Coordinate Autophagy and mTOR Pathway Inhibition Enhances Cell Death in Melanoma

The phosphatidylinositol 3-kinase/AKT/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway promotes melanoma tumor growth and survival while suppressing autophagy, a catabolic process through which cells collect and recycle cellular components to sustain energy homeostasis in starvation. Conversely, inhibitors of the PI3K/AKT/mTOR pathway, in particular the mTOR inhibitor temsirolimus (CCI-779), induce autophagy, which can promote tumor survival and thus, these agents potentially limit their own efficacy. We hypothesized that inhibition of autophagy in combination with mTOR inhibition would block this tumor survival mechanism and hence improve the cytotoxicity of mTOR inhibitors in melanoma. Here we found that melanoma cell lines of multiple genotypes exhibit high basal levels of autophagy. Knockdown of expression of the essential autophagy gene product ATG7 resulted in cell death, indicating that survival of melanoma cells is autophagy-dependent. We also found that the lysosomotropic agent and autophagy inhibitor hydroxychloroquine (HCQ) synergizes with CCI-779 and led to melanoma cell death via apoptosis. Combination treatment with CCI-779 and HCQ suppressed melanoma growth and induced cell death both in 3-dimensional (3D) spheroid cultures and in tumor xenografts. These data suggest that coordinate inhibition of the mTOR and autophagy pathways promotes apoptosis and could be a new therapeutic paradigm for the treatment of melanoma.     

NADPH oxidases contribute to autophagy regulation

Reactive oxygen species (ROS) are emerging as regulators of autophagy in various cellular contexts. There are many cellular sources of ROS in eukaryotic cells. In phagocytes, the critical immune cells for host defense, the Nox2 NADPH oxidase generates ROS during phagocytosis and plays a central role in microbial killing. Toll-like receptors (TLRs) are important membrane microbial sensing receptors, which can activate Nox2,¹ and were recently demonstrated to signal autophagy targeting of phagosomes to promote their maturation.² Our recent study reveals that Nox2 activity and its generated ROS are key signals that induce TLR-activated autophagy of phagosomes. Our results provide the first evidence that ROS from the Nox2 NADPH oxidase can contribute to regulating autophagy in host defense against bacteria. The association of TLR, Nox2 and autophagy with inflammatory bowel disease (IBD) suggests a significant role of this antibacterial pathway in these diseases.     

Autophagy Is a Protective Mechanism for Human Melanoma Cells under Acidic Stress

Cyclic hypoxia and alterations in oncogenic signaling contribute to switch cancer cell metabolism from oxidative phosphorylation to aerobic glycolysis. A major consequence of up-regulated glycolysis is the increased production of metabolic acids responsible for the presence of acidic areas within solid tumors. Tumor acidosis is an important determinant of tumor progression and tumor pH regulation is being investigated as a therapeutic target. Autophagy is a cellular catabolic pathway leading to lysosomal degradation and recycling of proteins and organelles, currently considered an important survival mechanism in cancer cells under metabolic stress or subjected to chemotherapy. We investigated the response of human melanoma cells cultured in acidic conditions in terms of survival and autophagy regulation. Melanoma cells exposed to acidic culture conditions (7.0 < pH < 6.2) promptly accumulated LC3+ autophagic vesicles. Immunoblot analysis showed a consistent increase of LC3-II in acidic culture conditions as compared with cells at normal pH. Inhibition of lysosomal acidification by bafilomycin A1 further increased LC3-II accumulation, suggesting an active autophagic flux in cells under acidic stress. Acute exposure to acidic stress induced rapid inhibition of the mammalian target of rapamycin signaling pathway detected by decreased phosphorylation of p70S6K and increased phosphorylation of AMP-activated protein kinase, associated with decreased ATP content and reduced glucose and leucine uptake. Inhibition of autophagy by knockdown of the autophagic gene ATG5 consistently reduced melanoma cell survival in low pH conditions. These observations indicate that induction of autophagy may represent an adaptation mechanism for cancer cells exposed to an acidic environment. Our data strengthen the validity of therapeutic strategies targeting tumor pH regulation and autophagy in progressive malignancies.     

