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Atrazine (ATR) is one of the most commonly used herbicides worldwide. As an endocrine disruptor, it causes ovarian dysfunction, but the mechanism is unclear. We hypothesized that ATR could affect ovarian steroidogenesis, oxidative stress, inflammation, and apoptosis. In the current study, rats aged 28 days were treated with PMSG and HCG to obtain amounts of corpora lutea. Then, rats were injected with ATR (50 mg/kg/day) or saline (0.9%) for 7 days. Sera were collected to detect biochemical indices and progesterone (P4) level, ovaries were collected for antioxidant status, HE, qPCR, and WB analysis. Results showed that ATR exposure affected growth performance as well as serum TP, GLB, and ALB levels, increased serum P4 level and ovarian mRNA and protein levels of StAR, CYP11A1, and HSD3B. ATR treatment increased ovarian mRNA and protein levels of CREB but not PKA expression. ATR treatment increased ovarian mRNA abundances of Nrf-2 and Nqo1, MDA level, and decreased SOD, GST, and T-AOC levels. ATR exposure increased the mRNA abundances of pro-inflammatory cytokines including Tnf-α, Il-1β, Il-6, Il-18, and Inos. ATR exposure increased the mRNA and protein level of Caspase 3 and the ratio of BAX/BCL-2. In conclusion, NRF-2/NQO1 signaling pathway and CREB might be involved in the regulation of ATR in luteal steroidogenesis, oxidative stress, inflammation, and apoptosis in rat ovary., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Chronic obstructive pulmonary disease (COPD) is the third leading cause of death worldwide. N-acetylcysteine (NAC) is well known for its antioxidant properties, along with potential protective effects on COPD. However, the molecular mechanism of NAC against the apoptosis of alveolar epithelial cells (AECs) in COPD remains unclear., Objective: This study aimed to explore the anti-apoptosis effect of NAC in COPD mice and alveolar epithelial cells., Methods: In the present study, the mouse model of COPD was established by cigarette smoke (CS), and mouse alveolar epithelial (MLE-12) cells were treated with cigarette smoke extract (CSE). TdT-mediated dUTP nick-end labeling (TUNEL) assay, reverse transcription polymerase chain reaction (RT-PCR), and western blot were performed to evaluate the effects of NAC on apoptosis, endoplasmic reticulum (ER) stress, and mitochondrial dysfunction. Meanwhile, Lbuthionine- sulfoximine (BSO), a glutathione (GSH) inhibitor, was used to uncover the mechanism of COPD treatment by NAC., Results: We found that NAC pretreatment could attenuate the protein levels of apoptosis, ER stress, and mitochondrial dysfunction-related genes caused by CS in vivo. Meanwhile, CSE could decrease MLE-12 cell viability, which was prevented by apoptosis inhibitor ZVAD-FMK but not necroptosis inhibitor necrostatin-1. Pretreatment of MLE-12 cells with NAC increased cellular GSH levels, inhibited cellular and mitochondrial reactive oxygen species (ROS) accumulation, and decreased protein level of apoptosis, ER stress, and mitochondrial dysfunction-related genes. Moreover, experiment results showed that BSO could completely reverse the beneficial effects of NAC., Conclusion: Our study confirmed that NAC can attenuate CS-induced AEC apoptosis via alleviating ROS-mediated ER stress and mitochondrial dysfunction pathway, and the mechanism was found to be related to replenishing the cellular GSH content., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Sepsis‑induced myocardial dysfunction is one of the features of multiple organ dysfunction in sepsis, which is associated with extremely high mortality and is characterized by impaired myocardial compliance. To date, there are few effective treatment options available to cure sepsis. Tannic acid (TA) is reportedly protective during sepsis; however, the underlying mechanisms by which TA protects against septic heart injury remain elusive. The present study investigated the potential effects and underlying mechanisms of TA in alleviating lipopolysaccharide (LPS)‑induced H9C2 cardiomyocyte cell apoptosis. H9C2 cells were treated with LPS (15  µ g/ml), TA (10  µ M) and TA + LPS; control cells were treated with medium only. Apoptosis was measured using flow cytometry, reverse transcription‑quantitative PCR (RT‑qPCR) and western blot analysis. Additionally, the levels of cellular reactive oxygen species (ROS), malondialdehyde and nicotinamide adenine dinucleotide phosphate were evaluated. Western blotting and RT‑qPCR were also employed to detect the expression levels of endoplasmic reticulum (ER) stress‑associated functional proteins. The present findings demonstrated that TA reduced the degree of LPS‑induced H9C2 cell injury, including inhibition of ROS production and ER stress (ERS)‑associated apoptosis. ERS‑associated functional proteins, including activating transcription factor 6, protein kinase‑like ER kinase, inositol‑requiring enzyme 1, spliced X box‑binding protein 1 and C/EBP‑homologous protein were suppressed in response to TA treatment. Furthermore, the expression levels of ERS‑associated apoptotic proteins, including c‑Jun N‑terminal kinase, Bax, cytochrome c, caspase‑3, caspase‑12 and caspase‑9 were reduced following treatment with TA. Additionally, the protective effects of TA on LPS‑induced H9C2 cells were partially inhibited following treatment with the ROS inhibitor N‑acetylcysteine, which demonstrated that ROS mediated ERS‑associated apoptosis and TA was able to decrease ROS‑mediated ERS‑associated apoptosis. Collectively, the present findings demonstrated that the protective effects of TA against LPS‑induced H9C2 cell apoptosis may be associated with the amelioration of ROS‑mediated ERS. These findings may assist the development of potential novel therapeutic methods to inhibit the progression of myocardial cell injury.

Schistosomiasis is a parasitic helminth disease that can cause organ lesions leading to health damage. During a schistosome infection, schistosome eggs can flow into the liver along the portal vein. Numerous inflammatory cells gather around the eggs, causing granulomas and fibrosis in the liver. In this process, many molecules are involved in the initiation and regulation of the fibrous scar formation. However, the precise molecular mechanisms responsible for the progression of granuloma formation and fibrosis initiation caused by schistosome infection have not been extensively studied. In this study, C57BL/6 wild-type mice and Stat3 flox/flox Alb-Cre mice were infected with cercariae of Schistosoma japonicum Liver injury, effector molecule levels, and RNA transcriptome resequencing of liver tissue were detected at 4, 5, and 6 weeks postinfection. We investigated the role of STAT3 (signal transducer and activator of transcription 3) in Schistosoma -induced liver injury in mice. After 6 weeks postinfection, there was obvious liver fibrosis. A sustained pathological process (inflammation, oxidative stress, proliferation, and apoptosis) occurred in S. japonicum -induced liver fibrosis initiation. Meanwhile, we observed activation of the STAT3 pathway in hepatic injury during S. japonicum infection by RNA transcriptome resequencing. Liver deficiency of phospho-STAT3 alleviated infection-induced liver dysfunction, hepatic granuloma formation, and fibrosis initiation. It also promoted STAT3-dependent apoptosis and reduced liver inflammation, oxidative stress, and proliferation. Our results suggest that STAT3 signal pathway and its mediating inflammation, oxidative stress, proliferation, and apoptosis are involved in S. japonicum -induced liver injury and may be a new potential guideline for the treatment of schistosomiasis., (Copyright © 2021 American Society for Microbiology.)

