Suppression of basal autophagy reduces lung cancer cell proliferation and enhances caspase-dependent and -independent apoptosis by stimulating ROS formation

Autophagy is a catabolic process involved in the turnover of organelles and macromolecules which, depending on conditions, may lead to cell death or preserve cell survival. We found that some lung cancer cell lines and tumor samples are characterized by increased levels of lipidated LC3. Inhibition of autophagy sensitized non-small cell lung carcinoma (NSCLC) cells to cisplatin-induced apoptosis; however, such response was attenuated in cells treated with etoposide. Inhibition of autophagy stimulated ROS formation and treatment with cisplatin had a synergistic effect on ROS accumulation. Using genetically encoded hydrogen peroxide probes directed to intracellular compartments we found that autophagy inhibition facilitated formation of hydrogen peroxide in the cytosol and mitochondria of cisplatin-treated cells. The enhancement of cell death under conditions of inhibited autophagy was partially dependent on caspases, however, antioxidant NAC or hydroxyl radical scavengers, but not the scavengers of superoxide or a MnSOD mimetic, reduced the release of cytochrome c and abolished the sensitization of the cells to cisplatin-induced apoptosis. Such inhibition of ROS prevented the processing and release of AIF (apoptosis-inducing factor) and HTRA2 from mitochondria. Furthermore, suppression of autophagy in NSCLC cells with active basal autophagy reduced their proliferation without significant effect on the cell-cycle distribution. Inhibition of cell proliferation delayed accumulation of cells in the S phase upon treatment with etoposide that could attenuate the execution stage of etoposide-induced apoptosis. These findings suggest that autophagy suppression leads to inhibition of NSCLC cell proliferation and sensitizes them to cisplatin-induced caspase-dependent and -independent apoptosis by stimulation of ROS formation.     

Resveratrol depletes mitochondrial DNA and inhibition of autophagy enhances resveratrol-induced caspase activation

We recently demonstrated that resveratrol induces caspase-dependent apoptosis in multiple cancer cell types. Whether apoptosis is also regulated by other cell death mechanisms such as autophagy is not clearly defined. Here we show that inhibition of autophagy enhanced resveratrol-induced caspase activation and apoptosis. Resveratrol inhibited colony formation and cell proliferation in multiple cancer cell types. Resveratrol treatment induced accumulation of LC3-II, which is a key marker for autophagy. Pretreatment with 3-methyladenine (3-MA), an autophagy inhibitor, increased resveratrol-mediated caspase activation and cell death in breast and colon cancer cells. Inhibition of autophagy by silencing key autophagy regulators such as ATG5 and Beclin-1 enhanced resveratrol-induced caspase activation. Mechanistic analysis revealed that Beclin-1 did not interact with proapoptotic proteins Bax and Bak; however, Beclin-1 was found to interact with p53 in the cytosol and mitochondria upon resveratrol treatment. Importantly, resveratrol depleted ATPase 8 gene, and thus, reduced mitochondrial DNA (mtDNA) content, suggesting that resveratrol induces damage to mtDNA causing accumulation of dysfunctional mitochondria triggering autophagy induction. Together, our findings indicate that induction of autophagy during resveratrol-induced apoptosis is an adaptive response.     

Receptor-interacting Protein 1 Increases Chemoresistance by Maintaining Inhibitor of Apoptosis Protein Levels and Reducing Reactive Oxygen Species through a microRNA-146a-mediated Catalase Pathway

