Lysosomal membrane permeabilization contributes to elemene emulsion-induced apoptosis in A549 cells

Elemene is a broad-spectrum antitumor agent. In the present study, lysosomal membrane permeabilization (LMP) was detected after short elemene emulsion--exposure (12 h) that preceded a decrease of the mitochondrial membrane potential and DNA damage (24 h) in A549 cells. At later time points (36 h) elemene emulsion caused the appearance of A549 cells with apoptotic features, including apoptotic morphology, phosphatidylserine exposure, and caspase-3 activation. A significant increase in protein expression for cathepsin D was also observed utilizing Western blot analysis after exposure to elemene emulsion for 12 h. The present study showed that elemene emulsion induced the increased levels of reactive oxygen species (ROS) and depletion of glutathione (GSH) in A549 cells. Cells treated with pepstatin A, an inhibitor for cathepsin D, showed a significant inhibition in DNA damage, mitochondrial membrane permeabilization, caspase-3 activation, and phosphatidylserine exposure. These results demonstrated that apoptosis induced by elemene emulsion in A549 cells is mediated in part through LMP and lysosomal protease cathepsin D.     

Lysosomal membrane permeabilization contributes to elemene emulsion-induced apoptosis in A549 cells

Abstract

Elemene is a broad-spectrum antitumor agent. In the present study, lysosomal membrane permeabilization (LMP) was detected after short elemene emulsion - exposure (12 h) that preceded a decrease of the mitochondrial membrane potential and DNA damage (24 h) in A549 cells. At later time points (36 h) elemene emulsion caused the appearance of A549 cells with apoptotic features, including apoptotic morphology, phosphatidylserine exposure, and caspase-3 activation. A significant increase in protein expression for cathepsin D was also observed utilizing Western blot analysis after exposure to elemene emulsion for 12 h. The present study showed that elemene emulsion induced the increased levels of reactive oxygen species (ROS) and depletion of glutathione (GSH) in A549 cells. Cells treated with pepstatin A, an inhibitor for cathepsin D, showed a significant inhibition in DNA damage, mitochondrial membrane permeabilization, caspase-3 activation, and phosphatidylserine exposure. These results demonstrated that apoptosis induced by elemene emulsion in A549 cells is mediated in part through LMP and lysosomal protease cathepsin D.     

Lithium enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells

Non-small cell lung cancer (NSCLC) A549 cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Therefore, combination therapy using sensitizing agents to overcome TRAIL resistance may provide new strategies for treatment of NSCLC. Here, we investigated whether lithium chloride (LiCl), a drug for mental illness, could sensitize A549 cells to TRAIL-induced apoptosis. We observed that LiCl significantly enhanced A549 cells apoptosis through up-regulation of death receptors DR4 and DR5 and activation of caspase cascades. In addition, G2/M arrest induced by LiCl also contributed to TRAIL-induced apoptosis. Concomitantly, LiCl strongly inhibited the activity of c-Jun N-terminal kinases (JNKs), and the inhibition of JNKs by SP600125 also induced G2/M arrest and augmented cell death caused by TRAIL or TRAIL plus LiCl. However, glycogen synthase kinase-3β (GSK3β) inhibition was not involved in TRAIL sensitization induced by LiCl. Collectively, these findings indicated that LiCl sensitized A549 cells to TRAIL-induced apoptosis through caspases-dependent apoptotic pathway via death receptors signaling and G2/M arrest induced by inhibition of JNK activation, but independent of GSK3β.     

Cathepsin L is involved in cathepsin D processing and regulation of apoptosis in A549 human lung epithelial cells

Cathepsins are implicated in a multitude of physiological and pathophysiological processes. The aim of the present study was to investigate the function of cathepsin L (catL) in the proteolytic network of human lung epithelial cells and its role in the regulation of apoptosis. We found that catL-deficient A549 cells as well as lung tissue extracts of catL(-/-) mice express increased amounts of single-chain cathepsin D (catD). Degradation experiments indicate that catL specifically degrades the single-chain isoform of catD. Furthermore, we found that catL-deficient cells showed increased sensitivity to apoptosis. Finally, we demonstrate that the inhibition of catD activity by pepstatin A decreased the number of apoptotic cells in catL-deficient A549 cells after anti-Fas treatment. In conclusion, catL is involved in catD processing and the accumulation of catD isoforms in catL-deficient cells is associated with increased rates of spontaneous and anti-Fas-induced apoptosis.     

