Simulated microgravity increases heavy ion radiation-induced apoptosis in human B lymphoblasts

Aims

Microgravity and radiation, common in space, are the main factors influencing astronauts' health in space flight, but their combined effects on immune cells are extremely limited. Therefore, the effect of simulated microgravity on heavy ion radiation-induced apoptosis, and reactive oxygen species (ROS)-sensitive apoptosis signaling were investigated in human B lymphoblast HMy2.CIR cells.

Main methods

Simulated microgravity was achieved using a Rotating Wall Vessel Bioreactor at 37 °C for 30 min. Heavy carbon-ion irradiation was carried out at 300 MeV/u, with a linear energy transfer (LET) value of 30 keV/μm and a dose rate of 1 Gy/min. Cell survival was evaluated using the Trypan blue exclusion assay. Apoptosis was indicated by Annexin V/propidium iodide staining. ROS production was assessed by cytometry with a fluorescent probe dichlorofluorescein. Malondialdehyde was detected using a kit. Extracellular signal-regulated kinase (ERK), mitogen-activated protein kinase phosphatase-1 (MKP-1) and caspase-3 activation were measured by immunoblotting.

Key findings

Simulated microgravity decreased heavy ion radiation-induced cell survival and increased apoptosis in HMy2.CIR cells. It also amplified heavy ion radiation-elicited intracellular ROS generation, which induced ROS-sensitive ERK/MKP-1/caspase-3 activation in HMy2.CIR cells. The above phenomena could be reversed by the antioxidants N-acetyl cysteine (NAC) and quercetin.

Significance

These results illustrated that simulated microgravity increased heavy ion radiation-induced cell apoptosis, mediated by a ROS-sensitive signal pathway in human B lymphoblasts. Further, the antioxidants NAC and quercetin, especially NAC, might be good candidate drugs for protecting astronauts' and space travelers' health and safety.     

5′-Nitro-indirubinoxime induces G1 cell cycle arrest and apoptosis in salivary gland adenocarcinoma cells through the inhibition of Notch-1 signaling

Background

5′-Nitro-indirubinoxime (5′-NIO) is a new derivative of indirubin that exhibits anti-cancer activity in a variety of human cancer cells. However, its mechanism has not been fully clarified.

Methods

Human salivary gland adenocarcinoma (SGT) cells were used in this study. Western blot and RT-PCR analyses were performed to determine cellular Notch levels. The cell cycle stage and level of apoptosis were analyzed using flow cytometry analysis.

Results

5′-NIO significantly inhibited the mRNA levels of Notch-1 and Notch-3 and their ligands (Delta1, 2, 3, and Jagged-2) in SGT cells. Immunocytochemistry analysis showed that 5′-NIO specifically decreased the level of Notch-1 in the nucleus. In addition, 5′-NIO induced G1 cell cycle arrest by reducing levels of CDK4 and CDK6 in SGT cells. Using flow cytometry and immunoblotting analysis, we found that 5′-NIO induces apoptosis following the secretion of cytochrome c and the activation of caspase-3 and caspase-7. Intracellular Notch-1 overexpression led to a decrease in G1 phase arrest and an inhibition of 5′-NIO-induced apoptosis.

Conclusion

These observations suggest that 5′-NIO induces cell cycle arrest and apoptosis by down-regulating Notch-1 signaling.

General significance

This study identifies a new mechanism of 5′-NIO-mediated anti-tumor properties. Thus, 5′-NIO could be used as a candidate for salivary gland adenocarcinoma therapeutics.     

Interplay between autophagy and apoptosis mediated by copper oxide nanoparticles in human breast cancer cells MCF7

Background

Metal oxide nanoparticles are well known to generate oxidative stress and deregulate normal cellular activities. Among these, transition metals copper oxide nanoparticles (CuO NPs) are more compelling than others and able to modulate different cellular responses.