Although receptor-interacting protein 1 (RIP1) is well known as a key mediator in cell survival and death signaling, whether RIP1 directly contributes to chemotherapy response in cancer has not been determined. In this report, we found that, in human lung cancer cells, knockdown of RIP1 substantially increased cytotoxicity induced by the frontline anticancer therapeutic drug cisplatin, which has been associated with robust cellular reactive oxygen species (ROS) accumulation and enhanced apoptosis. Scavenging ROS dramatically protected RIP1 knockdown cells against cisplatin-induced cytotoxicity. Furthermore, we found that, in RIP1 knockdown cells, the expression of the hydrogen peroxide-reducing enzyme catalase was dramatically reduced, which was associated with increased miR-146a expression. Inhibition of microRNA-146a restored catalase expression, suppressed ROS induction, and protected against cytotoxicity in cisplatin-treated RIP1 knockdown cells, suggesting that RIP1 maintains catalase expression to restrain ROS levels in therapy response in cancer cells. Additionally, cisplatin significantly triggered the proteasomal degradation of cellular inhibitor of apoptosis protein 1 and 2 (c-IAP1 and c-IAP2), and X-linked inhibitor of apoptosis (XIAP) in a ROS-dependent manner, and in RIP1 knockdown cells, ectopic expression of c-IAP2 attenuated cisplatin-induced cytotoxicity. Thus, our results establish a chemoresistant role for RIP1 that maintains inhibitor of apoptosis protein (IAP) expression by release of microRNA-146a-mediated catalase suppression, where intervention within this pathway may be exploited for chemosensitization.     

Hydrogen peroxide inhibits non-small cell lung cancer cell anoikis through the inhibition of caveolin-1 degradation

Anoikis or detachment-induced apoptosis plays an essential role in the regulation of cancer cell metastasis. Caveolin-1 (Cav-1) is a key protein involved in tumor metastasis, but its role in anoikis and its regulation during cell detachment are unclear. We report here that Cav-1 plays a key role as a negative regulator of anoikis through a reactive oxygen species (ROS)-dependent mechanism in human lung carcinoma H460 cells. During cell detachment, Cav-1 is downregulated, whereas ROS generation is upregulated. Hydrogen peroxide and hydroxyl radical are two key ROS produced by cells during detachment. Treatment of the cells with hydrogen peroxide scavengers, catalase and N-acetylcysteine, promoted Cav-1 downregulation and anoikis during cell detachment, indicating that produced hydrogen peroxide plays a primary role in preventing anoikis by stabilizing Cav-1 protein. Catalase and N-acetylcysteine promoted ubiquitination and proteasomal degradation of Cav-1, which is a major pathway of its downregulation during cell anoikis. Furthermore, addition of hydrogen peroxide exogenously to the cells inhibited Cav-1 downregulation by preventing the formation of Cav-1-ubiquitin complex, supporting the inhibitory role of endogenous hydrogen peroxide in Cav-1 degradation during cell detachment. Together, these results indicate a novel role of hydrogen peroxide as an endogenous suppressor of cell anoikis through its stabilizing effect on Cav-1.     

Suppressed translation as a mechanism of initiation of CASP8 (caspase 8)-dependent apoptosis in autophagy-deficient NSCLC cells under nutrient limitation

Macroautophagy/autophagy inhibition under stress conditions is often associated with increased cell death. We found that under nutrient limitation, activation of CASP8/caspase-8 was significantly increased in autophagy-deficient lung cancer cells, which precedes mitochondria outer membrane permeabilization (MOMP), CYCS/cytochrome c release, and activation of CASP9/caspase-9, indicating that under such conditions the activation of CASP8 is a primary event in the initiation of apoptosis as well as essential to reduce clonogenic survival of autophagy-deficient cells. Starvation leads to suppression of CFLAR proteosynthesis and accumulation of CASP8 in SQSTM1 puncta. Overexpression of CFLARs reduces CASP8 activation and apoptosis during starvation, while its silencing promotes efficient activation of CASP8 and apoptosis in autophagy-deficient U1810 lung cancer cells even under nutrient-rich conditions. Similar to starvation, inhibition of protein translation leads to efficient activation of CASP8 and cell death in autophagy-deficient lung cancer cells. Thus, here for the first time we report that suppressed translation leads to activation of CASP8-dependent apoptosis in autophagy-deficient NSCLC cells under conditions of nutrient limitation. Our data suggest that targeting translational machinery can be beneficial for elimination of autophagy-deficient cells via the CASP8-dependent apoptotic pathway.     