Triptolide, a major active component of Triptergium wilfordii Hook. f, is used in the treatment of autoimmune disease. However, triptolide is associated with severe adverse reactions, especially hepatotoxicity, which limits its clinical application. To examine the underlying mechanism of triptolide-induced liver injury, a combination of dose- and time-dependent toxic effects, RNA-seq and metabolomics were employed. Triptolide-induced toxicity occurred in a dose- and time-dependent manners and was characterized by apoptosis and not necroptosis. Transcriptomics profiles of the dose-dependent response to triptolide suggested that PI3K/AKT, MAPK, TNFα and p53 signaling pathways were the vital steps in triptolide-induced hepatocyte apoptosis. Metabolomics further revealed that glycerophospholipid, fatty acid, leukotriene, purine and pyrimidine metabolism were the major metabolic alterations after triptolide exposure. Finally, acylcarnitines were identified as potential biomarkers for the early detection of triptolide-induced liver injury., Competing Interests: Declaration of Competing Interest The authors report no declarations of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Emerging evidence suggests that both apoptosis and autophagy contribute to global cerebral ischemia‑reperfusion (GCIR)‑induced neuronal death, which results from cardiac arrest (CA). However, the mechanism of how GCIR may affect the balance between apoptosis and autophagy resulting from CA remains to be elucidated. Additionally, the role of adiponectin (APN) in reversing the apoptosis and autophagy induced by GCIR following cardiac arrest‑cardiopulmonary resuscitation (CA‑CPR) is unclear. Thus, the aim of the present study was to investigate how GCIR affect the apoptosis and autophagy in response to CA and to clarify whether APN may alter the apoptosis and autophagy of neuronal death in GCIR‑injured brain post‑CA‑CPR. Using normal controls (Sham group) and two experimental groups [CA‑CPR‑induced GCIR injury (PCAS) group and exogenous treatment with adiponectin post‑CA‑CPR (APN group)], it was demonstrated that both apoptosis and autophagy were observed simultaneously in the brain subjected to GCIR, but apoptosis appeared to be more apparent. Exogenous administration of APN significantly reduced the formation of malondialdehyde, a marker of oxidative stress and increased the expression of superoxide dismutase, an anti‑oxidative enzyme, resulting in the stimulation of autophagy, inhibition of apoptosis and reduced brain tissue injury (P<0.05 vs. PCAS). APN treatment increased the expression of APN receptor 1 (AdipR1) and the phosphorylation of AMP‑activated protein kinase (AMPK; Ser182) in brain tissues. In conclusion, GCIR induced apoptosis and inhibited autophagy, contributing to brain injury in CA‑CPR. By contrast, APN reduced the brain injury by reversing the changes of neuronal autophagy and apoptosis induced by GCIR. The possible mechanism might owe to its effects on the activation of AMPK after combining with AdipR1 on neurons, which suggests a novel intervention against GCIR injury in CA‑CPR conditions.

The complement 1q binding protein C ( C1QBP ), also known as p32 , is highly expressed in rapidly growing tissues and plays a crucial role in cell proliferation and apoptosis. However, there are no data interpreting its mechanisms in muscle development. To investigate the role of p32 in sheep muscle development, an 856 bp cDNA fragment of p32 containing an 837 bp coding sequence that encodes 278 amino acids was analyzed. We then revealed that the expression of p32 in the longissimus and quadricep muscles of fetal sheep was more significantly up-regulated than expression at other developmental stages. Furthermore, we found that the expression of p32 was increased during myoblasts differentiation in vitro. Additionally, the knockdown of p32 in sheep myoblasts effectively inhibited myoblast differentiation, proliferation, and promoted cell apoptosis in vitro. The interference of p32 also changed the energy metabolism from Oxidative Phosphorylation (OXPHOS) to glycolysis and activated AMP-activated protein kinase ( AMPK ) phosphorylation in sheep myoblasts in vitro. Taken together, our data suggest that p32 plays a vital role in the development of sheep muscle and provides a potential direction for future research on muscle development and some muscle diseases.

Sertoli cells (SCs) are necessary for proper germ cell development and viability. Unc-51 like autophagy activating kinase (ULK1) protein kinase is an important regulator of autophagy activation. This study aims to investigate the role of autophagy promoter ULK1 on cell viability of goat SCs. Our results showed that ULK1 knockdown in goat SCs decreased autophagy activation, which was confirmed by decreased expression of autophagy-related markers including LC3, Beclin1, Atg5, and Atg7 (P < 0.05). Meanwhile, lower ULK1 levels resulted in decreased expressions of goat SC marker genes ABP, AMH, FASL, and GATA4. However, a reverse trend of these parameters occurred when the goat SCs were transfected with ULK1 overexpression construct; higher ULK1 levels in goat SCs also decreased the ratio of Bax/Bcl-2. Moreover, ULK1 overexpression in goat SCs activated the autophagy levels when cells were exposed to an environmental contaminant bisphenol A (BPA). The above results indicated that ULK1 gene might play important roles in goat SC function by regulating cell viability.

Cisplatin remains the standard first-line chemotherapeutic agent in the treatment of many types of cancers, but its clinical application is hindered by its severe nephrotoxicity. Previous studies reported that scutellarin enhanced the anti-cancer activity of cisplatin in lung cancer cells, with no confirmation on cisplatin-induced renal damage. Here, we investigated the nephroprotective effect of scutellarin on cisplatin-induced renal injury and its underlying mechanisms. Renal function, histological change, inflammation, apoptosis, autophagy and involved pathways were investigated. Pretreatment with scutellarin prevented cisplatin-induced decline of renal function including BUN, CRE, and histological damage. Scutellarin also reduced renal inflammation by suppressing the levels of pro-inflammatory cytokine, TNF-α and IL-6. Similarly, scutellarin administration inhibited apoptosis triggered by cisplatin through reducing the expressions of Cleaved caspase-3, Cleaved PARP, p53, and the ratio of Bax/Bcl-2. Moreover, scutellarin prevented cisplatin-induced inhibition of autophagy via enhancing LC3-II/LC3-I and Atg7, and inhibition of p62. Of note, the activations of JNK, ERK, p38 and stat3 induced by cisplatin were strikingly attenuated in scutellarin-treated mice. Thus, these results provide compelling evidence that scutellarin is a novel nephroprotectant against cisplatin-induced renal toxicity., (Copyright © 2019 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Oxidative stress induced disc cell apoptosis plays an important role in intervertebral disc (IVD) degeneration. The present study aims to investigate effects of resveratrol (RV), a natural polyphenol compound, on sodium nitroprusside (SNP) induced nucleus pulposus (NP) cell apoptosis and related mechanism. Rat NP cells were pretreated with RV, N-acetyl cysteine (NAC) and carboxy-PTIO (PTIO) before SNP treatment. Cell Counting Kit-8 assay was carried out for cell viability evaluation. Annexin V/propidium iodide (PI), Hoechst 33258 and Actin‑Tracker Green and Tubulin-Tracker Red staining were conducted to detect NP cell apoptosis and apoptotic structural changes. Mitochondrial membrane potential (ΔΨm) was analyzed with tetramethylrhodamine methyl ester staining. DCFH-DA and DAF-FM DA staining was used to determine intracellular reactive oxygen species (ROS) and nitric oxide (NO) levels. An ex vivo experiment was also carried out followed by TUNEL assay of sections of discs. SNP induced NP cell apoptosis, excessive production of intracellular ROS and NO, reduction of ΔΨm as well as disruption of cytoskeletal and morphological structure. Meanwhile, organ culture results showed that SNP induced NP cell apoptosis ex vivo. RV and NAC siginificantly inhibited SNP induced NP cell apoptosis, production of intracellular ROS, deline of ΔΨm as well as disruption of cytoskeletal and morphological structure, while RV did not suppress NO production. RV and NAC could also suppress SNP induced NP cell apoptosis ex vivo. However, PTIO did not prevent SNP induced NP cell apoptosis, though it scavenged NO significantly. In conclusion, RV protects against SNP induced NP cell apoptosis by scavenging ROS but not NO, suggesting a promising prospect of RV in IVD degeneration retardation.