Inhibition of autophagosome-lysosome fusion by ginsenoside Ro via the ESR2-NCF1-ROS pathway sensitizes esophageal cancer cells to 5-fluorouracil-induced cell death via the CHEK1-mediated DNA damage checkpoint

Modulation of autophagy has been increasingly regarded as a promising cancer therapeutic approach. In this study, we screened several ginsenosides extracted from Panax ginseng and identified ginsenoside Ro (Ro) as a novel autophagy inhibitor. Ro blocked the autophagosome-lysosome fusion process by raising lysosomal pH and attenuating lysosomal cathepsin activity, resulting in the accumulation of the autophagosome marker MAP1LC3B/LC3B and SQSTM1/p62 (sequestosome 1) in various esophageal cancer cell lines. More detailed studies demonstrated that Ro activated ESR2 (estrogen receptor 2), which led to the activation of NCF1/p47^PHOX (neutrophil cytosolic factor 1), a subunit of NADPH oxidase, and subsequent reactive oxygen species (ROS) production. Treatment with siRNAs or inhibitors of the ESR2-NCF1-ROS axis, such as N-acetyl-L-cysteine (NAC), diphenyleneiodonium chloride (DPI), apocynin (ACN), Tiron, and Fulvestrant apparently decreased Ro-induced LC3B-II, GFP-LC3B puncta, and SQSTM1, indicating that ROS instigates autophagic flux inhibition triggered by Ro. More importantly, suppression of autophagy by Ro sensitized 5-fluorouracil (5-Fu)-induced cell death in chemoresistant esophageal cancer cells. 5-Fu induced prosurvival autophagy, and by inhibiting such autophagy, siRNAs against BECN1/beclin 1, ATG5, ATG7, and LC3B enhanced 5-Fu-induced autophagy-associated and apoptosis-independent cell death. We observed that Ro potentiates 5-Fu cytotoxicity via delaying CHEK1 (checkpoint kinase 1) degradation and downregulating DNA replication process, resulting in the delayed DNA repair and the accumulation of DNA damage. In summary, these data suggest that Ro is a novel autophagy inhibitor and could function as a potent anticancer agent in combination therapy to overcome chemoresistance.     

Autophagy-Regulated ROS from Xanthine Oxidase Acts as an Early Effector for Triggering Late Mitochondria-Dependent Apoptosis in Cathepsin S-Targeted Tumor Cells

Cathepsin S (CTSS), which is highly expressed in various malignant tumor cells, has been proposed to promote tumor progression, migration, and invasion. CTSS inhibition not only blocks tumor cell invasion and endothelial tube formation but also induces cellular cytotoxicity. In our previous studies, we have observed that CTSS inhibition induces autophagy, which is responsible for up-regulating xanthine oxidase for early ROS generation and consequent cell death. However, whether the autophagy-regulated early ROS triggers apoptosis remains unclear. We conducted a long-term follow-up study to investigate the relationship between early autophagy and late mitochondria-dependent apoptosis. We demonstrated that early ROS generation is critical for mitochondria damage and the activation of intrinsic apoptotic pathway. Attenuating the early ROS level diminished later mitochondrial damage and downstream apoptotic signaling. Collectively, mitochondria-dependent apoptosis is regulated by autophagy-regulated early ROS, which serves as an early effector that triggers mitochondrial signaling for late apoptosis. The data emphasize the essential role of autophagy-regulated early ROS in triggering late apoptotic signaling.     