Methods

In this work, we have synthesized and characterized CuO NPs by various biophysical methods. These CuO NPs (~ 30 nm) induce autophagy in human breast cancer cell line, MCF7 in a time- and dose-dependent manner. Cellular autophagy was tested by MDC staining, induction of green fluorescent protein-light chain 3 (GFP-LC3B) foci by confocal microscopy, transfection of pBABE-puro mCherry-EGFP-LC3B plasmid and Western blotting of autophagy marker proteins LC3B, beclin1 and ATG5. Further, inhibition of autophagy by 3-MA decreased LD₅₀ doses of CuO NPs. Such cell death was associated with the induction of apoptosis as revealed by FACS analysis, cleavage of PARP, de-phosphorylation of Bad and increased cleavage product of caspase 3. siRNA mediated inhibition of autophagy related gene beclin1 also demonstrated similar results. Finally induction of apoptosis by 3-MA in CuO NP treated cells was observed by TEM.

Results

This study indicates that CuO NPs are a potent inducer of autophagy which may be a cellular defense against the CuO NP mediated toxicity and inhibition of autophagy switches the cellular response into apoptosis.

Conclusions

A combination of CuO NPs with the autophagy inhibitor is essential to induce apoptosis in breast cancer cells.

General significance

CuO NP induced autophagy is a survival strategy of MCF7 cells and inhibition of autophagy renders cellular fate to apoptosis.     

AD-1, a novel ginsenoside derivative,shows anti-lung cancer activity via activation of p38 MAPK pathway and generation of reactive oxygen species

Background

Ginseng is a traditional Chinese herb that has been used for thousands of years. In the present study, effects and mechanisms of AD-1 were evaluated for its development as a novel anti-lung cancer drug.

Methods

The cytotoxic activity was evaluated by MTT assay. Flow cytometry was employed to detect cell cycle, apoptosis and ROS. Western blot and immunohistochemistry were used to analyze signaling pathways. Lung cancer xenograft models were established by subcutaneous implantation of A549 or H292 cells into nude mice.

Results

AD-1 concentration-dependently reduces lung cancer cell viability without affecting normal human lung epithelial cell viability. In A549 and H292 lung cancer cells, AD-1 induces G₀/G₁ cell cycle arrest, apoptosis and ROS production. The apoptosis can be attenuated by a ROS scavenger — N-acetylcysteine (NAC). In addition, AD-1 up-regulates the expression of p38 and ERK phosphorylation. Addition of a p38 inhibitor SB203580, suppresses the AD-1-induced decrease in cell viability. Furthermore, genetic silencing of p38 attenuates the expression of p38 and decreases the AD-1-induced apoptosis. Treatment with NAC reduces AD-1-induced p38 phosphorylation, which indicates that ROS generation is involved in the AD-1-induced p38 activation. In mice, oral administration of AD-1 (10–40 mg/kg) dose-dependently inhibited the growth of xenograft tumors without affecting body weight and decreases the expression of VEGF, MMP-9 and CD34 in tumor tissue. TUNEL staining confirms that the tumors from AD-1 treated mice exhibit a markedly higher apoptotic index.

Conclusions and general significance

These data support development of AD-1 as a potential agent for lung cancer therapy.     

Stimulation of autophagic activity in human glioma cells by anti-proliferative ardipusilloside I isolated from Ardisia pusilla

Aims

Ardipusilloside I (ADS-I), a triterpenoid saponin isolated from Ardisia pusilla A.DC (Myrsinaceae), has been recently tested for cancer treatment including brain cancer. However, the mechanism of its action remains elusive. The present study was to investigate the role of autophagy activation in the anti-tumor activities of ADS-I in human glioma cells.

Main methods

The tetrazolium dye (MTT) colorimetric assay was used for the measurement of cell proliferation in cultured glioma cells, transmission electron microscopy (TEM) for the examination of autophagic activity, flow cytometric analysis for the determination of cell cycle and apoptotic cells, and immunocytochemistry and Western blot for protein expression of microtubule-associated protein light-chain 3 (LC3) and Beclin 1.