Although mesenchymal stem cells (MSCs) transplantation into the IVD (intervertebral disc) may be beneficial in inhibiting apoptosis of nucleus pulposus cells (NPCs) and alleviating IVD degeneration, the underlying mechanism of this therapeutic process has not been fully explained. The purpose of this study was to explore the protective effect of MSC-derived exosomes (MSC-exosomes) on NPC apoptosis and IVD degeneration and investigate the regulatory effect of miRNAs in MSC-exosomes and associated mechanisms for NPC apoptosis. MSC-exosomes were isolated from MSC medium, and its anti-apoptotic effect was assessed in a cell and rat model. The down-regulated miRNAs in apoptotic NPCs were identified, and their contents in MSC-exosomes were detected. The target genes of eligible miRNAs and possible downstream pathway were investigated. Purified MSC-exosomes were taken up by NPCs and suppressed NPC apoptosis. The levels of miR-21 were down-regulated in apoptotic NPCs while MSC-exosomes were enriched in miR-21. The exosomal miR-21 could be transferred into NPCs and alleviated TNF-α induced NPC apoptosis by targeting phosphatase and tensin homolog (PTEN) through phosphatidylinositol 3-kinase (PI3K)-Akt pathway. Intradiscal injection of MSC-exosomes alleviated the NPC apoptosis and IVD degeneration in the rat model. In conclusion, MSC-derived exosomes prevent NPCs from apoptotic process and alleviate IVD degeneration, at least partly, via miR-21 contained in exosomes. Exosomal miR-21 restrains PTEN and thus activates PI3K/Akt pathway in apoptotic NPCs. Our work confers a promising therapeutic strategy for IVD degeneration., (© 2017 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

Objectives. To investigate whether high glucose-induced oxidative stress is implicated in apoptosis of rat nucleus pulposus cells (NPCs) and abnormal expression of critical genes involved in the metabolic balance of extracellular matrix (ECM). Methods. NPCs were cultured with various concentrations of glucose to detect cell viability and apoptosis. Cells cultured with high glucose (25 mM) were untreated or pretreated with N-acetylcysteine or a p38 MAPK inhibitor SB 202190. Reactive oxygen species (ROS) production was evaluated. Activation of p38 MAPK was measured by Western blot. The expression of ECM metabolism-related genes, including type II collagen, aggrecan, SRY-related high-mobility-group box 9 (Sox-9), matrix metalloproteinase 3 (MMP-3), and tissue inhibitor of metalloproteinase 1 (TIMP-1), was analyzed by semiquantitative RT-PCR. Results. High glucose reduced viability of NPCs and induced apoptosis. High glucose resulted in increased ROS generation and p38 MAPK activation. In addition, it negatively regulated the expression of type II collagen, aggrecan, Sox-9, and TIMP-1 and positively regulated MMP-3 expression. These results were changed by pretreatment with N-acetylcysteine or SB 202190. Conclusions. High glucose might promote apoptosis of NPCs, trigger ECM catabolic pathways, and inhibit its anabolic activities, possibly through a p38 MAPK-dependent oxidative stress mechanism.

Chinese scorpion Buthus martensii Karsch (BmK) venom is a rich source of neurotoxins which bind to various ion channels with high affinity and specificity and thus widely used as compounds to modulate channel gating or channel currents. To promote the insecticidal effects of Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV), the gene encoding an excitatory insect toxin, BmK IT, was inserted into the genome of AcMNPV to construct a recombinant baculovirus, AcMNPV-BmK IT. Spodopter frugiperda 9 (Sf9) cells were infected with AcMNPV and AcMNPV-BmK IT respectively for 24 h. Results from the MTT assay, TUNEL assay, analysis of the expression level of apoptosis-related proteins (c-Myc, cleaved-Caspase3, Bcl-2 and Bax) of Sf9 cells, the transcription level of key genes (38K, C42, P78, F) of AcMNPV, and viral propagation assay demonstrated that AcMNPV-mediated expression of BmK IT promoted the apoptosis of Sf9 cells and replication of AcMNPV. The results laid a foundation for further structural and functional analysis of BmK IT.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease. Semen Crotonis Pulveratum (SCP) has been used as a traditional medicine for the treatment of UC. However, its molecular mechanisms of action have not yet been elucidated. In the present study, we aimed to investigate the preliminary mechanisms of the role of SCP on trinitrobenzene sulphonic acid (TNBS)-induced UC in rats and hydrogen peroxide (H2O2)-induced intestinal cell apoptosis in vitro. Wistar rats (n=9 per group) were randomly divided into 4 groups: the normal control group, the UC group, the UC + SCP group and the UC + sulfasalazine group as a positive control. The proportion of CD4+CD25+ T cells and CD4+CD25+Foxp3+ Tregs, and the expression levels of interleukin (IL)-6 and IL-10 in the peripheral blood, as well as the expression levels of cyclooxygenase-2 (COX-2) and intercellular adhesion molecule-1 (ICAM-1) in the colon tissues were determined by flow cytometry, ELISA and immunohistochemical staining, respectively. Rat intestinal epithelial (IEC-6) cell apoptosis induced by H2O2 was determined by TUNEL assay, flow cytometry using Annexin V/propidium iodide (PI) staining and western blot analysis of caspase-3 activation, respectively. Significantly higher proportions of circulating CD4+CD25+ T cells and CD4+CD25+Foxp3+ Tregs were present in the UC + SCP group compared with the UC group. A significantly decreased expression of IL-6 and an increased expression of IL-10 were also observed in the UC + SCP group compared with UC group. SCP significantly reduced the UC-induced increase in the expression of COX-2 and ICAM-1 in the colon tissues. SCP inhibited cell apoptosis and caspase-3 activation induced by H2O2 in the ICE-6 cells. Our data thus indicate that SCP inhibits inflammation in UC by increasing the proportion of circulating Tregs, altering cytokine production and decreasing COX-2 and ICAM-1 expression. In addition it protects against H2O2-induced intestinal cell apoptosis in vitro.

Objective: To explore the status of apoptosis in human striatum derived neural stem cells (NSCs) aft er accutase dissociation and passage., Methods: The NSCs were isolated from fetuses obtained through spontaneous abortion at 13- 18 weeks of pregnancy, which formed neurospheres in vitro. At passages of 3-5, the neurospheres were disassociated into single cell by accutase digestion and then passaged. At 1, 24 and 72 h after passage, the apoptosis of NSCs was measured by several methods, including active caspase-3 or TUNEL staining for fixed cells, Annexin V, Hoechst or PI staining for live cells., Results: At all of the 3 time points, the staining of TUNEL and active caspase-3 overlapped perfectly. The apoptosis rate of NSCs increased significantly from 20%-25% at 1 h to 75%-80% at 24 h after passage (P<0.01). At 72 h, the apoptosis rate was significantly decreased as compared to that at 24 h time point because of the self-renewal and proliferation of survived NSCs (P<0.01)., Conclusion: Many cells in the neurospheres formed by human striatum-derived NSCs underwent apoptosis soon after accutase disassociation. For NSCs cultured in vitro, anti-apoptosis treatments might be a good method to increase the self-renewal and the proliferation of NSCs.

Objective: To explore the alterations of apoptosis factor Bcl-2/Bax in the early Parkinson's disease (PD) rats and the protective effect of scorpion venom derived bioactive peptide., Methods: Healthy male SD rats (180-220 g) were randomly divided into 4 groups (n = 10): early PD model group, sham operation group, scorpion venom derived bioactive peptide control group, scorpion venom derived bioactive peptide therapy group. 6-hydroxydopamine (6-OHDA) was used to prepare the early PD rat model. The immunohistochemistry was used to detect the expression of Bax and Bcl-2 and further explore the mechanism of anti-apoptosis regarding the neuroprotective effect of scorpion venom derived bioactive peptide., Results: The results indicated that compared with the control rats, the immunostaining of Bax in the brain increased significantly while that of Bcl-2 decreased significantly in the lesion side of 6-OHDA treated rats. Interestingly, scorpion venom derived bioactive peptide could attenuate the above abnormal changes., Conclusion: Up-regulation of Bax and down-regulation of Bcl-2 could participate in the early stage of PD and the anti-apoptotic mechanism could be involved in the neuroprotective effect exerted by scorpion venom derived activity peptide regarding the dopaminergic neuron in the early stage.