Lysosomal membrane permeabilization is involved in oxidative stress-induced apoptotic cell death in LAMP2-deficient iPSCs-derived cerebral cortical neurons

Patients with Danon disease may suffer from severe cardiomyopathy, skeletal muscle dysfunction as well as varying degrees of mental retardation, in which the primary deficiency of lysosomal membrane-associated protein-2 (LAMP2) is considerably associated. Owing to the scarcity of human neurons, the pathological role of LAMP2 deficiency in neural injury of humans remains largely elusive. However, the application of induced pluripotent stem cells (iPSCs) may shed light on overcoming such scarcity.In this study, we obtained iPSCs derived from a patient carrying a mutated LAMP2 gene that is associated with Danon disease. By differentiating such LAMP2-deficient iPSCs into cerebral cortical neurons and with the aid of various biochemical assays, we demonstrated that the LAMP2-deficient neurons are more susceptible to mild oxidative stress-induced injury.The data from MTT assay and apoptotic analysis demonstrated that there was no notable difference in cellular viability between the normal and LAMP2-deficient neurons under non-stressed condition. When exposed to mild oxidative stress (10 μM H₂O₂), the LAMP2-deficient neurons exhibited a significant increase in apoptosis. Surprisingly, we did not observe any aberrant accumulation of autophagic materials in the LAMP2-deficient neurons under such stress condition.Our results from cellular fractionation and inhibitor blockade experiments further revealed that oxidative stress-induced apoptosis in the LAMP2-deficient cortical neurons was caused by increased abundance of cytosolic cathepsin L. These results suggest the involvement of lysosomal membrane permeabilization in the LAMP2 deficiency associated neural injury.     

Acetate-induced apoptosis in colorectal carcinoma cells involves lysosomal membrane permeabilization and cathepsin D release

Colorectal carcinoma (CRC) is one of the most common causes of cancer-related mortality. Short-chain fatty acids secreted by dietary propionibacteria from the intestine, such as acetate, induce apoptosis in CRC cells and may therefore be relevant in CRC prevention and therapy. We previously reported that acetic acid-induced apoptosis in Saccharomyces cerevisiae cells involves partial vacuole permeabilization and release of Pep4p, the yeast cathepsin D (CatD), which has a protective role in this process. In cancer cells, lysosomes have emerged as key players in apoptosis through selective lysosomal membrane permeabilization (LMP) and release of cathepsins. However, the role of CatD in CRC survival is controversial and has not been assessed in response to acetate. We aimed to ascertain whether LMP and CatD are involved in acetate-induced apoptosis in CRC cells. We showed that acetate per se inhibits proliferation and induces apoptosis. More importantly, we uncovered that acetate triggers LMP and CatD release to the cytosol. Pepstatin A (a CatD inhibitor) but not E64d (a cathepsin B and L inhibitor) increased acetate-induced apoptosis of CRC cells, suggesting that CatD has a protective role in this process. Our data indicate that acetate induces LMP and subsequent release of CatD in CRC cells undergoing apoptosis, and suggest exploiting novel strategies using acetate as a prevention/therapeutic agent in CRC, through simultaneous treatment with CatD inhibitors.     

Mechanisms of curcumin-induced apoptosis of Ehrlich's ascites carcinoma cells.

Curcumin, the active ingredient from the spice turmeric (Curcuma longa Linn), is a potent antioxidant and anti-inflammatory agent. It has been recently demonstrated to possess discrete chemopreventive activities. However, the molecular mechanisms underlying such anticancer properties of curcumin still remain unrealized, although it has been postulated that induction of apoptosis in cancer cells might be a probable explanation. In the current study, curcumin was found to decrease the Ehrlich's ascites carcinoma (EAC) cell number by the induction of apoptosis in the tumor cells as evident from flow-cytometric analysis of cell cycle phase distribution of nuclear DNA and oligonucleosomal fragmentation. Probing further into the molecular signals leading to apoptosis of EAC cells, we observed that curcumin is causing tumor cell death by the up-regulation of the proto-oncoprotein Bax, release of cytochrome c from the mitochondria, and activation of caspase-3. The status of Bcl-2 remains unchanged in EAC, which would signify that curcumin is bypassing the Bcl-2 checkpoint and overriding its protective effect on apoptosis.     