Key findings

ADS-I significantly inhibited the proliferation of both U373 and T98G glioma cells in cultures in a dose-dependent manner. The cytotoxic activity of ADS-I against glioma cell growth was associated not only with the induction of cell cycle arrest at G₂/M phase and cell apoptosis in flow cytometric analysis, but also with the activation of autophagy, indicated by the formation of autophagosomes and up-regulated expression of both autophagic protein Beclin 1 and LC3 in glioma cells. Additionally, the treatment with chloroquine, an autophagy inhibitor, reduced ADS-1-mediated cell death.

Significance

These data suggest that the anti-proliferative activity of ADS-I in human glioma cells is associated with the activation of autophagy in addition to cell cycle arrest and apoptosis, and the antagonistic effect of chloroquine suggests an important role of autophagy in ADS-I-mediated cell death against tumor growth.     

High glucose-induced Ca overload and oxidative stress contribute to apoptosis of cardiac cells through mitochondrial dependent and independent pathways

Background

Cardiac cell apoptosis is the initiating factor of cardiac complications especially diabetic cardiomyopathy. Mitochondria are susceptible to the damaging effects of elevated glucose condition. Calcium overload and oxidative insult are the two mutually non-exclusive phenomena suggested to cause cardiac dysfunction. Here, we examined the effect of high-glucose induced calcium overload in calpain-1 mediated cardiac apoptosis in an in vitro setting.

Methods

H9c2, rat ventricular myoblast cell line was treated with elevated glucose condition and the cellular consequences were studied. Intracellular calcium trafficking, ROS generation, calpain-1 activation and caspase-12 and caspase-9 pathway were studied using flow cytometry, confocal microscopy and Western blot analysis.

Results

High-glucose treatment resulted in increased intracellular calcium ([Ca^2 +]i) which was mobilized to the mitochondria. Concomitant intra-mitochondrial calcium ([Ca^2 +]m) increase resulted in enhanced reactive oxygen and nitrogen species generation. These events led to mitochondrial dysfunction and apoptosis. Cardiomyocyte death exhibited several classical markers of apoptosis, including activation of caspases, appearance of annexin V on the outer plasma membrane, increased population of cells with sub-G₀/G₁ DNA content and nuclear condensation. Key findings include elucidation of cell signaling mechanism of high-glucose induced calcium-dependent cysteine protease calpain-1 activation, which triggers non-conventional caspases as alternate mode of cell death.

Conclusion

This information increases the understanding of cardiac cell death under hyperglycemic condition and can possibly be extended for designing new therapeutic strategies for diabetic cardiomyopathy.

General significance

The novel findings of the study reveal that high glucose induces apoptosis by both mitochondria-dependent and independent pathways via concomitant rise in intracellular calcium.     

Low ATP level is sufficient to maintain the uncommitted state of multipotent mesenchymal stem cells

Background

Multipotent mesenchymal stromal cells (MMSCs) are minimally differentiated precursors with great potential to transdifferentiate. These cells are quite resistant to oxygen limitation, suggesting that a hypoxic milieu can be physiological for MMSCs.

Methods

Human MMSCs isolated from adipose tissue were grown at various oxygen concentrations. Alteration in cell immunophenotype was determined by flow cytometry after staining with specific antibodies. Concentrations of glucose and lactate were determined using the Biocon colorimetric test. Cellular respiration was assessed using oxygen electrode. The modes of cell death were analyzed by flow cytometry after staining with Annexin V and propidium iodide.

Results

We found that permanent oxygen deprivation attenuated cellular ATP levels in these cells, diminishing mitochondrial ATP production but stimulating glycolytic ATP production. At the same time, permanent hypoxia did not affect MMSCs' viability, stimulated their proliferation and reduced their capacity to differentiate. Further, permanent hypoxia decreased spontaneous cell death by MMSCs.

Conclusions

Under hypoxic conditions glycolysis provides sufficient energy to maintain MMSCs in an uncommitted state.

General significance

These findings are of interest not only for scientific reasons, but also in practical terms. Oxygen concentration makes an essential contribution to MMSC physiology and should be taken into account in the setting of protocols for cellular therapy.     

