigate the effect of p38 MAPK signal pathway on cerulein-treated pancreatic acinar AR42J cells.METHODS: AR42J cells were divided into control group, cerulein group (treated with 10^-8 mol/L of cerulein), and SB203580 group (treated with 10 μmol/L of SB203580 and 10^-8mol/L of cerulein).The cells were harvested 3 h after treatment.Secretion rate of amylase was measured.The translocation of p-p38 MAPK to nuclei was imaged by immunofluorescence.The protein expression levels of p-p38 MAPK and TNF-α were detected by Western blotting.The activation of NF-κB was measured by electrophoretic mobility assay.RESULTS: Compared with control group, cerulein resulted in increases in the secretion rate of amylase and protein level of TNF-α (P<0.01), as well as the expression levels of p-p38 MAPK and NF-κB (P<0.01).Cerulein induced nuclear translocation of p-p38 MAPK.Compared with cerulein group, the secretion rate of amylase and protein level of TNF-α in SB203580 group decreased significantly (P<0.01).The expression of p-p38 MAPK and NF-κB also decreased greatly (P<0.05).Nuclear translocation of p-p38 MAPK was inhibited by SB203580.CONCLUSION: The p38 MAPK pathway involves in cerulein-induced pancreatic inflammatory response via regulating NF-κB.     

Berberine promotes anti-proliferative effects of doxorubicin in T24 bladder cancer cells by enhancing apoptosis

AIM: To observe the effect of berberine (Ber) on doxorubicin (DOX)-induced apoptosis in bladder cancer T24 cells. METHODS: The cells were exposed to DOX in the presence or absence of different concentrations of Ber. The viability of the cells was determined by CCK-8 assay. The apoptosis was measured by Hoechst 33258 staining and the protein levels of cleaved caspase-3, cleaved caspase-9, Bcl-2 and Bax were detected by Western blotting.RESULTS: Ber enhanced the inhibitory effect of DOX on the viability of T24 cells and promoted DOX-induced apoptosis in T24 cells. DOX increased the protein levels of cleaved caspase-3, cleaved caspase-9 and Bax, all of which were enhanced by treatment with Ber. In contrast, Ber exposure further decreased the expression of Bcl-2 in DOX-treated T24 cells.CONCLUSION: Ber enhances the anti-proliferative effects of DOX through promoting apoptosis in bladder cancer cells.     

Effect of Panax notoginoside on pulmonary arterial pressure and expression of p38 MAPK in lung tissues of hypoxic rats

AIM: To observe the effect of Panax notoginoside (PNS) on the pulmonary artery pressure and the p38 mitogen-activated protein kinase(p38 MAPK) in lung tissues of rats treated with hypoxia. METHODS: Thirty adult male SD rats were randomly divided into 3 groups. The rats in normal control group were exposed to normal conditions, the rats in hypoxia group were exposed to isobaric hypoxia, and the rats in hypoxia+PNS group were treated with PNS under the condition of hypoxia. After 4 weeks of treatment, the mean pulmonary arterial pressure (mPAP) and the mean carotid arterial pressure (mCAP) were measured by cardiac catheterization. The heart was isolated, and the right ventricle (RV), left ventricle plus ventricular septum (LV+S) were weighed to calculate the ratio of RV/(LV+S).The quantity of phospho-p38 MAPK(p-p38 MAPK) in rat pulmonary arterioles was determined by the method of immunohistochemistry and the mRNA content of p38 MAPK was tested by RT-PCR. RESULTS: The mPAP and RV/(LV+S) in hypoxia group were higher than those in normal control group. The expression of p-p38 MAPK in rat pulmonary arterioles and p38 MAPK mRNA in the lung tissues were higher than those in normal control group (P<0.05). The mPAP, RV/(LV+S), the expression of p-p38 MAPK in rat pulmonary arterioles and p38 MAPK mRNA in the lung tissues in hypoxia+PNS group were significantly lower than those in hypoxia group (P<0.05).CONCLUSION: PNS possesses the preventive and therapeutic effect on hypoxic pulmonary hypertension by decreasing p-p38 MAPK and down-regulation of p38 MAPK mRNA in the lungs.     

Metoprolol inhibits norepinephrine-induced rat cardiomyocyte apoptosis and connexin 43 phosphorylation