Hydroxyl radical mediates cisplatin-induced apoptosis in human hair follicle dermal papilla cells and keratinocytes through Bcl-2-dependent mechanism

Induction of massive apoptosis of hair follicle cells by chemotherapy has been implicated in the pathogenesis of chemotherapy-induced alopecia (CIA), but the underlying mechanisms of regulation are not well understood. The present study investigated the apoptotic effect of cisplatin in human hair follicle dermal papilla cells and HaCaT keratinocytes, and determined the identity and role of specific reactive oxygen species (ROS) involved in the process. Treatment of the cells with cisplatin induced ROS generation and a parallel increase in caspase activation and apoptotic cell death. Inhibition of ROS generation by antioxidants inhibited the apoptotic effect of cisplatin, indicating the role of ROS in the process. Studies using specific ROS scavengers further showed that hydroxyl radical, but not hydrogen peroxide or superoxide anion, is the primary oxidative species responsible for the apoptotic effect of cisplatin. Electron spin resonance studies confirmed the formation of hydroxyl radicals induced by cisplatin. The mechanism by which hydroxyl radical mediates the apoptotic effect of cisplatin was shown to involve down-regulation of the anti-apoptotic protein Bcl-2 through ubiquitin-proteasomal degradation. Bcl-2 was also shown to have a negative regulatory role on hydroxyl radical. Together, our results indicate an essential role of hydroxyl radical in cisplatin-induced cell death of hair follicle cells through Bcl-2 regulation. Since CIA is a major side effect of cisplatin and many other chemotherapeutic agents with no known effective treatments, the knowledge gained from this study could be useful in the design of preventive treatment strategies for CIA through localized therapy without compromising the chemotherapy efficacy.     

IscU2对非小细胞肺癌增殖、迁移、侵袭能力的影响及其作用机制的研究

该文通过shRNA干扰技术敲低IscU2干扰细胞IscU2的表达,研究了干扰IscU2对非小细胞肺癌(NSCLC)细胞NCI-H520增殖、迁移及侵袭能力的影响。构建了稳定低表达IscU2的非小细胞肺癌细胞系NCI-H520;采用CCK-8和平板克隆实验检测细胞的增殖能力;流式细胞仪检测细胞周期、凋亡、ROS、线粒体膜电位变化情况;Transwell实验检测细胞迁移及侵袭能力;Western blot检测相关蛋白的表达。结果表明,干扰IscU2后,非小细胞肺癌细胞的增殖及克隆形成能力降低;细胞周期停滞在G1/G0期,同时伴随有p-AKT和Cyclin D1蛋白含量的下降;细胞晚期凋亡率明显增加,凋亡蛋白Cleaved-caspase3和Cleaved-PARP表达上调;细胞迁移和侵袭能力降低,上皮标志物E-Cadherin表达上调,间质标志物N-Cadherin和Snail表达下调;细胞ROS积累和线粒体膜电位下降。该研究结果表明,干扰IscU2显著抑制非小细胞肺癌的增殖、迁移、侵袭能力和上皮–间质转化,这为非小细胞肺癌的诊断和治疗提供了新的潜在靶点和视角。     

Inhibition of Autophagic Flux by Salinomycin Results in Anti-Cancer Effect in Hepatocellular Carcinoma Cells

Salinomycin raised hope to be effective in anti-cancer therapies due to its capability to overcome apoptosis-resistance in several types of cancer cells. Recently, its effectiveness against human hepatocellular carcinoma (HCC) cells both in vitro and in vivo was demonstrated. However, the mechanism of action remained unclear. Latest studies implicated interference with the degradation pathway of autophagy. This study aimed to determine the impact of Salinomycin on HCC-autophagy and whether primary human hepatocytes (PHH) likewise are affected. Following exposure of HCC cell lines HepG2 and Huh7 to varying concentrations of Salinomycin (0–10 µM), comprehensive analysis of autophagic activity using western-blotting and flow-cytometry was performed. Drug effects were analyzed in the settings of autophagy stimulation by starvation or PP242-treatment and correlated with cell viability, proliferation, apoptosis induction, mitochondrial mass accumulation and reactive oxygen species (ROS) formation. Impact on apoptosis induction and cell function of PHH was analyzed.Constitutive and stimulated autophagic activities both were effectively suppressed in HCC by Salinomycin. This inhibition was associated with dysfunctional mitochondria accumulation, increased apoptosis and decreased proliferation and cell viability. Effects of Salinomycin were dose and time dependent and could readily be replicated by pharmacological and genetic inhibition of HCC-autophagy alone. Salinomycin exposure to PHH resulted in transient impairment of synthesis function and cell viability without apoptosis induction. In conclusion, our data suggest that Salinomycin suppresses late stages of HCC-autophagy, leading to impaired recycling and accumulation of dysfunctional mitochondria with increased ROS-production all of which are associated with induction of apoptosis.     

Autophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity

One of the major side effects of cisplatin chemotherapy is toxic acute kidney injury due to preferential accumulation of cisplatin in renal proximal tubule epithelial cells and the subsequent injury to these cells. Apoptosis is known as a major mechanism of cisplatin-induced cell death in renal tubular cells. We have also recently demonstrated that autophagy induction is an immediate response of renal tubular epithelial cell exposure to cisplatin. Inhibition of cisplatin-induced autophagy blocks the formation of autophagosomes and enhances cisplatin-induced caspase-3, -6, and -7 activation, nuclear fragmentation and apoptosis. The switch from autophagy to apoptosis by autophagic inhibitors suggests that autophagy induction was responsible for a pre-apoptotic lag phase observed on exposure of renal tubular cells to cisplatin. Our studies provide evidence that autophagy induction in response to cisplatin mounts an adaptive response that suppresses and delays apoptosis. The beneficial effect of autophagy has a potential clinical significance in minimizing or preventing cisplatin nephrotoxicity.     

Inactivated Sendai virus strain Tianjin induces apoptosis and autophagy through reactive oxygen species production in osteosarcoma MG-63 cells

Sendai virus strain Tianjin, a novel genotype of Sendai virus, has been proven to possess potent antitumor effect on certain cancer cell types although inactivated by ultraviolet (UV). This study was carried out to investigate the in vitro anticancer properties of UV-inactivated Sendai virus strain Tianjin (UV-Tianjin) on human osteosarcoma cells and the underlying molecular mechanism. Our studies demonstrated UV-Tianjin significantly inhibited the viability of human osteosarcoma cell lines and triggered apoptosis through activation of both extrinsic and intrinsic pathways in MG-63 cells. Meanwhile, autophagy occurred in UV-Tianjin-treated cells. Blockade of autophagy with 3-methyladenine remarkably attenuated the inhibition of cell proliferation by UV-Tianjin, suggesting that UV-Tianjin-induced autophagy may be contributing to cell death. Furthermore, UV-Tianjin induced reactive oxygen species (ROS) production, which was involved in the execution of MG-63 cell apoptosis and autophagy, as evidenced by the result that treatment of N-acetyl-L-cysteine, a ROS scavenger, attenuated both apoptosis and autophagy. In addition, inhibition of apoptosis promoted autophagy, whereas suppression of autophagy attenuated apoptosis. Our results suggest that UV-Tianjin triggers apoptosis and autophagic cell death via generation of the ROS in MG-63 cells, which might provide important insights into the effectiveness of novel strategies for osteosarcoma therapy.     

Autophagy delays apoptosis in renal tubular epithelial cells in cisplatin cytotoxicity

One of the major side effects of cisplatin chemotherapy is toxic acute kidney injury due to preferential accumulation of cisplatin in renal proximal tubule epithelial cells and the subsequent injury to these cells. Apoptosis is known as a major mechanism of cisplatin-induced cell death in renal tubular cells. We have also recently demonstrated that autophagy induction is an immediate response of renal tubular epithelial cell exposure to cisplatin. Inhibition of cisplatin-induced autophagy blocks the formation of autophagosomes and enhances cisplatin-induced caspase-3, -6, and -7 activation, nuclear fragmentation, and apoptosis. The switch from autophagy to apoptosis by autophagic inhibitors suggests that autophagy induction was responsible for a pre-apoptotic lag phase observed on exposure of renal tubular cells to cisplatin. Our studies provide evidence that autophagy induction in response to cisplatin mounts an adaptive response that suppresses and delays apoptosis. The beneficial effect of autophagy has a potential clinical significance in minimizing or preventing cisplatin nephrotoxicity.