Objective: To investigate the process of apoptosis in lungs and liver induced by crushing hindlimbs of rat, and study the mechanism of crush injury., Methods: The rat experimental model of hindlimbs crush injury was established. The cell apoptosis in lungs and liver was detected by TUNEL assay, and the expression of Bax, Bcl-2 and caspase-3 apoptin was examined by immunohistochemistry., Results: Compared with the control group, the partial muscle injury of rat's hindlimbs was more serious with more apoptosis observed in lungs and liver (P < 0.05). The expression of Bax was up-regulated and Bcl-2 was down-regulated, whereas caspase-3 expression was activated (P < 0.05)., Conclusion: The cell apoptosis has increased significantly in lungs and liver after crush injury of hindlimbs in rat. The correlation factor released during tissue injury may mediate apoptosis process.

Objective: To observe the apoptosis of neural stem cells (NSCs) at differential time points after the dissociation of neurospheres by Accutase or trypsin., Methods: The NSCs were isolated from striatum of human fetals that suffered abortion at 12-16 weeks of pregnancy. The 3(rd)-5(th) passages of NSCs were digested by Accutase or trypsin. Only vortexing was applied, and the triturating by Pasteur pipette was avoided to attenuate the injury to the cells during the dissociation. The single cells were then stained by Annexin V/propidium iodide and Hoechst 33342. The apoptosis rates 2 and 24 hours after passaging were evaluated., Results: The trypan blue staining confirmed that immediately after the dissociation,the viability of cells digested by trypsin was (83.10 ± 6.76)%, which was significantly lower than that digested by Accutase,which was (91.65 ± 4.43)% (P<0.05). The apoptosis of the NSCs digested by Accutase was higher than that digested by trypsin at both 2 and 24 hours after passaging (P<0.01). Four days after the passaging, both the new clone formation rate and diameter of new spheres after trypsin digestion were significantly higher than those after Accutase digestion (P<0.01)., Conclusions: Although the viability of NSCs immediately after the disassociation by trypsin is lower than that digested by Accutase, the apoptosis of NSCs subsequently caused by trypsin is lower than that caused by Accutase. Trypan blue test immediately after the disassociation can not be used as an indicator in estimating the apoptosis of NSCs during the expanding.

Nucleophosmin (NPM1) is a multifunctional protein that functions as a molecular chaperone, shuttling between the nucleolus and the cytoplasm. In up to one third of patients with acute myeloid leukemia, mutation of NPM1 results in the aberrant cytoplasmic accumulation of mutant protein and is thought to be responsible for leukemogenesis. Deguelin, a rotenoid isolated from several plant species, has been shown to be a strong anti-tumor agent. Human leukemia cell lines were used for in vitro studies. Drug efficacy was evaluated by apoptosis and differentiation assays, and associated molecular events were assessed by Western blot. Gene silencing was performed using small interfering RNA (siRNA). Deguelin exhibited strong cytotoxic activity in the cell line of OCI-AML3 and selectively down-regulated the NPM1 mutant protein, which was accompanied by up-regulation of the activity of caspase-6 and caspase-8 in high concentrations. Deguelin induced differentiation of OCI-AML3 cells at a nontoxic concentration which was associated with a decrease in expression of activated caspase-8, p53, p21, and the 30-kD form of CCAAT/enhancer binding protein α (C/EBPα), whereas no effects were found in OCIM2 cells expressing NPM-wt. Moreover, treatment with siRNA in the NPM mutant cell line OCI-AML3 decreased expression of p53, p21, pro-caspase-8, and the 30-kD form of C/EBPα, and it inhibited proliferation and induced differentiation of the OCI-AML3 cells. In conclusion, deguelin is a potent in vitro inhibitor of the mutant form of NPM1, which provides the molecular basis for its anti-leukemia activities in NPM1 mutant acute myeloid leukemia cells.

As the principal active ingredient in the Chinese herb Tripterygium wilfordii Hook.F (TwHF), triptolide has been shown to have very strong antitumor properties. The trimethylation of lysine 4 on histone H3 (H3K4me3) has been proposed to promote gene expression, and the accumulation of H3K4me3 at the transcriptional start sites of oncogenes is involved in carcinogenesis. To identify the association between the reduction of H3K4me3 and the apoptosis of MM cells induced by triptolide, we investigated the global patterns of H3K4me3 occupancy in the MM cell genome. Combined analyses using ChIP-on-chip and western blotting showed that H3K4me3 were highly enriched on the gene promoters of c-Myc and VEGFA and were associated with the up-regulation of both genes. Treatment of KM3 cells with triptolide and siRNA targeting ASH2L reduced the expression of c-Myc and VEGFA. These results suggest that triptolide can down-regulate c-Myc and VEGFA expression by blocking the accumulation of H3K4me3 on their promoters,and thus play an important role in anti-MM mechanism.

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Unfortunately, treatment failures are common due to the metastasis and chemoresistance, but the underlying molecular mechanism remains unclear. Accumulating evidence indicated that the deregulation of DNA-binding protein high-mobility group box 1 (HMGB1) was associated with the development of cancer. This study aimed to explore the expression of HMGB1 in osteosarcoma tissues and its correlation to the clinical pathology of osteosarcoma and to discuss the role of HMGB1 in the development of osteosarcoma. The results from RT-PCR and Western blot showed that the expression rate of HMGB1 messenger RNA (mRNA) and the expression of HMGB1 in the osteosarcoma tissues were significantly higher than those in normal bone tissue (p < 0.05), the expression rate of HMGB1 mRNA and the expression of HMGB1 in the carcinoma tissues with positive lung metastasis were significantly higher than those without lung metastasis (p < 0.05), and with increasing Enneking stage, the expression rate of HMGB1 mRNA and the expression of HMGB1 also increased (p < 0.05). In order to explore the role of HMGB1 in osteosarcoma, the expression of HMGB1 in the human osteosarcoma MG-63 cell line was downregulated by the technique of RNA interference. Western blot results showed that the protein expression of HMGB1 was significantly decreased in the MG-63 cells from HMGB1-siRNA transfection group (p < 0.05), which suggested that HMGB1 was successfully downregulated in the MG-63 cells. Then the changes in proliferation, apoptosis, and invasion of MG-63 cells were examined by MTT test, PI staining, annexin V staining, and transwell chamber assay. Results showed that the abilities of proliferation and invasion were suppressed in HMGB1 knockdown MG-63 cells, and the abilities of apoptosis were enhanced in HMGB1 knockdown MG-63 cells. The expression of cyclin D1, MMP-9 was downregulated in HMGB1 knockdown MG-63 cells, and the expression of caspase-3 was upregulated in HMGB1 knockdown MG-63 cells. Taken together, the overexpression of HMGB1 in osteosarcoma might be related to the tumorigenesis, invasion, and metastasis of osteosarcoma, which might be a potential target for the treatment of osteosarcoma.

Objective: To investigate the roles of cytochrome c (Cyt-c), caspase-9, and caspase-3 in pentavalent vanadium-induced neuronal apoptosis and to provide a basis for mechanism research., Methods: Neurons from rats aged 1-3 days were cultured and treated with vanadium pentoxide (V2O5) at 5, 10, or 20 mmol/L. Neuronal apoptosis was detected by TdT-mediated dUTP-biotin nick end labeling (TUNEL). The protein levels of Cyt-c, caspase-9, and caspase-3 were determined by Western blot., Results: Apoptosis bodies were detected in the nuclei of neurons by TUNEL. The number of neurons with apoptosis bodies increased with increasing dose of V2O5 The apoptosis index (AI) was significantly higher in the 10 and 20 mm/L exposure groups than in the control group (P < 0.05 or P < 0.01). Western blot showed that the protein expression levels of Cyt-c and caspase-3 significantly increased in the 5 mmol/L exposure group as compared with the control group (P < 0.05). In the 10 and 20 mmol/L exposure groups, the protein expression of Cyt-c, caspase-9, and caspase-3 all increased as compared with the control group (P < 0.01). Neuronal AI was positively correlated with Cyt-c, caspase-9, and caspase-3 (r = 0.954, P < 0.01; r = 0.938, P < 0.01; r = 0.943, P < 0.01)., Conclusion: Pentavalent vanadium may induce neuronal apoptosis. The protein expression of Cyt-c, caspase-9, and caspase-3 may play an important role in neuronal apoptosis induced by pentavalent vanadium.