NOX4 regulates autophagy during energy deprivation

NADPH oxidase is a cellular enzyme devoted to the production of reactive oxygen species (ROS). NOX4 and NOX2 are the main isoforms of NADPH oxidase in the cardiovascular system. In our recent study, we demonstrated that NOX4, but not NOX2, is a critical mediator of the cardiomyocyte adaptive response to energy stress. NOX4 activity and protein levels are increased in the endoplasmic reticulum (ER) but not in mitochondria of cardiomyocytes during the early phase of energy deprivation. NOX4-derived production of ROS in the ER is a critical event that activates autophagy through stimulation of the EIF2AK3/PERK-EIF2S1/eIF-2α-ATF4 pathway. NOX4-dependent autophagy is an important mechanism to preserve cellular energy and limit cell death in energy-deprived cardiomyocytes. Aside from elucidating a crucial physiological function of NOX4 during cellular energy stress, our study dissects a novel signaling mechanism that regulates autophagy under this condition.     

NADPH氧化酶与ROS信号区域化

最近有关活性氧物质 (ROS)的研究取得了突飞猛进的进展,尤其是其作为第二信使介导了许多生理性与病理性细胞事件,包括细胞分化、过度生长、增殖及凋亡.为了避免ROS的毒性产生特异性的信号转导,ROS的产生与代谢必须被严格调控;其具体的调控机制一直是人们关注的焦点. 最近有关ROS区域化观点的提出解决了这一问题. NADPH是生成ROS的主要来源. 研究发现,NADPH氧化酶及其衍生的ROS存在于机体的多种组织内,且在细胞中呈区域化分布,对细胞内信号的精确调控具有至关重要的作用. NADPH一方面通过小窝/脂筏组装成功能型复合物,从而产生ROS区域化;另一方面,NADPH通过其不同亚细胞定位亚基与各种靶蛋白之间的相互作用,产生ROS特异性. 本文系统综述了NADPH衍生的ROS信号区域化,为进一步理解ROS信号在各种生理或病理过程的分子调控机制提供理论依据.     

Increasing Glucose 6-Phosphate Dehydrogenase Activity Restores Redox Balance in Vascular Endothelial Cells Exposed to High Glucose

Previous studies have shown that high glucose increases reactive oxygen species (ROS) in endothelial cells that contributes to vascular dysfunction and atherosclerosis. Accumulation of ROS is due to dysregulated redox balance between ROS-producing systems and antioxidant systems. Previous research from our laboratory has shown that high glucose decreases the principal cellular reductant, NADPH by impairing the activity of glucose 6-phosphate dehydrogenase (G6PD). We and others also have shown that the high glucose-induced decrease in G6PD activity is mediated, at least in part, by cAMP-dependent protein kinase A (PKA). As both the major antioxidant enzymes and NADPH oxidase, a major source of ROS, use NADPH as substrate, we explored whether G6PD activity was a critical mediator of redox balance. We found that overexpression of G6PD by pAD-G6PD infection restored redox balance. Moreover inhibition of PKA decreased ROS accumulation and increased redox enzymes, while not altering the protein expression level of redox enzymes. Interestingly, high glucose stimulated an increase in NADPH oxidase (NOX) and colocalization of G6PD with NOX, which was inhibited by the PKA inhibitor. Lastly, inhibition of PKA ameliorated high glucose mediated increase in cell death and inhibition of cell growth. These studies illustrate that increasing G6PD activity restores redox balance in endothelial cells exposed to high glucose, which is a potentially important therapeutic target to protect ECs from the deleterious effects of high glucose.     

Carotenoid deficiency triggers autophagy in the model green alga Chlamydomonas reinhardtii

All aerobic organisms have developed sophisticated mechanisms to prevent, detect and respond to cell damage caused by the unavoidable production of reactive oxygen species (ROS). Plants and algae are able to synthesize specific pigments in the chloroplast called carotenoids to prevent photo-oxidative damage caused by highly reactive by-products of photosynthesis. In this study we used the unicellular green alga Chlamydomonas reinhardtii to demonstrate that defects in carotenoid biosynthesis lead to the activation of autophagy, a membrane-trafficking process that participates in the recycling and degradation of damaged or toxic cellular components. Carotenoid depletion caused by either the mutation of phytoene synthase or the inhibition of phytoene desaturase by the herbicide norflurazon, resulted in a strong induction of autophagy. We found that high light transiently activates autophagy in wild-type Chlamydomonas cells as part of an adaptation response to this stress. Our results showed that a Chlamydomonas mutant defective in the synthesis of specific carotenoids that accumulate during high light stress exhibits constitutive autophagy. Moreover, inhibition of the ROS-generating NADPH oxidase partially reduced the autophagy induction associated to carotenoid deficiency, which revealed a link between photo-oxidative damage, ROS accumulation and autophagy activation in Chlamydomonas cells with a reduced carotenoid content.     