MicroRNA-34a induces apoptosis in the human glioma cell line,A172, through enhanced ROS production and NOX2 expression

Background

MicroRNA is a type of non-coding small RNA involved in regulating genes and signaling pathways through incomplete complementation with target genes. Recent research supports key roles of miRNA in the formation and development of human glioma.

Methods

The relative quantity of miR-34a was initially determined in human glioma A172 cells and glioma tissues. Next, we analyzed the impact of miR-34a on A172 cell viability with the MTT assay. The effects of miR-34a overexpression on apoptosis were confirmed with flow cytometry and Hoechst staining experiments. We further defined the target genes of miR-34a using immunofluorescence and Western blot.

Results

MiR-34a expression was significantly reduced in human glioma A172 cells and glioma tissue, compared with normal glial cells and tissue samples. Our MTT data suggest that up-regulation of miR-34a inhibits cell viability while suppression of miR-34a enhances cell viability. Flow cytometry and Hoechst staining results revealed increased rates of apoptosis in A172 human glioma cells overexpressing miR-34a. Using immunofluorescence and Western blot analyses, we identified NOX2 as a target of miR-34a in A172 cells.

Conclusion

MiR-34a serves as a tumor suppressor in human glioma mainly by decreasing NOX2 expression.     

Curcusone D,a novel ubiquitin–proteasome pathway inhibitor via ROS-induced DUB inhibition,is synergistic with bortezomib against multiple myeloma cell growth

Background

Ubiquitin–proteasome pathway (UPP) plays a very important role in the degradation of proteins. Finding novel UPP inhibitors is a promising strategy for treating multiple myeloma (MM).

Methods

Ub-YFP reporter assays were used as cellular UPP models. MM cell growth, apoptosis and overall death were evaluated with the MTS assay, Annexin V/PI dual-staining flow cytometry, poly (ADP-ribose) polymerase (PARP) cleavage, and PI uptake, respectively. The mechanism of UPP inhibition was analyzed by western blotting for ubiquitin, in vitro and cellular proteasomal and deubiquitinases (DUBs) activity assays. Cellular reactive oxygen species (ROS) were measured with H₂DCFDA.

Results

Curcusone D, identified as a novel UPP inhibitor, causes cell growth inhibition and apoptosis in MM cells. Curcusone D induced the accumulation of poly-ubiquitin-conjugated proteins but could not inhibit proteasomal activity in vitro or in cells. Interestingly, the mono-ubiquitin level and the total cellular DUB activity were significantly downregulated following curcusone D treatment. Furthermore, curcusone D could induce ROS, which were closely correlated with DUB inhibition that could be nearly completely reversed by NAC. Finally, curcusone D and the proteasomal inhibitor bortezomib showed a strong synergistic effect against MM cells.

Conclusions

Curcusone D is novel UPP inhibitor that acts via the ROS-induced inhibition of DUBs to produce strong growth inhibition and apoptosis of MM cells and synergize with bortezomib.

General significance

The anti-MM molecular mechanism study of curcusone D will promote combination therapies with different UPP inhibitors against MM and further support the concept of oxidative stress regulating the activity of DUBs.     

Circulating hormone adrenomedullin and its binding protein protect neural cells from hypoxia-induced apoptosis

Background

Brain ischemia is the underlying cause of neuron death during stroke and brain trauma. Neural cells exposed to ischemia can undergo apoptosis. Adrenomedullin (AM) in combination with its enhancing binding protein, AMBP-1, has been shown to reduce tissue damage in inflammation.

Methods

To evaluate a beneficial effect of AM/AMBP-1 administration in brain ischemia, we employed an in vitro model of neuronal hypoxia using differentiated human neuroblastoma SH-SY5Y cells.

Results

After exposure to 1% O₂ for 20 h, neural cells were injured with decreased ATP levels and increased LDH release. Pre-administration of AM/AMBP-1 significantly reduced hypoxia-induced cell injury. Moreover, AM/AMBP-1 treatment reduced the number of TUNEL-positive cells and activation of caspase-3, compared to cells exposed to hypoxia alone. AM/AMBP-1 prevented a reduction of cAMP levels and protein kinase A (PKA) activity in neural cells after hypoxia exposure. Correspondingly, an elevation of cAMP levels by forskolin protected neural cells from hypoxia-induced injury. Inhibition of PKA by KT5720 abolished the protective effect of AM/AMBP-1 on hypoxia-induced apoptosis.