AMP-activated protein kinase (AMPK) activation is involved in chrysin-induced growth inhibition and apoptosis in cultured A549 lung cancer cells

Here we show that chrysin induces growth inhibition and apoptosis in cultured lung cancer A549 cells, and activation of AMP-activated protein kinase (AMPK) may contribute to this process. Our Western-blots results demonstrated a significant AMPK activation after chrysin treatment in A549 cells. Inhibition of AMPK by shRNA-mediated gene silencing, or by its inhibitor, diminished chrysin-induced A549 cell growth inhibition and apoptosis. Forced activation of AMPK by introducing a constitutively active form of AMPKα (CA-AMPKα), or by its activators, mimicked chrysin's effect. For mechanism analysis, we found chrysin inhibited Akt/mammalian target of rapamycin (mTOR) activation, and knocking-down of AMPK by shRNA almost reversed this effect. Finally, we observed that a relative low dose of chrysin enhanced doxorubicin-induced AMPK activation to promote A549 cell apoptosis. Our study suggests that activation of AMPK by chrysin contributes to Akt suppression, growth inhibition and apoptosis in human lung cancer cells, and agents that could activate AMPK may serve as useful adjuvants for traditional chemotherapy against lung cancer.     

Effect of halothane on lung carcinoma cells A 549

The halogenated hydrocarbons, such as halothane, are widely used as anesthetics in clinical practice; however their application is often accompanied with metabolic, cardiovascular and respiratory complications. One of the possible factors for this negative outcome might be the severe toxicity of these agents. In this paper, we investigate in vitro effects of halothane on human lung carcinoma A 549 cells, namely on their cytotoxicity, adhesive properties and metabolic activity. The cytotoxicity response of lung carcinoma A 549 cells to halothane was determined by lactate dehydrogenase (LDH) assay (for cytotoxicity), by detachment assay after adhesion to type IV collagen (for cell adhesive properties) and by surface tension measurements of culture medium (for cell metabolic activity). Regarding the cytotoxicity, the determined maximal non-toxic concentration of halothane on A 549 cells, given here as volume percentages (vol.%) was 0.7 vol.% expressed as aqueous concentration in the culture medium. Direct measurement of the actual halothane concentration in the culture medium showed that 0.7 vol.% corresponds to 1.05 mM and 5.25 aqueous-phase minimum alveolar concentration (MAC). Concentrations equal or higher than 1.4 vol.% (2.1 mM; 10.5 MAC) of halothane provoked complete detachment (cell death), or reduction of initial adhesion to collagen IV in half of the cell population. Surfactant production of A 549 cells, registered up to 48 h after halothane treatment, was inhibited by halothane concentrations as low as 0.6 vol.% (0.9 mM; 4.5 MAC). Our results demonstrate that sub toxic halothane concentrations of 0.6 vol.% inhibits surfactant production; concentrations in the range 0.8-1.4 vol.% induce membrane damages and concentrations equal and higher than 1.4 vol.%--cell death of approximately 50% of the cells.     

Photodynamic therapy-induced apoptosis in epidermoid carcinoma cells. Reactive oxygen species and mitochondrial inner membrane permeabilization

Photodynamic therapy (PDT), a novel and promising cancer treatment that employs a combination of a photosensitizing chemical and visible light, induces apoptosis in human epidermoid carcinoma A431 cells. However, the precise mechanism of PDT-induced apoptosis is not well characterized. To dissect the pathways of PDT-induced apoptosis, we investigated the involvement of mitochondrial damage by examining a second generation photosensitizer, the silicon phthalocyanine 4 (Pc 4). By using laser-scanning confocal microscopy, we found that Pc 4 localized to cytosolic membranes primarily, but not exclusively, in mitochondria. Formation of mitochondrial reactive oxygen species (ROS) was detected within minutes when cells were exposed to Pc 4 and 670-675 nm light. This was followed by mitochondrial inner membrane permeabilization, depolarization and swelling, cytochrome c release, and apoptotic death. Desferrioxamine prevented mitochondrial ROS production and the events thereafter. Cyclosporin A plus trifluoperazine, blockers of the mitochondrial permeability transition, inhibited mitochondrial inner membrane permeabilization and depolarization without affecting mitochondrial ROS generation. These data indicate that the mitochondrial ROS are critical in initiating mitochondrial inner membrane permeabilization, which leads to mitochondrial swelling, cytochrome c release to the cytosol, and apoptotic death during PDT with Pc 4.     

