Cadmium Activates Reactive Oxygen Species-dependent AKT/mT OR and Mitochondrial Apoptotic Pathways in Neuronal Cells

Objective To examine the role of Cd-induced reactive oxygen species（ROS） generation in the apoptosis of neuronal cells. Methods Neuronal cells（primary rat cerebral cortical neurons and PC12 cells） were incubated with or without Cd post-pretreatment with rapamycin（Rap） or N-acetyl-L-cysteine（NAC）. Cell viability was determined by MTT assay, apoptosis was examined using flow cytometry and fluorescence microscopy, and the activation of phosphoinositide 3’-kinase/protein kinase B（Akt）/mammalian target of rapamycin（m TOR） and mitochondrial apoptotic pathways were measured by western blotting or immunofluorescence assays. Results Cd-induced activation of Akt/m TOR signaling, including Akt, m TOR, p70 S6 kinase（p70 S6K）, and eukaryotic initiation factor 4E binding protein 1（4E-BP1）. Rap, an m TOR inhibitor and NAC, a ROS scavenger, blocked Cd-induced activation of Akt/m TOR signaling and apoptosis of neuronal cells. Furthermore, NAC blocked the decrease of B-cell lymphoma 2/Bcl-2 associated X protein（Bcl-2/Bax） ratio, release of cytochrome c, cleavage of caspase-3 and poly（ADP-ribose） polymerase（PARP）, and nuclear translocation of apoptosis-inducing factor（AIF） and endonuclease G（Endo G）. Conclusion Cd-induced ROS generation activates Akt/m TOR and mitochondrial pathways, leading to apoptosis of neuronal cells. Our findings suggest that m TOR inhibitors or antioxidants have potential for preventing Cd-induced neurodegenerative diseases.

Amyloid precursor protein regulates 5-fluorouracil resistance in human hepatocellular carcinoma cells by inhibiting the mitochondrial apoptotic pathway

Hepatocellular carcinoma(HCC)is a malignant tumor with high morbidity and mortality globally.It accounts for the majority of primary liver cancer cases.Amyloid precursor protein(APP),a cell membrane protein,plays a vital role in the pathogenesis of Alzheimer’s disease,and has been found to be implicated in tumor growth and metastasis.Therefore,to understand the relationship between APP and 5-fluorouracil(5-FU)resistance in liver cancer,Cell Counting Kit-8,apoptosis and cell cycle assays,western blotting,and reverse transcription-quantitative polymerase chain reaction(q PCR)analysis were performed.The results demonstrated that APP expression in Bel7402-5-FU cells was significantly up-regulated,as compared with that in Bel7402 cells.Through successful construction of APP-silenced(si APP)and overexpressed(OE)Bel7402 cell lines,data revealed that the Bel7402-APP751-OE cell line was insensitive,while the Bel7402-si APP cell line was sensitive to 5-FU in comparison to the matched control group.Furthermore,APP overexpression decreased,while APP silencing increased 5-FU-induced apoptosis in Bel7402 cells.Mechanistically,APP overexpression and silencing can regulate the mitochondrial apoptotic pathway and the expression of apoptotic suppressor genes(B-cell lymphoma-2(Bcl-2)and B-cell lymphoma-extra large(Bcl-xl)).Taken together,these results preliminarily revealed that APP overexpression contributes to the resistance of liver cancer cells to 5-FU,providing a new perspective for drug resistance.