(?)-Epigallocatechin-3-Gallate Induces Non-Apoptotic Cell Death in Human Cancer Cells via ROS-Mediated Lysosomal Membrane Permeabilization

(−)-Epigallocatechin-3-gallate (EGCG) is the most extensive studied tea polyphenol for its anti-cancer function. In this study, we report a novel mechanism of action for EGCG-mediated cell death by identifying the critical role of lysosomal membrane permeabilization (LMP). First, EGCG-induced cell death in human cancer cells (both HepG2 and HeLa) was found to be caspase-independent and accompanied by evident cytosolic vacuolization, only observable when cells were treated in serum-free medium. The cytosolic vacuolization observed in EGCG-treated cells was most probably caused by lysosomal dilation. Interestingly, EGCG was able to disrupt autophagic flux at the degradation stage by impairment of lysosomal function, and EGCG-induced cell death was independent of Atg5 or autophagy. The key finding of this study is that EGCG is able to trigger LMP, as evidenced by Lyso-Tracker Red staining, cathepsin D cytosolic translocation and cytosolic acidification. Consistently, a lysosomotropic agent, chloroquine, effectively rescues the cell death via suppressing LMP-caused cytosolic acidification. Lastly, we found that EGCG promotes production of intracellular ROS upstream of LMP and cell death, as evidenced by increased level of ROS in cells treated with EGCG and the protective effects of antioxidant N-acetylcysteine (NAC) against EGCG-mediated LMP and cell death. Taken together, data from our study reveal a novel mechanism underlying EGCG-induced cell death involving ROS and LMP. Therefore, understanding this lysosome-associated cell death pathway shed new lights on the anti-cancer effects of EGCG.     

Carotenoid deficiency triggers autophagy in the model green alga Chlamydomonas reinhardtii

All aerobic organisms have developed sophisticated mechanisms to prevent, detect and respond to cell damage caused by the unavoidable production of reactive oxygen species (ROS). Plants and algae are able to synthesize specific pigments in the chloroplast called carotenoids to prevent photo-oxidative damage caused by highly reactive by-products of photosynthesis. In this study we used the unicellular green alga Chlamydomonas reinhardtii to demonstrate that defects in carotenoid biosynthesis lead to the activation of autophagy, a membrane-trafficking process that participates in the recycling and degradation of damaged or toxic cellular components. Carotenoid depletion caused by either the mutation of phytoene synthase or the inhibition of phytoene desaturase by the herbicide norflurazon, resulted in a strong induction of autophagy. We found that high light transiently activates autophagy in wild-type Chlamydomonas cells as part of an adaptation response to this stress. Our results showed that a Chlamydomonas mutant defective in the synthesis of specific carotenoids that accumulate during high light stress exhibits constitutive autophagy. Moreover, inhibition of the ROS-generating NADPH oxidase partially reduced the autophagy induction associated to carotenoid deficiency, which revealed a link between photo-oxidative damage, ROS accumulation and autophagy activation in Chlamydomonas cells with a reduced carotenoid content.     

Inhibition of Oxidative Phosphorylation Induces a Rapid Death of GA-Pretreated Aleurone Cells,But Not of ABA-Pretreated Aleurone Cells