Conclusions

AM/AMBP-1 elevates cAMP levels, followed by activating PKA, to protect neural cells from the injury caused by hypoxia.

General significance

AM/AMBP-1 may be used as therapeutic agents to prevent neuron damage from brain ischemia.     

Endoplasmic reticulum stress plays a role in the advanced glycation end product-induced inflammatory response in endothelial cells

Aims

Both advanced glycation end products (AGEs) and endoplasmic reticulum (ER) stress play important roles in the development of various diseases. This study aimed to clarify the consequence of AGE-induced ER stress and its underlying mechanisms in human umbilical venous endothelial cells (HUVECs).

Main methods

AGE-induced ER stress was assessed by the increased expression and activation of the ER stress marker proteins GRP78, IRE1α and JNK, which were detected using Western blot. NF-κB translocation was revealed using Western blot and immunofluorescent staining in IRE1α-knockdown HUVECs. The mechanism of AGE-induced ER stress was also explored by inhibiting the effect of reactive oxygen species (ROS) using NADPH oxidase 4 (Nox4) siRNA and the antioxidant reduced glutathione (GSH). The cellular ROS level was measured using flow cytometry.

Key findings

AGEs time- and dose-dependently enhanced the expression of GRP78 and increased the phosphorylation of IRE1α and its downstream signal JNK in HUVECs. siRNA-induced IRE1α down-regulation suppressed AGE-induced NF-κB p65 nuclear translocation. Inhibiting the ROS production using Nox4 siRNA or antagonizing ROS using GSH reduced cellular ROS level and attenuated AGE-induced GRP78 expression and IRE1α and JNK activation.

Significance

This study confirms that AGE-induced ER stress in HUVECs focuses on the ER stress-enhanced inflammatory response through JNK and NF-κB activation. It further reveals the involvement of ROS in the AGE-induced ER stress mechanism.     

The antiangiogenic activity of Kushecarpin D,a novel flavonoid isolated from Sophora flavescens Ait

Aims

Kushecarpin D (KD) is a novel flavonoid isolated from the traditional Chinese herbal medicine Kushen (the dried root of Sophora flavescens Ait). As part of our continuous effort to explore Chinese traditional medicinal herbs and to identify novel natural anticancer products, the antiangiogenic properties of KD were examined in vitro using a human umbilical vein endothelial cell line (ECV304).

Main methods

The SRB and Trypan Blue exclusion assays were used to evaluate the effect of KD on cell proliferation. The antiangiogenic activities of KD were evaluated through studies of cell migration, cell adhesion, and tube formation. DCFH-DA and DHE fluorescent assays were used to detect the reactive oxygen species (ROS) levels. Catalase activity was detected using the colorimetric ammonium molybdate method. Cell cycle and apoptosis were measured using flow cytometry and the Hoechst 33258 staining assay.

Key findings

The results indicated that KD showed antiangiogenic activity via inhibitory effects on cell proliferation, cell migration, cell adhesion, and tube formation. ROS levels were down-regulated and catalase activity was up-regulated after treatment with KD. The cell cycle was arrested at the G2/M phase, while no apoptosis was observed using the Hoechst 33258 staining assay or following the flow cytometric analysis of the sub-G1 proportion.

Significance

The antiangiogenic properties of KD, in combination with its anti-proliferative effect and ability to induce cell cycle arrest without inducing apoptosis, make it a good candidate for development as antitumor agent. However, further studies are essential to elucidate its mechanism of action.     

Selenoprotein W serves as an antioxidant in chicken myoblasts

Background

Selenoprotein W (SelW) was thought to play an antioxidant role in mammals. Because chicken SelW has no cysteine (Cys) at the residue 37 (Cys37) that is required for the presumed antioxidant function in mammals, this study was conducted to determine whether chicken SelW possessed the same function.