AIM: To study the effects of metoprolol (Meto) on the apoptosis of neonatal rat cardiomyocytes and the phosphorylation of connexin 43 (Cx43) induced by norepinephrine (NE). METHODS: Neonatal SD rat cardiomyocytes were divided into the following five groups (n=6 in each group): (1) control (Con) group: no treatment; (2) NE group: treatment with NE at 0.1 μmol/L for 24 h; (3) NE+Meto group: simultaneous treatment with NE and Meto both at 01 μmol/L for 24 h; (4) NE+Meto+PD98059 group: pretreatment with extracellular signal-regulated kinase (ERK) phosphorylation inhibitor PD98059 at 10 μmol/L for 30 min and then treatment with NE and Meto both at 01 μmol/L for 24 h; (5) NE+PD98059 group: pretreatment with PD98059 at 10 μmol/L for 30 min and then treatment with NE at 01 μmol/L for 24 h. The beating rates of cardiomyocytes in various groups were calculated, and the viability of cardiomyocytes was assayed by MTT method. The Cx43 mRNA expression was detected by RT-PCR, and the protein expression of phosphorylated Cx43 (p-Cx43), phosphorylated ERK1/2 (p-ERK1/2) and cleaved caspase-3 was detected by Western blotting. RESULTS: (1) Separate NE treatment could significantly increased the beating rate of cardiomyocytes and reduced cell viability, while Meto showed the opposite effects. PD98059 treatment had no significant effect on cardiomyocyte beating rate, but suppressed Meto to improve cell viability to some extent. (2) Compared with Con group, separate NE treatment significantly increased the Cx43 mRNA expression (P<001). Compared with NE group, Meto or PD98059 intervention could significantly inhibited Cx43 mRNA expression (both P<001), and simultaneous treatment with Meto and PD98059 could further suppress Cx43 mRNA expression up-regulated by NE (P<001). (3) Compared with NE group, Meto significantly inhibited the increased p-Cx43, p-ERK1/2 and cleaved caspase-3 expression induced by NE (P<001), and simultaneous treatment with Meto and PD98059 could further enhance the inhibition of p-Cx43, p-ERK1/2 and cleaved caspase-3 expression by Meto (P<001). PD98059 treatment had no significant effect on the increased p-Cx43 and cleaved caspase-3 expression induced by NE (P>005). CONCLUSION: The inhibitory effect of Meto on NE-induced cardiomyocyte apoptosis is related to the inhibition of Cx43 phosphorylation, which may be partly mediated via ERK1/2 pathway.     

Effects of PDGFRα on melanocyte apoptosis induced by hydrogen peroxide

AIM: To investigate the effects of platelet-derived growth factor receptor α (PDGFRα) on melanocyte apoptosis induced by hydrogen peroxide (H₂O₂). METHODS: Melanocyte PIGI was used as the research object. After exposed to H₂O₂ at different concentrations, the cell viability was detected by MTT assay. The PIGI cells were transfec-ted with empty vector pCMV6 or PDGFRα over-expression vector pCMV6-PDGFRα. The transfection efficiency was determined by RT-qPCR and Western blot. The effect of H₂O₂ on the viability of the PIGI cells after over-expression of PDGFRα was measured by MTT assay. The cell apoptosis was analyzed by flow cytometry. The protein levels of p38, p-p38 and cleaved caspase-3 in the cells were detected by Western blot. DCDHF-DA was used to estemate the generation of reactive oxygen species (ROS) in the cells. RESULTS: The viability of PIGI cells decreased after exposed to H₂O₂ (P<0.05), and the half maximal inhibitory concentration of H₂O₂ was 0.7 mmol/L. Transfection with PDGFRα over-expression vector successfully induced high expression of PDGFRα at mRNA and protein levels in the PIGI cells, and increased the viability of the cells with H₂O₂ treatment (P<0.05). Over-expression of PDGFRα decreased the apoptotic rate of PIGI cells treated with H₂O₂ (P<0.05), and the level of ROS in the cells (P<0.05). The protein levels of cleaved caspase-3 and p-p38 were also decreased (P<0.05). CONCLUSION: PDGFRα inhibits the apoptosis of melanocytes induced by H₂O₂, partially reverses the growth inhibition of melanocytes by H₂O₂, and decreases the ROS level. The mechanism may be related to regulating the protein levels of p-p38 and cleaved caspase-3 in the cells.     

Insulin-like growth factor I protects neonatal rat cardiomyocytes from apoptosis through improvement of mitochondrial function

AIM：To investigate whether mitochondrial mechanism is involved in the anti-apoptotic effect of insulin-like growth factor I (IGF-I) on cardiomyocytes. METHODS：Primary neonatal rat cardiomyocytes (NRCMs) were cultured and treated with 200 μmol/L hydrogen peroxide (H2O2) to induce apoptosis. Kruppel-like factor 9 (KLF9)-specific siRNA was transfected into the cells by Lipofectamine 2000. The mitochondrial function was measured by JC-1 mitochondrial membrane potential (MMP) assay. The mitochondrial morphology was observed by transmission electron microscopy. Myocardial cell apoptosis was detected by Annexin V-FITC/PI dual staining, caspase-3 activity assay, DNA-ladder analysis and Hoechst 33258 staining. RESULTS：The apoptosis of NRCMs was induced by H2O2, with MMP decreased by (24.0±1.6)% compared with control group. The fall rates of MMP in IGF-I group and KLF9 siRNA group were (18.3±1.2)% and (15.2±1.2)%, respectively (both P<0.01 vs H2O2 group), and improved mitochondrial morphology, decreased caspase-3 activity, attenuated DNA fragmentation and reduced apoptotic bodies were also observed in these two groups. The apoptotic rates of NRCMs in IGF-I group and KLF9 siRNA group were (22.4±4.2)% and (32.5±3.5)%, respectively, both lower than that in H2O2 group ［(42.5±1.8)%, P<0.01］. The anti-apoptotic effect of KLF9 silencing on NRCMs was consistent with that of IGF-I treatment. CONCLUSION：IGF-I protects NRCMs from apoptosis through down-regulating KLF9 expression and improving mitochondrial function.     