Reactive oxygen species (ROS) mediate programmed cell death in aleurone cells, which is promoted by gibberellic acid (GA) and prevented by abscisic acid (ABA). Plant mitochondria contain two distinct respiratory pathways: respiration through cytochrome c oxidase increases ROS production, whereas respiration through the alternative oxidase pathway lowers it. While studying the effects of GA and ABA on partitioning of respiration between those two pathways during the germinating process, we discovered that oxidative phosphorylation inhibitors like sodium azide and 2, 4-dinitrophenol induce rapid death of GA-pretreated aleurone cells but not of ABA-pretreated cells. Functional aerobic respiration was required for GA signaling, and 6 to 12 hours of GA signaling altered the cellular state of aleurone cells to be extremely susceptible to inhibition of oxidative phosphorylation. Anaerobic conditions were also able to mimic the effects of respiratory inhibitors in specifically inducing cell death in GA-treated cells, but cell death was provoked much more slowly. Cotreatment with various antioxidants did not prevent this process at all, suggesting that no ROS are responsible for this respiratory inhibitor-induced cell death. Our observation implicates that GA may partition all the electrons produced during mitochondrial respiration only to the cytochrome oxidase pathway, which would at least partly contribute to cellular accumulation of ROS.     

Cadmium affects tobacco cells by a series of three waves of reactive oxygen species that contribute to cytotoxicity

Cadmium is suspected to exert its toxic action on cells through oxidative damage. However, the transition metal is unable to directly generate reactive oxygen species (ROS) via redox reactions with molecular oxygen in a biological environment. Here, we show that bright yellow-2 (BY-2) tobacco cells exposed to millimolar concentrations of CdCl(2) developed cell death within 2-3 h. The death process was preceded by two successive waves of ROS differing in their nature and subcellular localization. Firstly, these consisted in the transient NADPH oxidase-dependent accumulation of H(2)O(2) followed by the accumulation of O(2) (-*) in mitochondria. A third wave of ROS consisting in fatty acid hydroperoxide accumulation was concomitant with cell death. Accumulation of H(2)O(2) was preceded by an increase in cytosolic free calcium concentration originating from internal pools that was essential to activate the NADPH oxidase. The cell line gp3, impaired in NADPH oxidase activity, and that was unable to accumulate H(2)O(2) in response to Cd(2+), was nevertheless poisoned by the metal. Therefore, this first wave of ROS was not sufficient to trigger all the cadmium-dependent deleterious effects. However, we show that the accumulation of O(2) (-*) of mitochondrial origin and membrane peroxidation are key players in Cd(2+)-induced cell death.     

Rac1-NADPH oxidase-regulated generation of reactive oxygen species mediates glutamate-induced apoptosis in SH-SY5Y human neuroblastoma cells

Reactive oxygen species (ROS) are known to play an important role in glutamate-induced neuronal cell death. In the present study, we examined whether NADPH oxidase serves as a source of ROS production and plays a role in glutamate-induced cell death in SH-SY5Y human neuroblastoma cells. Stimulation of the cells with glutamate (100 mM) induced apoptotic cell death and increase in the level of ROS, and these effects of glutamate were significantly suppressed by the inhibitors of the NADPH oxidase, diphenylene iodonium, apocynin, and neopterine. In addition, RT-PCR revealed that SH-SY5Y cells expressed mRNA of gp91^phox, p22^phox and cytosolic p47^phox, p67^phox and p40^phox, the components of the plasma membrane NADPH oxidase. Treatment with glutamate also resulted in activation and translocation of Rac1 to the plasma membrane. Moreover, the expression of Rac1N17, a dominant negative mutant of Rac1, significantly blocked the glutamate-induced ROS generation and cell death. Collectively, these results suggest that the plasma membrane-bound NADPH oxidase complex may play an essential role in the glutamate-induced apoptotic cell death through increased production of ROS.     