Addedum to: Yang C, Kaushal V, Shah SV, Kaushal GP. Autophagy and apoptosis are associated in cisplatin injury to renal tubular epithelial cell injury. Am J Physiol Renal Physiol 2008; 294:F777-87.     

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.     

ROS accumulation contributes to abamectin‐induced apoptosis and autophagy via the inactivation of PI3K/AKT/mTOR pathway in TM3 Leydig cells

Abamectin (ABA) as one of the worldwide used compounds in agriculture has raised safety concerns on nontarget organism toxicity. However, the study of male reproductive system damage caused by ABA remains unclear. Our aim is to investigate the effect of ABA‐induced cytotoxicity in TM3 Leydig cells and their underlying mechanisms. ABA inhibits TM3 cell viability and proliferation via cell cycle arrested in the G0/G1 phase. In addition, ABA‐induced mitochondrial depolarization leads to an imbalance in Bcl‐2 family expression, causing caspase‐dependent apoptosis in TM3 cells. The increased ratio of cells expression LC3 protein and LC3‐II to LC3‐I indicated the activation of autophagy potentially. Further experiments revealed ABA treatment reduced phosphatidylinositol 3‐kinase (PI3K), protein kinase B (AKT) phosphorylation, and mammalian target of rapamycin (mTOR) phosphorylation. Pretreatment with a PI3K/AKT inhibitor, LY294002, mimicked the ABA‐mediated effects on cytotoxicity. Pretreatment with a PI3K/AKT agonist, insulin‐like growth factor‐1, reversed the effects of ABA. ABA caused the accumulation of intracellular reactive oxygen species (ROS) by increased intensity of the ROS indicator. However, N‐acetylcysteine as ROS scavengers inhibited ABA‐induced apoptosis and autophagy and reversed these ABA‐mediated effects on PI3K/AKT/mTOR pathway. On the basis of the above results, it is suggested that ABA exposure induces apoptosis and autophagy in TM3 cells by ROS accumulation to mediate PI3K/AKT/mTOR signaling pathway suppression.     

Paradoxical role of autophagy in the dysplastic and tumor-forming stages of hepatocarcinoma development in rats

Many reports have shown that autophagy has a role as both a promoter and inhibitor in tumor development. However, the mechanism of this paradox is unknown. Tumor development is a multistep process. Therefore, we investigated whether the role of autophagy in hepatocarcinoma formation depended on the stage of tumor development. Based on our results, autophagy inhibition by chloroquine had a tumor-promotive effect in the rat model with N-diethylnitrosamine-induced hepatocarcinogenesis in its dysplastic stage (Ds) and a tumor-suppressive effect in its tumor-forming stage (Ts). In the Ds, autophagy inhibition enhanced cell proliferation, DNA damage and inflammatory cytokines expression in liver. These changes were dependent on the upregulation of reactive oxygen species (ROS) that was resulted from autophagy inhibition, and ultimately accelerated the process of hepatocarcinogenesis. However, in the Ts, autophagy inhibition restrained tumor formation by decreasing tumor cell survival and proliferation. In this stage, autophagy inhibition led to excessive ROS accumulation in the tumor, which promoted cell apoptosis, and prominently suppressed tumor cell metabolism. Taken together, our data suggested that autophagy suppressed hepatocarcinogenesis in the Ds by protecting normal cell stability and promoted hepatocarcinogenesis in the Ts by supporting tumor cells growth. Autophagy always had a role as a protector throughout the process of hepatocarcinoma development.     