Notch-1 gene silencing promotes phosphorylations of JNK1 and p53 in human breast cancer MCF-7 cells

AIM: To investigate the effect of Notch1 gene silencing on phosphorylations of JNK1 and p53 in human breast cancer MCF-7 cells.METHODS: shRNA-Notch1 eukaryotic expression plasmid was constructed and transfected into MCF-7 cells. The expression of Notch1 and Hes-1 was observed by Western blotting after transfction. Apoptosis and mitochondrial membrane potential were detected by flow cytometry. Western blotting was also used to determine the protein levels of p-JNK1, p-p53, PUMA, NOXA and cleaved caspase-3 after Notch1 silencing was performed in MCF-7 cells.RESULTS: Silencing of Notch1 significantly reduced the expression of Notch1 and Hes-1 in MCF-7 cells (P<0.01). In shNotch1 group, the number of apoptotic cells was much higher (P<0.01) and mitochondrial membrane potential was much lower (P<0.05) than those in shControl group. The protein levels of p-JNK1, p-p53, PUMA, NOXA and cleaved caspase-3 increased obviously after silencing of Notch1 was performed in MCF-7 cells (P<0.05).CONCLUSION: Notch1 silencing induces apoptosis of human breast cancer MCF-7 cells through promoting phosphorylations of JNK1 and p53, and increasing the production of PUMA, NOXA and cleaved caspase-3.     

Celastrol induces apoptosis of multiple myeloma H929 cells

AIM: To investigate the effect of celastrol on the apoptosis of human multiple myeloma H929 cells and its molecular mechanism. METHODS: The H929 cells were cultured in vitro and treated with celastrol at different concentrations (0.5, 1, 5 and 10 mg/L). The viability of H929 cells was analyzed by CCK8 assay. Annexin V-PE/7-AAD staining was used to analyzed the effect of celastrol on apoptosis of H929 cells, and mitochondrial membrane potential was observed by flow cytometry. The effect of celastrol on DNA damage was detected by comet assay. The protein levels of apoptosis-related molecules P53, XIAP, cleaved PARP-1 and cleaved caspase-3, and the release of mitochondrial cytochrome C in the H929 cells treated with celastrol were determined by Western blot. RESULTS: The viability of H929 cells was significantly inhibited by different concentrations of celastrol in a concentration-dependent and time-dependent manner. Apoptosis and decreased mitochondrial membrane potential of H929 cells in a concentration-dependent manner were observed after treatment with celastrol (P<0.05). The results of comet assay showed that celastrol induced DNA damage in the H929 cells. The protein levels of apoptotic molecules P53, cleaved PARP-1 and cleaved caspase-3 were significantly increased and the expression level of anti-apoptotic protein XIAP was significantly decreased in the H929 cells treated with celastrol (P<0.05). Celastrol promoted the release of cytochrome C in mitochondria, and activated caspase-3 in dependence on caspase-9. CONCLUSION: Celastrol has an apoptosis-inducing effect on multiple myeloma H929 cells. Its mechamism may be related to activation of mitochondrial apoptosis pathway by inducing DNA damage.     

Osthole enhances doxorubicin-induced apoptosis in p53-wildtype prostate cancer cells by down-regulating SIRT1 expression

AIM: To investigate the effect and mechanism of osthole on increasing the cytotoxicity of doxorubicin (DOX) to prostate cancer cells. METHODS: MTT assay was performed to evaluate the viability of LNCaP cells treated with osthole and DOX. The protein expression of silent information regulator 1 (SIRT1), p53, acetylated p53 and Puma, as well as release of cytochrome C and activation of caspase-9 and caspase-3 in the LNCaP cells treated with osthole and DOX were determined by Western blot. The apoptosis of the LNCaP cells treated with osthole and DOX was analyzed by flow cytometry. RESULTS: Osthole significantly increased the cytotoxicity of DOX against p53-wildtype prostate cancer cell line LNCaP. Osthole significantly inhibited the expression of SIRT1 in the LNCaP cells. Transfection with SIRT1 plasmid decreased the cytotoxicity of osthole and DOX co-treatment against LNCaP cells. Combination with osthole and DOX significantly induced the over-expression and acetylation of p53. Transfection with p53 siRNA significantly decreased the synergistic effect of osthole on cytotoxicity of DOX-treated LNCaP cells. Combination with osthole and DOX significantly induced the release of cytochrome C into the cytoplasm from mitochondria, followed by activation of caspase-9 and its downstream molecule caspase-3, thus leading to cell apoptosis in the LNCaP cells. CONCLUSION: Osthole promotes the p53-dependent apoptosis in DOX-treated prostate cancer LNCaP cells by down-regulating the expression of SIRT1.