Aim: Skewed cytoplasmic accumulation of NPM mutant protein (NPM1c+) is close related to leukemia pathogenesis. The aim of this study was to investigate whether oridonin, a diterpenoid isolated from the Chinese traditional medicine Rabdosia rubescens, was able to interfere with NPM1c+ protein trafficking and induce apoptosis in NPM1c+ acute myeloid leukemia cells in vitro., Methods: OCI-AML3 cell line harboring a NPM1 gene mutation was examined. Cell growth was detected by MTT assay. Cell apoptosis was evaluated using flow cytometry and Hoechst 33258 staining. The expression and subcellular localization of relevant proteins were detected by Western blot and immunofluorescent staining. The mRNA expression was detected by RT-PCR., Results: Oridonin (2-12 μmol/L) dose-dependently inhibited the viability of OCI-AML3 cells (the IC50 value was 3.27±0.23 μmol/L at 24 h). Moreover, oridonin induced OCI-AML3 cell apoptosis accompanied by activation of caspase-3 and nuclear translocation of NPM1c+ protein. Oridonin did not change the expression of Crm1 (the export receptor for nuclear export signal-containing proteins), but induced nuclear translocation of Crm1. Oridonin markedly increased the expression of nucleoporin98 (Nup98), which had an important role in Crm1-mediated nuclear protein export, and induced nuclear accumulation of Nup98. Furthermore, oridonin markedly increased the expression of p14arf and p53., Conclusion: In NPM1c+ leukemia cells, oridonin induces NPM1c+ protein translocation into the nucleus possibly via nuclear accumulation of Crm1; the compound markedly increases p53 and p14arf expression, which may contribute to cell apoptosis.

The mixed lineage kinase domain-like protein (MLKL) has recently been identified as a key RIP3 (receptor interacting protein 3) downstream component of tumour necrosis factor (TNF)-induced necroptosis. MLKL is phosphorylated by RIP3 and is recruited to the necrosome through its interaction with RIP3. However, it is still unknown how MLKL mediates TNF-induced necroptosis. Here, we report that MLKL forms a homotrimer through its amino-terminal coiled-coil domain and locates to the cell plasma membrane during TNF-induced necroptosis. By generating different MLKL mutants, we demonstrated that the plasma membrane localization of trimerized MLKL is critical for mediating necroptosis. Importantly, we found that the membrane localization of MLKL is essential for Ca(2+) influx, which is an early event of TNF-induced necroptosis. Furthermore, we identified that TRPM7 (transient receptor potential melastatin related 7) is a MLKL downstream target for the mediation of Ca(2+) influx and TNF-induced necroptosis. Hence, our study reveals a crucial mechanism of MLKL-mediated TNF-induced necroptosis.

Deregulated expression of protein tyrosine phosphorylation has been implicated in testicular response to different stimuli. Herein, YES1, a nonreceptor protein tyrosine kinase, was found to be significantly up-regulated in pachytene spermatocytes (PS) during early recovery from a transient testicular heat stress. Coculture of PS with Sertoli cells (SCs) could enhance the hyperthermia-induced YES1 activation, indicative of a positive regulation of the paracrine signaling. Moreover, SU6656, a selective YES1 inhibitor, was shown to effectively block YES1 activity, thereafter resulting in a dramatic increase of heat stress-induced apoptosis in primary cultured PS. Mechanistically, the antiapoptotic effect of YES1 activation in response to testicular heat insult may mediate via the regulation of extracellular signal-regulated kinase (ERK)/metastasis-associated 1 (MTA1) cascade. From a clinical standpoint, a notably higher level of YES1 expression was observed in the pathological testis from varicocele patients as compared to a negligible staining in the control group. Taken together, our present results provide the first evidence that the YES1/ERK/MTA1/p53 cascade may serve as a naturally occurring, indispensable self-defensive mechanism maintaining apoptotic balance during meiotic heat stress. Our study may have also partially answered the question of how activation of signal pathways at the cell membrane surface interacts with the key regulatory events occurring in the nucleus during testicular heat shock.

The present study was undertaken to study the effects of exogenous hydrogen sulfide (H(2)S) on global cerebral ischemia-reperfusion(I/R) and the underlying mechanisms. After a 24h I/R, administration of NaHS, an exogenous donor for H(2)S, at the dose of 0.2 or 0.4 μmol/kg significantly decreased the apoplexy index, neurological symptom scoring, and brain infarcted area as compared to the I/R group in a dose dependent manner. At the same time, NaHS-treated rats displayed significant reduction of MDA content, and striking increase of SOD activity in the brain tissues compared with I/R group. The up-regulated mRNA levels of p47(phox) and gp91(phox) subunits of NADPH oxidase were also suppressed by NaHS treatment. In this study, the pro-inflammatory markers TNF-α and MCP-1 in I/R group were markedly increased by 24h I/R, which were significantly attenuated by NaHS in a dose-dependent manner. In contrast, the anti-inflammatory factor IL-10 was markedly induced by NaHS administration. In addition, the expression of the anti-apoptotic protein Bcl-2 was significantly decreased in I/R group compared with the sham-operated group. This reduction was significantly blunted in NaHS-treated group. On the contrary, the pro-apoptotic protein Bax content in brain tissues of I/R group was markedly elevated compared with sham-operated animals. And such an induction of Bax content was significantly ameliorated by NaHS. Taken together, our results suggest that hydrogen sulfide has potent protective effect against a severe cerebral injury induced by a global I/R possibly through the inhibition of oxidative stress, inflammation and apoptosis., (Copyright © 2012 Elsevier B.V. All rights reserved.)

A series of carbamate derivatives of 4'-demethylepipodophyllotoxin have been synthesized, and their cytotoxicities against several human cancer cell lines, including HeLa, A549, HCT-8, and HL-60 cells, evaluated. Some of these compounds exhibited higher levels of cytotoxicity than the anticancer drug etoposide. 4β-4'-Demethylepipodophyllotoxin 1-(4-nitrophenyl) piperazinyl carbamate (19) was found to be the most potent compound of those synthesized in the current study, and induced cell cycle arrest in the G2/M phase in HeLa cells, which was accompanied by apoptosis. Furthermore, this compound activated the expression of Bax, p53 and caspase-3 in HeLa cells, leading to changes in the conformation of calf thymus DNA from the B-form to a more compact C-form., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Aim: To investigate the effects of betulinic acid (BA) on apoptosis and autophagic flux in multiple myeloma cells and the relationship between the two processes., Methods: The proliferation of human multiple myeloma KM3 cells was measured with MTT assay. FITC/PI double-labeled flow cytometry (FCM) and Hoechst 33258 staining were used to analyze the cell apoptosis. Caspase 3, PARP, Beclin1, LC3-II, and P62 were detected using Western blotting., Results: Treatment of KM3 cells with BA (5-25 μg/mL) suppressed the cell proliferation in time- and dose-dependent manners. The IC(50) values at 12, 24, and 36 h were 22.29, 17.36, and 13.06 μg/mL, respectively. BA treatment dose-dependently induced apoptosis of KM3 cells, which was associated with the activation of caspase 3. However, Z-DEVD-FMK, a specific inhibitor of caspase 3, did not decrease, but rather sensitized the cells to BA-induced apoptosis, suggesting an alternative mechanism involved. On other hand, BA treatment dose-dependently increased the accumulation of LC3-II and P62 in KM3 cells, representing the inhibition of autophagic flux. Furthermore, BA treatment dose-dependently downregulated the expression of Beclin 1, an important inducer of autophagy, in KM3 cells. In the presence of BA, Z-DEVD-FMK induced autophagy and increased the amount of LC3-II in KM3 cells, which may occur via attenuating BA-induced decrease in the level of Beclin 1. Similarly, rapamycin, an autophagy inducer, increased the amount of LC3-II in KM3 cells. In the presence of BA, rapamycin caused further increase in the amount of LC3-II. Furthermore, rapamycin sensitized BA-treated KM3 cells to apoptosis., Conclusion: The results demonstrate that BA induces apoptosis and blocks autophagic flux in KM3 cells. Furthermore, in addition to activation of caspase 3, the inhibition of autophagic flux also contributes to the BA-mediated apoptosis of KM3 cells.