Autophagy protects auditory hair cells against neomycin-induced damage

Aminoglycosides are toxic to sensory hair cells (HCs). Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. However, the role of autophagy in aminoglycoside-induced HC injury is unknown. Here, we first found that autophagy activity was significantly increased, including enhanced autophagosome-lysosome fusion, in both cochlear HCs and HEI-OC-1 cells after neomycin or gentamicin injury, suggesting that autophagy might be correlated with aminoglycoside-induced cell death. We then used rapamycin, an autophagy activator, to increase the autophagy activity and found that the ROS levels, apoptosis, and cell death were significantly decreased after neomycin or gentamicin injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) or knockdown of autophagy-related (ATG) proteins resulted in reduced autophagy activity and significantly increased ROS levels, apoptosis, and cell death after neomycin or gentamicin injury. Finally, after neomycin injury, the antioxidant N-acetylcysteine could successfully prevent the increased apoptosis and HC loss induced by 3-MA treatment or ATG knockdown, suggesting that autophagy protects against neomycin-induced HC damage by inhibiting oxidative stress. We also found that the dysfunctional mitochondria were not eliminated by selective autophagy (mitophagy) in HEI-OC-1 cells after neomycin treatment, suggesting that autophagy might not directly target the damaged mitochondria for degradation. This study demonstrates that moderate ROS levels can promote autophagy to recycle damaged cellular constituents and maintain cellular homeostasis, while the induction of autophagy can inhibit apoptosis and protect the HCs by suppressing ROS accumulation after aminoglycoside injury.     

Oxidatively truncated phospholipids are required agents of tumor necrosis factor α (TNFα)-induced apoptosis

TNFα generates reactive oxygen species (ROS) at the cell surface that induce cell death, but how ROS communicate to mitochondria and their specific apoptotic action(s) are both undefined. ROS oxidize phospholipids to hydroperoxides that are friable and fragment adjacent to the (hydro)peroxide function, forming truncated phospholipids, such as azelaoyl phosphatidylcholine (Az-PC). Az-PC is relatively soluble, and exogenous Az-PC rapidly enters cells to damage mitochondrial integrity and initiate intrinsic apoptosis. We determined whether this toxic phospholipid is formed within cells during TNFα stimulation in sufficient quantities to induce apoptosis and if they are essential in TNFα-induced cytotoxicity. We found that TNFα induced ROS formation and phospholipid peroxidation in Jurkat cells, and either chemical interference with NADPH oxidase activity or siRNA suppression of the NADPH oxidase-4 subunit blocked ROS accumulation and phospholipid peroxidation. Mass spectrometry showed that phospholipid peroxides and then Az-PC increased after TNFα exposure, whereas ROS inhibition abolished Az-PC accumulation and TNFα-induced cell death. Glutathione peroxidase-4 (GPx4), which specifically metabolizes lipid hydroperoxides, fell in TNFα-stimulated cells prior to death. Ectopic GPx4 overcame this, reduced peroxidized phospholipid accumulation, blocked Az-PC accumulation, and prevented death. Conversely, GPx4 siRNA knockdown enhanced phospholipid peroxidation, increasing TNFα-stimulated Az-PC formation and apoptosis. Truncated phospholipids were essential elements of TNFα-induced apoptosis because overexpression of PAFAH2 (a phospholipase A(2) that selectively hydrolyzes truncated phospholipids) blocked TNFα-induced Az-PC accumulation without affecting phospholipid peroxidation. PAFAH2 also abolished apoptosis. Thus, phospholipid oxidation and truncation to apoptotic phospholipids comprise an essential element connecting TNFα receptor signaling to mitochondrial damage and apoptotic death.     

Mycobacterium tuberculosis eis regulates autophagy, inflammation, and cell death through redox-dependent signaling

The "enhanced intracellular survival" (eis) gene of Mycobacterium tuberculosis (Mtb) is involved in the intracellular survival of M. smegmatis. However, its exact effects on host cell function remain elusive. We herein report that Mtb Eis plays essential roles in modulating macrophage autophagy, inflammatory responses, and cell death via a reactive oxygen species (ROS)-dependent pathway. Macrophages infected with an Mtb eis-deletion mutant H37Rv (Mtb-Δeis) displayed markedly increased accumulation of massive autophagic vacuoles and formation of autophagosomes in vitro and in vivo. Infection of macrophages with Mtb-Δeis increased the production of tumor necrosis factor-α and interleukin-6 over the levels produced by infection with wild-type or complemented strains. Elevated ROS generation in macrophages infected with Mtb-Δeis (for which NADPH oxidase and mitochondria were largely responsible) rendered the cells highly sensitive to autophagy activation and cytokine production. Despite considerable activation of autophagy and proinflammatory responses, macrophages infected with Mtb-Δeis underwent caspase-independent cell death. This cell death was significantly inhibited by blockade of autophagy and c-Jun N-terminal kinase-ROS signaling, suggesting that excessive autophagy and oxidative stress are detrimental to cell survival. Finally, artificial over-expression of Eis or pretreatment with recombinant Eis abrogated production of both ROS and proinflammatory cytokines, which depends on the N-acetyltransferase domain of the Eis protein. Collectively, these data indicate that Mtb Eis suppresses host innate immune defenses by modulating autophagy, inflammation, and cell death in a redox-dependent manner.     