Methods

Small interfering RNAs (siRNAs) technology was applied to suppress the SelW expression in chicken embryonic myoblasts. Thereafter, these myoblasts were treated with different concentrations of H₂O₂ and assayed for cell viability, apoptosis rate, reactive oxygen species (ROS) status, and expression levels of apoptosis-related genes and proteins (Bax, Bcl-2, and caspase-3).

Results

Silencing of the myoblast SelW gene decreased their cell viability, and increased their apoptosis rate and susceptibility to H₂O₂. While the knockout down of SelW up-regulated Bax and caspase-3 and down-regulated Bcl-2, the induced oxidative injuries were alleviated by treatment with a ROS scavenger, N-acetyl-l-cysteine (NAC).

Conclusion

Chicken SelW protected embryonic myoblasts against cell apoptosis mediated by endogenous and exogenous H₂O₂.

General significance

Chicken SelW possesses antioxidant function similar to the mammalian homologues despite the lack of Cys37 in the peptide.     

Prohibitin 1 modulates mitochondrial stress-related autophagy in human colonic epithelial cells

Introduction

Autophagy is an adaptive response to extracellular and intracellular stress by which cytoplasmic components and organelles, including damaged mitochondria, are degraded to promote cell survival and restore cell homeostasis. Certain genes involved in autophagy confer susceptibility to Crohn''s disease. Reactive oxygen species and pro-inflammatory cytokines such as tumor necrosis factor α (TNFα), both of which are increased during active inflammatory bowel disease, promote cellular injury and autophagy via mitochondrial damage. Prohibitin (PHB), which plays a role in maintaining normal mitochondrial respiratory function, is decreased during active inflammatory bowel disease. Restoration of colonic epithelial PHB expression protects mice from experimental colitis and combats oxidative stress. In this study, we investigated the potential role of PHB in modulating mitochondrial stress-related autophagy in intestinal epithelial cells.

Methods

We measured autophagy activation in response to knockdown of PHB expression by RNA interference in Caco2-BBE and HCT116 WT and p53 null cells. The effect of exogenous PHB expression on TNFα- and IFNγ-induced autophagy was assessed. Autophagy was inhibited using Bafilomycin A₁ or siATG16L1 during PHB knockdown and the affect on intracellular oxidative stress, mitochondrial membrane potential, and cell viability were determined. The requirement of intracellular ROS in siPHB-induced autophagy was assessed using the ROS scavenger N-acetyl-L-cysteine.

Results

TNFα and IFNγ-induced autophagy inversely correlated with PHB protein expression. Exogenous PHB expression reduced basal autophagy and TNFα-induced autophagy. Gene silencing of PHB in epithelial cells induces mitochondrial autophagy via increased intracellular ROS. Inhibition of autophagy during PHB knockdown exacerbates mitochondrial depolarization and reduces cell viability.

Conclusions

Decreased PHB levels coupled with dysfunctional autophagy renders intestinal epithelial cells susceptible to mitochondrial damage and cytotoxicity. Repletion of PHB may represent a therapeutic approach to combat oxidant and cytokine-induced mitochondrial damage in diseases such as inflammatory bowel disease.     

Stimulation by P2X7 receptors of calcium-dependent production of reactive oxygen species (ROS) in rat submandibular glands

Background

Agonists of P2X₇ receptors increase the production of reactive oxygen species (ROS) in immunocytes. In this work we tested this response and its effect on mitochondrial inner membrane potential (Δψ_m) in exocrine glands.

Methods

The production of ROS by rat submandibular glands was investigated by measuring the oxidation of dichlorodihydrofluorescein (DCFH), a fluorescent probe. The Δψ_m was estimated with tetramethylrhodamine.

Results

Activation of P2X₇ receptors by ATP or Bz-ATP increased the production of ROS. This response was not modified by inhibitors of phospholipase A2 or of various kinases. The effect of ATP was calcium-dependent and was blocked by diphenyliodonium, an inhibitor of flavoproteins. It was not affected by rotenone, an inhibitor of the complex I of the mitochondrial electron transfer chain. Scavengers of ROS had no effect on the dissipation of Δψ_m by ATP.