Objective: To observe the effects of Benzo(a)pyrene (BaP) on apoptosis of neuronal cells and expression of Bcl-2 and Bax proteins and to explore the mechanism of neurotoxicity induced by BaP in rats., Methods: A total of 32 SD rats were divided randomly into 4 groups, i.e. 3 BaP (126.2, 63.1 and 31.5 µg/kg) groups and a solvent control (50 µg/kg olive oil) group. All rats were exposed to BaP or olive oil by lateral cerebral ventricle micro-injection 1 time a week for 3 weeks. The apoptosis of neuronal cells was detected with TdT-mediated dUTP-biotin nicked labeling (TUNEL) assay and the expression levels of Bcl-2 and Bax were measured with SABC immunohistochemistry in the cerebral cortex and hippocampus tissues of rats., Results: The results of TUNEL assay showed that apoptosis bodies on the surface of the neurons in the cerebral cortex and hippocampus were clearly observed and the number of apoptosis bodies increased with BaP. Apoptosis indexes (AIs) of the rat cerebral cortex and hippocampus in high exposure group were significantly higher than those in control group (P < 0.05 or P < 0.01). The analysis of immunohistochemistry showed that the Bcl-2 expression levels significantly decreased, the Bax expression levels obviously increased and the ratio of Bcl-2 to Bax decreased in the rat cerebral cortex and hippocampus of medium and high exposure groups, as compared with control group (P < 0.05 or P < 0.01). In the rat cerebral cortex and hippocampus, there were the negative correlation (r = -0.927, P < 0.01; r = -0.934, P < 0.01) between AI and Bcl-2, the positive correlation (r = 0.858, P < 0.01; r = 0.847, P < 0.01) between AI and Bax and the negative correlation (r = -0.939, P < 0.01; r = -0.942, P < 0.01) between AI and Bcl-2/Bax., Conclusion: BaP could induce the apoptosis of neuronal cells in the rat cerebral cortex and hippocampus. Bcl-2 and Bax protein expression may play an important role in the apoptosis of neuronal cells induced by BaP.

It has been reported by us and other groups that the expression of small GTP binding protein RhoB can be induced by genotoxic stressors and glucocorticoid (GC), a stress hormone that plays a key role in stress response. Until now stress-induced genes that confer cytoprotection under stressed conditions are largely unknown. In this study, we investigated the effects and mechanism of non-genotoxic stressors, including scalding in vivo and heat stress in vitro on the expression of RhoB. We found for the first time that both scalding, which could induce typical neuroendocrine responses of acute stress and cellular heat stress significantly increased the expression of RhoB at mRNA and protein levels. Moreover, in vitro experiments in human lung epithelial cells (A549) showed that induction of RhoB by heat stress was in a glucocorticoid receptor (GR)-independent manner and through multiple pathways including stabilization of RhoB mRNA and activation of p38 MAPK. Further experiments demonstrated that up-regulation of RhoB significantly inhibited heat stress-induced apoptosis and elevated transcriptional activity of NF-κB, but did not affect the expression of Hsp70 in A549 cells. In conclusion, we showed for the first time that RhoB was up-regulated by scalding in vivo and heat stress in vitro and played an important cytoprotective role during heat stress-induced apoptotic cell death., (Copyright © 2010 Wiley-Liss, Inc.)

Background: Deregulated thermal factors have been frequently implicated in the pathogenesis of male infertility, but the molecular basis through which certain responses are directed remain largely unknown. We previously reported that overexpression of exogenous Metastasis-associated protein 1 (MTA1) protects spermatogenic tumor cells GC-2spd (ts) against heat-induced apoptosis. To further dissect the underlying mechanism, we addressed here the fine coordination between MTA1 and p53 in pachytene spermatocytes upon hyperthermal stimulation., Methodology/principal Findings: High level of MTA1 expression sustained for 1.5 h in primary spermatocytes after heat stress before a notable decrease was detected conversely correlated to the gradual increase of acetylation status of p53 and of p21 level. Knockdown of the endogenous MTA1 in GC-2spd (ts) elevated the acetylation of p53 by diminishing the recruitment of HDAC2 and thereafter led to a dramatic increase of apoptosis after heat treatment. Consistent with this, in vivo interference of MTA1 expression in the testes of C57BL/6 mice also urged an impairment of the differentiation of spermatocytes and a disruption of Sertoli cell function due to the elevated apoptotic rate after heat stress. Finally, attenuated expression of MTA1 of pachytene spermatocytes was observed in arrested testes (at the round spermatid level) of human varicocele patients., Conclusions: These data underscore a transient protective effect of this histone modifier in primary spermatocytes against heat-stress, which may operate as a negative coregulator of p53 in maintenance of apoptotic balance during early phase after hyperthermal stress.

K562 cells and peripheral blood mononuclear cells were treated with hydrogen peroxide (H(2)O(2)) to determine the expression of Krüppel-like factor (KLF) 4. A full-length complementary DNA or an anti-sense oligonucleotide of KLF4 was transfected into cells, and expressions of B-cell lymphoma/leukemia-2 (bcl-2) and bcl-2-associated X (bax) proteins were analyzed. The results showed that H(2)O(2) treatment of cells resulted in an increase in KLF4 levels; KLF4 induced apoptosis and slowed cell growth, potentially resulting from up-regulation of bax and down-regulation of bcl-2. Transcriptional activities on bcl-2 and bax were promoted following KLF4 overexpression potentially through KLF4 binding sites on corresponding promoters. All results indicate that KLF4 induces apoptosis in leukemia cells involving the bcl-2/bax pathway during H(2)O(2) stimulation, suggesting a potential mechanism for research on drug-induced apoptosis.

Objective: To investigate the changes of mitochondria membrane potential and cytoplasma cytochrome C as the mechanism of neuron apoptosis induced by B(a)P., Methods: Primary neurons were dissociated from cerebral cortex of 1 - 3 days old SD rats and cultured with DMEM incubator at 37 degrees C. After 5 days' cultivation, the neurons were added S9 and B(a)P, and the concentrations of treated B(a)P were 0, 10, 20 and 40 micromol/L respectively. After administering of B(a)P, the neurons were cultivated for 40 hours. Apoptosis rate was measured by flow cytometry using Annexin V-FITC and propidium iodide (PI) staining, and the changes in mitochondrial potential (DeltaPsim) were tested with Rhodamine fluorescence (R2123) technique. Preparation of cytosolic extracts by centrifugation. Western blotting analysis was used to evaluate the level of cytochrome C of cytoplasm., Results: The apoptotic rate of neuron increased in both the middle dose group and the high dose group compared with controls, and had a dose-response tendency with the concentration of B(a)P. Moreover mitochondrial potential decreased in a dose dependent manner. There was a negative correlation between DeltaPsim and the apoptotic rate of neurons (r = -0.763, P < 0.05); Western blotting analysis showed cytoplasmic cytochrome C level increased significantly, which was positively related with neuron apoptosis (r = 0.831, P < 0.01)., Conclusion: Loss of mitochondria membrane potential and increase of cytoplasma cytochrome C may be the main cause of neuron apoptosis induced by B(a)P.