Nitric oxide-mediated cytosolic glucose-6-phosphate dehydrogenase is involved in aluminum toxicity of soybean under high aluminum concentration

Aims

Glucose-6-phosphate dehydrogenase (G6PDH) has been reported to be involved in resistance to various environmental stresses. However, the role of G6PDH in aluminum (Al) toxicity remains unclear.

Methods

Physiological and biochemical methods together with histochemical analysis were used to investigate the participation of G6PDH in Al-induced inhibition of root growth.

Results

Exposure to high Al concentration caused a significant increase in the activities of total and cytosolic G6PDH in roots of soybean. Al-induced inhibition of root growth and oxidative stress were alleviated by a G6PDH inhibitor. Reactive oxygen species (ROS) accumulation in Al-treated root apexes could be abolished by a NADPH oxidase inhibitor. Furthermore, treatment with a G6PDH inhibitor reduced NADPH content and NADPH oxidase activity in Al-treated root apexes. Further investigation demonstrates that nitric oxide (NO) mediates Al-induced increase in cytosolic G6PDH activity by modulating the expression of genes encoding cytosolic G6PDH. In addition, nitrate reductase pathway is mainly responsible for Al-induced NO production in root apexes.

Conclusions

These results indicate that NADPH produced by NO-modulated cytosolic G6PDH in root apexes is responsible for ROS accumulation mediated by NADPH oxidase under Al stress, subsequently suffering from oxidative stress and thus causing the inhibition of root elongation.

     

Blue LED causes autophagic cell death in human osteosarcoma by increasing ROS generation and dephosphorylating EGFR

Osteosarcoma (OS) is the most common primary malignant bone tumour in adolescence. Lately, light-emitting diodes (LED)-based therapy has emerged as a new promising approach for several diseases. However, it remains unknown in human OS. Here, we found that the blue LED irradiation significantly suppressed the proliferation, migration and invasion of human OS cells, while we observed blue LED irradiation increased ROS production through increased NADPH oxidase enzymes NOX2 and NOX4, as well as decreased Catalase (CAT) expression levels. Furthermore, we revealed blue LED irradiation-induced autophagy characterized by alterations in autophagy protein markers including Beclin-1, LC3-II/LC3-I and P62. Moreover, we demonstrated an enhanced autophagic flux. The blockage of autophagy displayed a remarkable attenuation of anti-tumour activities of blue LED irradiation. Next, ROS scavenger N-acetyl-L-cysteine (NAC) and NOX inhibitor diphenyleneiodonium (DPI) blocked suppression of OS cell growth, indicating that ROS accumulation might play an essential role in blue LED-induced autophagic OS cell death. Additionally, we observed blue LED irradiation decreased EGFR activation (phosphorylation), which in turn led to Beclin-1 release and subsequent autophagy activation in OS cells. Analysis of EGFR colocalization with Beclin-1 and EGFR-immunoprecipitation (IP) assay further revealed the decreased interaction of EGFR and Beclin-1 upon blue LED irradiation in OS cells. In addition, Beclin-1 down-regulation abolished the effects of blue LED irradiation on OS cells. Collectively, we concluded that blue LED irradiation exhibited anti-tumour effects on OS by triggering ROS and EGFR/Beclin-1-mediated autophagy signalling pathway, representing a potential approach for human OS treatment.