6-formylpterin, a xanthine oxidase inhibitor, intracellularly generates reactive oxygen species involved in apoptosis and cell proliferation

The chemical property of 6-formylpterin and its biological functions were examined. Polarographic studies revealed that 6-formylpterin reacted with NAD(P)H and consumed oxygen. In contrast, other conjugated pterins, such as biopterin and neopterin, showed no consumption of oxygen. The production analysis using high-performance liquid chromatography documented that 6-formylpterin catalyzes the conversion from NADH to NAD. Electroparamagnetic resonance spin trapping experiments demonstrated that this reaction is accompanied with the generation of reactive oxygen species (ROS), superoxide anion and hydrogen peroxide. When 6-formylpterin was administered to HL-60 cells, intracellular ROS generation was observed and apoptosis was induced. In contrast, other conjugated pterins induced neither intracellular ROS generation nor apoptosis in HL-60 cells. The intracellular ROS generation by 6-formylpterin was observed in other cells, such as PanC-1 cells and Jurkat cells. 6-formylpterin suppressed cell proliferation in PanC-1 cells and inhibited Fas-mediated apoptosis in Jurkat cells. These findings indicate that, among conjugated pterins, 6-formylpterin has the unique property to transfer electron from NAD(P)H to oxygen and that the property brings about intracellular ROS generation, which exerts various biological functions such as induction of apoptosis, suppression of cell proliferation, and inhibition of Fas-mediated apoptosis.     

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.     

Inhibition of autophagy by autophagic inhibitors enhances apoptosis induced by bortezomib in non-small cell lung cancer cells

Bortezomib is a novel proteasome inhibitor that has promising antitumor activity against various cancer cells. We have assessed its antitumor activity in non-small cell lung cancer (NSCLC) A549 and H157 cells in vitro where it inhibited cell growth and induced apoptosis, which was associated with cytochrome c release and caspase-3 activation. Bortezomib upregulated autophagic-related proteins, the Atg12–Atg5 complex and LC3-II, which indicated autophagy had occurred. The combination of bortezomib with autophagic inhibitor 3-methyladenine or chloroquine significantly enhanced suppression of cell growth and apoptosis induced by bortezomib in A549 and H157 cells. Our study indicated that inhibition of both proteasome and autophagy has great potential for NSCLC treatment.     

Expression of inhibitor of apoptosis proteins in small- and non-small-cell lung carcinoma cells

Small-cell lung cancer (SCLC) and non-small-cell lung cancer (NSCLC) cells both initiate apoptotic signaling, resulting in caspase activation, after treatment with anti-cancer agents. However, in contrast to SCLC cells, NSCLC cells do not fully execute apoptosis. The apoptotic process in NSCLC cells seems to be blocked downstream of caspase activation, thus the failure of NSCLC cells to execute apoptosis could result from inhibition of active caspases by inhibitor of apoptosis proteins (IAPs). Here we investigate the mRNA and protein expression of IAPs in a panel of SCLC and NSCLC cell lines. The NSCLC cell lines had a stronger cIAP-2 expression at both mRNA and protein levels, while the SCLC cell lines had a higher level of XIAP protein. Expression of cIAP-1, cIAP-2, and XIAP, the most potent caspase inhibitors, was further investigated in three lung carcinoma cell lines after treatment with 8 Gy of ionizing radiation or etoposide (VP16). In response to treatment, the level of IAPs was not altered in a way that explained the differences in cellular chemo- and radiosensitivity. The intracellular localization of IAPs was analyzed in untreated and treated lung cancer cells. Surprisingly, we found that cIAP-2 was mainly detected in the mitochondrial fraction, although the function of this protein in mitochondria is unknown. No major relocalization of IAPs was observed after treatment. Taken together, these results indicate that IAPs alone are not the main factor responsible for the resistance of NSCLC cells to treatment.     

Autophagy, reactive oxygen species and the fate of mammalian cells

The paper reviews the rapidly expanding pool of information on cellular and molecular mechanisms of autophagy, including autophagy types, macroautophagy induction, formation of autophagosomes and cross-talk between autophagy and apoptosis. Special attention is given to generation of reactive oxygen species (ROS) in various cellular compartments of cells under stress conditions inducing autophagy. The roles of hydrogen peroxide and superoxide in autophagy are analysed based on the recent experimental work. The relation between ROS and life span prolongation is briefly discussed, with the final conclusion that the paradox of dual role of ROS in life and death may be solved to a considerable extent due to research on autophagy.