Multiple lines of evidence have demonstrated that gambogic acid (GA) is an efficient apoptosis inducing agent. However, the mechanisms of GA induced apoptosis have been controversial, despite the tremendous effort made during recent years. Here we report a novel mechanism through which GA induces cell apoptosis. Instead of dealing with tumor cells directly, GA first activates inactive T lymphocytes, which in turn triggers cancer cell apoptosis. This is supported by the observation that GA inhibited tumor growth and extended the survival time of mice bearing H22 tumor. cDNA microarray analysis indicated that 22.92% of the 48 genes that were affected with GA treatment were immune related genes. RT-PCR assay revealed that GA up-regulated MHC-II and TCR transcriptions, implicating that GA activates T lymphocytes to induce tumor cell apoptosis in vivo. HE staining showed that T lymphocytes penetrated into tumor tissues after GA administration. Western blotting revealed that GA enhanced CD4+ and CD8+ expressions. Annexin-V/PI double-staining and DNA ladder assays confirmed that GA induced tumor cell apoptosis. In summary, this report demonstrated, for the first time, that GA mainly activates T lymphocytes to induce cancer cell apoptosis in H22 transplanted mice.

Gambogic acid (GA) is a natural product with potent apoptotic activity. Here, we showed that GA broadly inhibited the growth of cancer cells that expressed wild-type p53 as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazol-iumbromide assay, (3)H-thymidine incorporation analysis, and an in vivo mouse xenograft model. GA induced massive cell apoptosis as judged by Annexin V and propidium iodide dual-staining experiments. Furthermore, we found that GA partially induced cancer cell growth inhibition in a p53-dependent manner because cell survival could be restored after endogenous p53 was attenuated by p53 transcriptional repressor pifithrin-alpha or p53 small interfering RNA. Interestingly, GA had no influence on p53 mRNA synthesis but dramatically enhanced its protein expression. This unique observation could be accounted for by the down-regulation of mdm2 at both mRNA and protein levels. It is concluded that GA enhances p53 protein level through inhibition of mdm2 expression and thereby hampers p53 harboring tumor growth.

This review illuminates the current approaches of ion channels and tumor. Potassium, calcium, chloride channnels and others are abnormally expressed in the membrane of tumor cells. They are crucial for tumor development and growth. By the regulation of membrane voltage, cell cycle, cell volume, Ca2+ signaling and cytosolic pH, they can adjust the cell proliferation and apoptosis. With further study, ion channels will become a new target for cancer therapy and diagnosis.

Cadmium (Cd) is a well-known toxic compound for the kidney in vivo and in vitro. It has been demonstrated to induce nephrotoxicity via in part by apoptotic cell death, but the precise mechanism is still unclear. Therefore, we have studied the effects of Cd on HEK 293 cells and investigated the mechanisms of Cd-induced apoptosis. Studies of morphology and oligonucleosomal DNA fragmentation demonstrated that 30-60 microM Cd induced apoptosis as early as 6-9h with strong effects on MTT activity, whereas 120 microM Cd revealed mainly necrosis, and the result of flow cytometry confirmed it. A concomitant time-dependent decrease of mitochondrial transmembrane potential (DeltaPsi(m)) and Bcl-2 expression was observed, subsequently, release of cytochrome c (Cyt c) and activation of caspase-3 were detected, suggesting a caspase-dependent pathway. Meanwhile, mitochondrial AIF was released to cytoplasm and nucleus, suggesting a caspase-independent pathway. Furthermore, when cells were transfected with pcDNA3/Bcl-2 before exposed to CdCl(2), alleviated apoptosis was assessed by part of the apoptotic features in this study. Taken together, our results showed that CdCl(2) caused time- and dose-dependent apoptosis or even necrosis in HEK 293 cells depending on the exposure conditions. The apoptotic events may involve mitochondrial disruption including both caspase-dependent and -independent pathways.

JNK signaling pathway is activated and involved in the selective neuronal death in the hippocampal CA1 subfield following cerebral ischemia. However, little is known about upstream partner controlling the pathway. Here we reported that ischemia/reperfusion significantly elevated Cdc42 activity, enhanced assembly of the Cdc42-MLK3 complex and activation of JNK pathway. Most importantly, knock-down endogenous Cdc42 selectively suppressed the MLK3/MKK7/JNK3 cascade, and subsequently blocked the phosphorylation of c-Jun and FasL expression. Meanwhile, Bcl-2 was inactivated and the release of cytochrome c was diminished. These alterations eventually perturbed the caspase-3 activation as well as post-ischemic neuronal cell death. Taken together, our findings strongly suggest that Cdc42 serves as an upstream activator and modulates JNK-mediated apoptosis machinery in vivo, which ultimately results in neuronal apoptosis via nuclear and non-nuclear pathways. Thus, Cdc42 may be a potential therapeutic target in ischemic brain injury.

HEK293 cell was chose to study the kidney damage of cadmium and to explore the significance of caspase 3,Bcl-2 and AIF (apoptosis inducing factor) in the apoptosis of cells induced by cadmium. Inhibition of the cell proliferation was measured by MTT assay. The structure of apoptotic cells was observed by light microscopy and electron microscopy; moreover, apoptotic cells were detected by DNA electrophoresis, flow cytometry and confocal laser microscopy. Furthermore,the expressions of Pro-caspase-3, Bcl-2 and the location of AIF in cells (mitochondria,cytoplasm or nuclei) were tested by western blot and immunofluorescence assay. CdCl2 exhibited anti-proliferative activity in dosage and time-dependent manner. DNA ladders of HEK293 cells were showed on agarose gel electrophoresis and the fragments of DNA were integral of 180-200 bp. 6-9 hours after 30 micromol/L CdCl2 treatment,DNA ladders were distinct. However, mistiness DNA ladder or smear was found when HEK293 cells were treated with CdCl2 on higher concentration or treated longer. It suggests that necrosis may happen, and flow cytometry results confirmed it. Morphological examination showed cell shrinkage, chromosomal condensation, karyotheca margination, nucleus cracking, vacuoles formed in cytoplasm and the presence of apoptotic bodies. At the same time,mitochondrial membrane potential (MMP) decreased, and the expression of Pro-caspase-3, Bcl-2 were decreased in time-dependent manner. Furthermore, AIF was released from mitochondria,and then traveled to nuclei. It suggests that CdCl2 may induce the apoptosis of HEK293 cells involving mitochondrial disruption including AIF migration and Cyt c release through both caspase-independent and -dependent pathways, and Bcl-2 and Caspase-3 are important factors which participate in the processes.

During the past two decades, the knowledge of the molecular mechanism by which estrogens exert various functions in different tissues and organs has evolved rapidly. Recent reports demonstrated that estrogen could decrease the cell growth in several types of cancer cells, including ovarian cancer cells. Though experiments explored the possible mechanism of the inhibitory effect, the exact mechanism is responsible for the effect, which remains unclear. The ovary is the main source of the estrogen, estrogen receptor is expressed in several ovarian cell types, including ovarian surface epithelium, the tissue of origin of approximately 90% of the ovarian cancers. It was of great interest to analyze the effects of 17beta-estradiol (E2) on apoptosis of ovarian cancer cells, and the identification of E2-regulated specific genes involved in epithelial proliferation apoptosis, thus may be a clue for understanding the progression of ovarian cancer and for the design of new target therapies. To elucidate the mechanism involved, effects of pharmacological concentrations of estrogen were studied in human ovarian cancer cell line 3AO cells. Inhibition of cellular growth of 3AO cells was seen with E2 at concentrations higher than 0.1 micromol/L. The estrogen receptor inhibitor ICI 182780 cannot block the inhibitory effect of E2. It was surprising to find that ICI 182780 itself can inhibit the growth of 3AO cells, and had a collaborative effect with E2. The decreased cell growth induced by E2 was shown to be apoptosis as analyzed by flow cytometry. ERbeta was detected in the 3AO ovarian cancer cell line but not ERalpha. The expression of ERbeta was weak, which may partially explain why high but not low dose of E2 needed to induce the apoptosis of 3AO cells. We also observed that membrane impermeable E2, E2-BSA have lost growth inhibitory on 3AO cells, which excluded the membrane effect of E2 as previously reported by many investigators. The p38 kinase inhibitor, SB203580 were partially protected 3AO cells against growth inhibition by E2, while inhibitor of JNK, SP600125 enhanced cell death induced by E2. These results showed that MAPK is implicated in cellular processes involving apoptosis.