Lysosomal membrane permeabilization causes oxidative stress and ferritin induction in macrophages

Moderate lysosomal membrane permeabilization (LMP) is an important inducer of apoptosis. Macrophages are professional scavengers and are rich in hydrolytic enzymes and iron. In the present study, we found that LMP by lysosomotropic detergent MSDH resulted in early up-regulation of lysosomal cathepsins, oxidative stress and ferritin up-regulation, and cell death. Lysosomotropic base NH₄Cl reduced the ferritin induction and oxidative stress in apoptotic cells induced by MSDH. Cysteine cathepsin inhibitors significantly protected cell death and oxidative stress, but had less effect on ferritin induction. We conclude that oxidative stress induced by lysosomal rupture causes ferritin induction with concomitant mitochondrial damage, which are the potential target for prevention of cellular oxidative stress and cell death induced by typical lysosomotropic substances in different disorders.     

Kaempferol-induced growth inhibition and apoptosis in A549 lung cancer cells is mediated by activation of MEK-MAPK

A vast variety of naturally occurring substances have been shown to protect against experimental carcinogenesis and an increasing amount of evidence suggests that kaempferol may have cancer chemopreventative properties. However, the precise underlying protective mechanisms are poorly understood. To elucidate these mechanisms, we challenged human lung cancer cell line A549 with kaempferol and investigated its effects upon cellular growth and signal transduction pathways. Treatment of A549 cells with kaempferol resulted in a dose- and time-dependent reduction in cell viability and DNA synthesis with the rate of apoptosis equivalent to 0.9+/-0.5, 5.2+/-1.5, 16.8+/-2.0, 25.4+/-2.6, and 37.8+/-4.5% on treatment with 0, 17.5, 35.0, 52.5, and 70.0 microM kaempferol, respectively. Concomitantly, kaempferol treatments led to a 1.2-, 2.7-, 3.3-, and 3.4-fold increase in Bax. Similar elevations were also observed in Bad which increased 1.2-, 3.3-, 3.7-, and 4.7-fold, respectively, as compared to control. Bcl-2 and Bcl-xL expression were inhibited in a dose-dependent fashion. While the Akt-1 and phosphorylated Akt-1 were inhibited, the mitogen-activated protein kinase (MAPK) was activated upon kaempferol treatment. Kaempferol induced apoptosis was associated with the cleavage of caspase-7 and poly ADP-ribose polymerase (PARP). Inhibition of MEK1/2 but not PI-3 kinase blocked kaempferol-induced cleavage of caspase-7, PARP cleavage, and apoptosis. The results suggest that inactivation of Akt-1 and alteration of Bcl-2 family of proteins are not sufficient for kaempferol to induce apoptosis and activation of MEK-MAPK is a requirement for kaempferol-induced cell death machinery in A549 cells.     

Induction of apoptosis in human lung carcinoma A549 epithelial cells with an ethanol extract of Tremella mesenterica

Tremella mesenterica (TM) is a common food and folk medicine widely used in several Asian countries as a tonic for the lungs. In the present study, we compared the effects of extracellular polysaccharides (EPS), intracellular polysaccharides (IPS), and ethanol extract (EE) of Tremella mesenterica on the induction of apoptosis into human lung carcinoma A549 epithelial cells. The EE, but not the EPS or the IPS, almost completely inhibited the growth of A549 cells. The results of Annexin V-FITC/PI staining and flow cytometric analysis indicated that the percentage of Annexin V(+)/PI(-) cells in EE-treated cells increased to 32.8%. The results of further investigation showed a disruption of mitochondrial transmembrane potential (DeltaPsi(m)), the production of reactive oxygen species (ROS), and the activation of caspase-3 protein in EE-treated cells. These findings suggest that EE can decrease cell viability and induce apoptosis in A549 cell lines by activating a mitochondrial pathway.     