Conclusions

We conclude that, in rat submandibular glands, P2X₇ receptors stimulate in a calcium-dependent manner an oxidase generating ROS, suggesting the involvement of the dual oxidase Duox2. The production of ROS does not contribute to the depolarization of mitochondria by purinergic agonists.

General significance

Purinergic receptors could be regulators of the bactericidal properties of saliva by promoting both the secretion of peroxidase from acinar cells and by activating Duox2.     

Pigment-Epithelium Derived Factor (PEDF) and the human ovary: A role in the generation of ROS in granulosa cells

Aims

Pigment Epithelium Derived Factor (PEDF) is a multifunctional factor, which was found in mouse ovary and in human ovarian follicular fluid (FF). Its ovarian functions include anti-angiogenic actions. This study aimed to explore other PEDF-actions and the sites of PEDF expression in the human ovary.

Materials and methods

We used paraffin-embedded human ovarian sections for PEDF-immunohistochemistry and IVF-derived human granulosa cells (GCs) for RT-PCR, Western blotting and functional studies, including measurement of cell viability (ATP-assay), apoptosis (caspase-assay) and reactive oxygen species (ROS).

Key findings

Immunohistochemistry revealed PEDF in the cytoplasm of GCs of avascular follicles from the preantral to the antral stage and in FF. PEDF was also found in luteinized GCs of the highly vascularized corpus luteum, a result not in line with a sole anti-angiogenic action. Like GCs in vivo, cultured human luteinizing GCs express PEDF. They also responded to exogenous recombinant PEDF. In low concentrations PEDF did not affect cell viability but caused generation ROS. ROS-induction by PEDF was a concentration-dependent process and may be due to the activity of NADPH oxidase (NOX) type 4 and/or 5, which as we found are expressed by GCs. An antioxidant and apocynin, which inhibits NOX, blocked ROS generation. High levels of exogenous recombinant PEDF induced apoptosis of GCs, which was prevented by antioxidants, implying involvement of ROS.

Significance

PEDF is emerging as an ovarian factor, which has unexpected ROS-augmenting activities in the human ovary. It may be involved in ovarian ROS homeostasis and may contribute to oxidative stress.     

Concurrence of autophagy with apoptosis in alveolar epithelial cells contributes to chronic pulmonary toxicity induced by methamphetamine

Objectives

Methamphetamine (MA) abuse evokes pulmonary toxicity. The aim of our study is to investigate if autophagy is induced by MA and if autophagy‐initiated apoptosis in alveolar epithelial cells is involved in MA‐induced chronic pulmonary toxicity.

Materials and Methods

The rats in Control group and MA group were tested by Doppler and HE staining. The alveolar epithelial cells were treated with MA, following by western blot, RT‐PCR and immunofluorescence assay.

Results

Chronic exposure to MA resulted in lower growth ratio of weight and in higher heart rate and peak blood flow velocity of the main pulmonary artery of rats. MA induced infiltration of inflammatory cells in lungs, more compact lung parenchyma, thickened alveolar septum and reduction in the number of alveolar sacs. In alveolar epithelial cells, the autophagy marker LC3 and per cent of cells containing LC3‐positive autophagosome were significantly increased. MA dose dependently suppressed the phosphorylation of mTOR to inactivate mTOR, elicited autophagy regulatory proteins LC3 and Beclin‐1, accelerated the transformation from LC3 I to LC3 II and initiated apoptosis by decreasing Bcl‐2 and increasing Bax, Bax/Bcl‐2 and cleaved Caspase 3. The above results suggest that sustained autophagy was induced by long‐term exposure to MA and that the increased Beclin‐1 autophagy initiated apoptosis in alveolar epithelial cells.

Conclusions

Concurrence of autophagy with apoptosis in alveolar epithelial cells contributes to chronic pulmonary toxicity induced by MA.