AIM: To investigate the effects of antitumor drug paclitaxel(PTX)on the proliferation, apoptosis, cell cycle, cell morphology, and related protein expression of Müller cells, and to evaluate its potential toxicity to the retina.METHODS:Müller cells were cultured in vitro and divided into two groups: control group(normal medium)and PTX group. Retinal Müller cells were treated with different concentrations of PTX(0.005, 0.05, 0.5 and 5mg/L)for varying durations(12, 24, 36, 48 and 72h). The CCK8 method was used to assess the effects of different concentrations of PTX and treatment duration on the proliferation Müller cells. Flow cytometry was employed to investigate the impact of different concentrations of PTX on Müller cells apoptosis and cell cycle arrest. Immunofluorescence was used to observe morphological changes in Müller cells. The effects of PTX on the expression of apoptosis-related proteins and aquaporins were analyzed by Western blot and qRT-PCR.RESULTS: PTX exhibits the ability to inhibit the proliferation of Müller cells when cultured in vitro. The efficacy of this inhibition was found to be dependent on both the concentration of the drug and the duration of the stimulation. Higher concentrations of the drug and longer stimulation times resulted in a weaker ability of the cells to proliferate. Additionally, PTX also induces apoptosis in Müller cells, with increased drug concentrations and longer stimulation times leading to higher apoptosis rates. Flow cytometry analysis demonstrates that PTX arrests Müller cells in the G2-M phase of the cell cycle. Moreover, there is a distinct change in cell morphology, with a shift from the typical appearance characterized by clear and slender fibrous structures to a rounder morphology, accompanied by a significant decrease in cell numbers. Further, our findings reveal that there is a transient increase in the expression of cytoinflammatory factors following drug treatment compared to the control group. However, discontinuation of drug stimulation can alleviate this heightened expression. In treated cells, the expression of the CA XIV protein is upregulated compared to the control group, while the expression of vascular endothelial growth factor(VEGF)is downregulated(P

Background: Hypothermia is an effective method of reducing brain injury caused by a variety of neurological insults. It is aimed to elucidate whether a change in the expression of PERK‐mediated pathway proteins is an indicator of the neuroprotective effect of mild hypothermia after cerebral ischaemia/reperfusion. Methods: One hundred and ninety‐two male C57BL/6 mice were randomly divided into three groups: a sham group, a cerebral normothermic ischaemia/reperfusion (I/R) group and a cerebral hypothermic I/R group. A cerebral ischaemia model was established by ligating the bilateral common carotid artery for 15 min. Mice in the hypothermia group stayed in a cage that was set at 33°C, sprayed with a spray of 70% ethanol, and blown with two high‐speed fans. The state of neurons was assessed on micropreparations stained with haematoxylin–eosin and TUNEL. The expressions of GRP78, p‐perk, p‐eif2α, ATF4 and CHOP were measured by western blot analysis 6, 12, 24 and 72 h after reperfusion. Results: The number of surviving cells was significantly higher in the hypothermia group than in the group without hypothermia (p <.05). The GRP78 expression in the hypothermia group was statistically higher (p <.05) than in the ischaemia/reperfusion group. Optical densities of p‐perk, p‐eif2α and ATF4 in hippocampus CA1 neurons ischaemia were statistically significantly lower in the hypothermia group than in the ischaemia/reperfusion group (p <.05). The CHOP expression in the hypothermia group was statistically lower (p <.05) than in the ischaemia/reperfusion group. Conclusion: Mild hypothermia for 6 h promoted moderate neuroprotection by mediating the expression of GRP78, p‐PERK, p‐eIF2α, ATF4 and CHOP. [ABSTRACT FROM AUTHOR]

Multiple myeloma (MM) is an incurable cancer that is characterized by malignant plasma cell proliferation. Approximately 10% of all blood cancers are MM, and there is no standard curative therapy. In this work, we intended to synthesize, characterize, and assess the anticancer effects of selenium/chitosan/polyethylene glycol–carvacrol nanocomposites (SCP‐Car‐NCs) on MM U266 cells in vitro. Various characterization techniques were used to characterize the synthesized SCP‐Car‐NCs. Several in vitro free radical scavenging experiments were conducted to test the ability of synthesized SCP‐Car‐NCs to scavenge the different free radicals. The cytotoxicity of SCP‐Car‐NCs was assessed on Vero and U266 cells using the 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5 diphenyl tetrazolium bromide (MTT) assay. By using various fluorescence staining techniques, the amount of reactive oxygen species (ROS) generation, MMP, and apoptosis were measured. Using commercial test kits, the levels of oxidative stress and apoptotic biomarkers in control and treated U266 cells were assessed. The highest peak in the UV spectral analysis was found to be at 271 nm, demonstrating the development of SCP‐Car‐NCs. Fourier transform infrared analysis showed that the synthesized SCP‐Car‐NCs contained a variety of stretching and bonding. The X‐ray diffraction study confirmed the crystallinity of SCP‐Car‐NCs. The dynamic light scattering analysis showed that the SCP‐Car‐NCs had an average size of 171 nm. The different free radicals, such as the 2,2‐diphenyl‐1‐picrylhydrazyl, hydroxyl, and peroxyl radicals, were significantly scavenged by the SCP‐Car‐NCs. According to the MTT assay results, the SCP‐Car‐NCs decreased the viability of U266 cells while having no impact on the proliferation of Vero cells. The SCP‐Car‐NCs significantly boosted ROS production, decreased the MMP level, and promoted apoptosis, as evidenced by the fluorescence staining experiments. In U266 cells treated with SCP‐Car‐NCs, the level of thiobarbituric acid reactive substances increased while superoxide dismutases and glutathione levels were reduced. In the SCP‐Car‐NCs treated U266 cells, it was found that the Bax, caspase‐3, and −9 activities had increased while the Bcl‐2 level had decreased. In conclusion, our findings show that SCP‐Car‐NCs treatment reduced the viability and increased apoptosis in the U266 cells, providing a new insight on SCP‐Car‐NCs' potential for usage in the future to treat MM. [ABSTRACT FROM AUTHOR]

Patients with cervical cancer have a significant risk of tumor recurrence and metastasis. The discovery of effective treatments for cervical cancer is urgently needed. Poly(lactic-co-glycolic acid) (PLGA) was recently discovered to offer a promising therapeutic drug carrier. Hence, we developed dual-loaded PLGA nanoparticles (NPs) as a drug carrier to combat this problem with current chemotherapeutic drugs for cervical cancer. We engineered PLGA NPs with epidermal growth factor (EGF) functionalization and co-loaded them with SN-38 and perfluorocarbon (PC) to treat cervical cancer selectively. Cell counting kit-8 test results reveal that newly fabricated NPs effectively induce cell proliferation in cervical cancer cells. Further, flow cytometry, Hoechst 33342 staining and acridine orange and propidium iodide staining were used to determine the apoptosis of SN38/PC@EGF-PLGA NPs in HeLa and CaSki cells. And the release was pH-dependent when tested in vitro. Cervical cancer cells took up targeted SN38/PC@EGF-PLGA NPs at a greater rate than untargeted NPs. Furthermore, HeLa and CaSki cells were more sensitive to apoptosis induction and cell viability suppression when exposed to SN38/PC@EGF-PLGA NPs than nontargeted NPs. The findings of this study improve the exploration of SN38/PC@EGF-PLGA NPs in the new development of effective drug candidates for highly invasive cervical cancer in future. [ABSTRACT FROM AUTHOR]