Effect of resveratrol treatment on apoptosis and apoptotic pathways during boar semen freezing

Resveratrol(3,5,4’-trihydroxystilbene,RSV) has been widely used in mammalian cells,but whether it can be used during freezing boar semen is still unknown.The effects of RSV treatment during boar semen freezing on its anti-freezing ability,apoptosis,and possible apoptotic pathways were observed in this study.Sperm motility,mitochondrial membrane potential(ΔΨm),adenosine triphosphate(ATP) content,terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL)-positive apoptotic state,and messenger RNA(mRNA) expression levels of apoptotic genes involved in different apoptotic pathways after freezing with or without RSV treatment were tested.The results showed that:(1) Compared with fresh sperm,the motility,normal acrosome rate,and plasma membrane integrity rate of frozen boar sperm decreased significantly(P<0.05),and RSV did not significantly increase the sperm motility(0.44 vs.0.40,P>0.05),but it did significantly improve the normal acrosome rate(57.65% vs.47.00%,P<0.05) and plasma membrane integrity rate(46.67% vs.38.85%,P<0.05).(2) After freezing,most boar sperm showed low mitochondrial ΔΨm.RSV treatment could increase the rate of high mitochondrial ΔΨm of boar sperm.(3) RSV treatment significantly decreased reactive oxygen species(ROS) levels(58.65% vs.88.41%,P<0.05)and increased the ATP content(0.49 μmol/L vs.0.25 μmol/L,P<0.05) of boar sperm during freezing.(4) The apoptotic rate of the freezing group(80.41%) with TUNEL detection increased significantly compared to the fresh group(9.70%,P<0.05),and RSV treatment greatly decreased the apoptotic rate(68.32%,P<0.05).(5) Real-time polymerase chain reaction(RT-PCR) showed that not only the genes from the death receptor-mediated apoptotic pathway(tumor necrosis factor-α(TNF-α),Fas ligand(FasL),and Caspase-8),but also the genes from the mitochondria-mediated apoptotic pathway(manganese superoxide dismutase(MnSOD),B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),and Caspase-9) were both significantly changed after freezing.RSV treatment during freezing greatly changed their expression levels.Although RSV treatment during boar semen freezing did not significantly increase motility after thawing,it still played an efficient antioxidant role,which could enhance the mitochondrial function and decrease the apoptotic level induced by both the death receptor-and mitochondria-mediated apoptotic pathways.

Thermotherapy enhances oxaliplatin-induced cytotoxicity in human colon carcinoma cells

AIM： To observe the synergistic effects of hyperthermia in oxaliplatin-induced cytotoxicity in human colon adenocarcinoma Lovo cells.

Mitochondrial superoxide anions induced by exogenous oxidative stress determine tumor cell fate: an individual cell-based study

Objective: Reactive oxygen species(ROS) are involved in a variety of biological phenomena and serve both deleterious and beneficial roles. ROS quantification and assessment of reaction networks are desirable but difficult because of their short half-life and high reactivity. Here, we describe a pro-oxidative model in a single human lung carcinoma SPC-A-1 cell that was created by application of extracellular H2O2 stimuli. Methods: Modified microfluidics and imaging techniques were used to determine O2·- levels and construct an O2^·- reaction network. To elucidate the consequences of increased O2^·- input, the mitochondria were given a central role in the oxidative stress mode, by manipulating mitochondria-interrelated cytosolic Ca2+ levels, mitochondrial Ca^2+ uptake, auto-amplification of intracellular ROS and the intrinsic apoptotic pathway. Results and conclusions: Results from a modified microchip demonstrated that 1 mmol/L H·-2 O2 induced a rapid increase in cellular O2 levels(>27 vs.>406 amol in 20 min), leading to increased cellular oxidizing power(evaluated by ROS levels) and decreased reducing power(evaluated by glutathione(GSH) levels). In addition, we examined the dynamics of cytosolic Ca^2+ and mitochondrial Ca^2+ by confocal laser scanning microscopy and confirmed that Ca^2+ stores in the endoplasmic reticulum were the primary source of H2O2-induced cytosolic Ca^2+ bursts. It is clear that mitochondria have pivotal roles in determining how exogenous oxidative stress affects cell fate. The stress response involves the transfer of Ca^2+ signals between organelles,ROS auto-amplification, mitochondrial dysfunction, and a caspase-dependent apoptotic pathway.