Glutathione content and growth in A549 human lung carcinoma cells

The relationship between glutathione content and cell growth was investigated in A549 human lung carcinoma cells. A decreased cellular glutathione content was achieved by exposing the cells to L-buthionine-SR-sulfoximine (BSO). It also occurred in these cells as they approached their plateau phase of growth. During exponential growth, a lower initial glutathione content correlated with a longer lag phase in subcultured cells. Further, depletion of cellular glutathione by BSO inhibited cell growth. This inhibition became apparent 36 h after the addition of BSO. These observations raise the possibility that a critical concentration of GSH may be required for optimal growth of A549 human lung carcinoma cells.     

Autophagy is a regulator of TRAIL-induced apoptosis in NSCLC A549 cells

Autophagy, a catabolic process by which cytoplasmic components are degraded in lysosomes, plays an important role in the maintenance of cellular homeostasis. Dysregulation of autophagy is associated with several diseases. However, few studies have addressed the role of autophagy in the lung, and its role in lung diseases remains unclear. In the present study, we examined the effect of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) on autophagy in A549 cells and explored the underlying mechanisms. We showed that TRAIL promoted autophagosome formation, as detected by the levels of LC3-II, and its effect on promoting autophagy was dependent on the expression of the autophagy related genes (ATGs) Atg5, Atg7, and beclin-1. TRAIL-induced ATG expression was attenuated by JNK silencing or treatment with the JNK inhibitor SP600125, indicating the involvement of the JNK pathway. Crosstalk between autophagy and apoptosis was demonstrated by silencing the autophagy related genes Atg5, Atg7, and beclin-1, and the dependence of TRAIL-induced apoptosis on autophagy-related gene expression. Taken together, our results indicate that TRAIL promotes autophagy in A549 cells via a mechanism involving the modulation of ATG expression through the JNK pathway. Inhibition of autophagy enhanced TRAIL-induced cell proliferative inhibition and apoptosis in A549 cells.     

葛根素对人非小细胞肺癌A549细胞凋亡的作用

目的：观察葛根素对人非小细胞肺癌A549细胞生长的抑制作用及其机制。方法：体外培养人非小细胞肺癌细胞（A549）,不同浓度（60 μg/ml,120 μg/ml,240 μg/ml）葛根素处理24 h后;采用CCK-8法观察葛根素对细胞的增值抑制作用;吖啶橙（AO）/溴化乙锭（EB）双染法及AnnexinⅤ-PI双染流式细胞术检测药物作用前后A549细胞的形态学变化及凋亡状况;Western blot法检测Apelin/APJ蛋白水平的变化。结果：CCK-8法检测结果说明葛根素能抑制A549细胞的增值,具有浓度和时间依赖关系;流式细胞术进一步证实葛根素具有诱导细胞凋亡的作用,与A549细胞组比较,葛根素各处理组Apelin/APJ蛋白水平均有不同程度下调。结论：葛根素可能通过调节Apelin/APJ蛋白的表达诱导A549细胞凋亡。     

Induction of apoptosis in arsenic trioxide-treated lung cancer A549 cells by buthionine sulfoximine

Arsenic trioxide (ATO) affects many biological processes such as cell proliferation, apoptosis, differentiation and angiogenesis. L-buthionine sulfoximine (BSO) is an inhibitor of GSH synthesis. We tested whether ATO reduced the viability of lung cancer A549 cells in vitro, and investigated the in vitro effect of the combination of ATO and BSO on cell viability in relation to apoptosis and the cell cycle. ATO caused a dose-dependant decrease of viability of A549 cells with an IC50 of more than 50 muM. Low doses of ATO or BSO (1~10 muM) alone did not induce cell death. However, combined treatment depleted GSH content and induced apoptosis, loss of mitochondrial transmembrane potential (DeltaPsi(m)) and cell cycle arrest in G2. Reactive oxygen species (ROS) increased or decreased depending on the concentration of ATO. In addition, BSO generally increased ROS in ATO-treated A549 cells. ROS levels were at least in part related to apoptosis in cells treated with ATO and/or BSO. In conclusion, we have demonstrated that A549 lung cells are very resistant to ATO, and that BSO synergizes with clinically achievable concentration of ATO. Our results suggest that combination treatment with ATO and BSO may be useful for treating lung cancer.