Molecular mechanisms of 3,3′‐dichlorobenzidine‐mediated toxicity in HepG2 cells

3,3′‐Dichlorobenzidine (DCB) (CAS 91–94‐1), a synthetic, chlorinated, primary aromatic amine, is typically used as an intermediate in the manufacturing of pigments for printing inks, textiles, paints, and plastics. In this study, we found that DCB could significantly inhibit the cell viability of HepG2 cells in a concentration‐dependent manner. Flow cytometry revealed that DCB induced G2/M‐phase arrest and apoptosis in HepG2 cells. DCB treatment dramatically induced the dissipation of mitochondrial membrane potential (Δψ_m) and enhanced the enzymatic activities of caspase‐9 and caspase‐3 whilst hardly affecting caspase‐8 activity. Furthermore, Western blotting indicated that DCB‐induced apoptosis was accompanied by the down‐regulation of Bcl‐2/Bax ratio. These results suggested that DCB led to cytotoxicity involving activation of mitochondrial‐dependent apoptosis through Bax/Bcl‐2 pathways in HepG2 cells. Furthermore, HepG2 cells treated with DCB showed significant DNA damage as supported by the concentration‐dependent increase in olive tail moments as determined by the comet assay and by concentration‐ and time‐dependent increase in histone H2AX phosphorylation (γ‐H2AX). Two‐dimensional‐difference gel electrophoresis (2D‐DIGE), combined with mass spectrometry (MS), was used to unveil the differences in protein expression between cells exposed to 25 µM or 100 µM of DCB for 24 hr and the control cells. Twenty‐seven differentially expressed proteins involved in DNA repair, unfolded protein response, metabolism, cell signaling, and apoptosis were identified. Among these, 14‐3‐3 theta, CGI‐46, and heat‐shock 70 protein 4 were confirmed using Western blot assay. Taken together, these data suggest that DCB is capable of inducing DNA damage and some cellular stress responses in HepG2 cells, thus eventually leading to cell death by apoptosis. Environ. Mol. Mutagen. 55:407–420, 2014. © 2014 Wiley Periodicals, Inc.     

5‐Nitro‐2‐(3‐Phenylpropylamino) Benzoic Acid Induced Drug Resistance to Cisplatin in Human Erythroleukemia Cell Lines

Mechanisms of cisplatin resistance in cancer cells are not fully understood. Here, we showed a critical role for the chloride channel‐3 (ClC‐3) in cisplatin resistance in human erythroleukemia K562 and RK562 cells. We found that a chloride channel blocker 5‐nitro‐2‐(3‐phenylpropylamino) benzoic acid (NPPB) could protect cells from cisplatin‐induced apoptosis. NPPB treatment decreased the mRNA and the protein expression of Bax/Bcl‐2, decreased the protein expressions of cytochrome C and caspase‐3, and increased the mRNA expressions of cyclin D1 and ClC‐3 in cells treated with cisplatin. The caspase‐3 activity was decreased significantly and the rate of cell apoptosis was decreased. NPPB treatment increased CIC‐3 expression, which could increase acidification of intracellular compartments, and increased sequestration of cisplatin, inducing decreased effective drug concentrations, and subsequently cell death. Collectively, our data indicate that NPPB can induce drug resistance to cisplatin by upregulating the expression of CIC‐3. NPPB‐induced CIC‐3 expression facilitates acidification of sequestrated cisplatin, and plays an important role in preventing cisplatin‐induced apoptosis in human erythroleukemia K562 and RK562 cells. Anat Rec,, 2011. © 2011 Wiley‐Liss, Inc.     

Induction of macrophage cell‐cycle arrest and apoptosis by humic acid

Humic acid (HA) in well water is associated with Blackfoot disease and various cancers. Previously, we reported that acute humic acid exposure (25–200 µg/mL for 24 hr) induces inflammation in RAW264.7 macrophages. In this study, we observed that prolonged (72 hr) HA exposure (25–200 µg/mL) induces cell‐cycle arrest and apoptosis in cultured RAW264.7 cells. We also observed that exposing macrophages to HA arrests cells in the G₂/M phase of the cell cycle by reducing cyclin A/B₁, Cdc2, and Cdc25C levels. Treating macrophages with HA triggers a sequence of events characteristic of apoptotic cell death including loss of cell viability, morphological changes, internucleosomal DNA fragmentation, sub‐G₁ accumulation. Molecular markers of apoptosis associated with mitochondrial dysfunction were similarly observed, including cytochrome c release, caspase‐3 or caspase‐9 activation, and Bcl‐2/Bax dysregulation. In addition to the mitochondrial pathway, HA‐induced apoptosis may also be mediated through the death receptor and ER stress pathways, as evidence by induction of Fas, caspase‐8, caspase‐4, and caspase‐12 activity. HA also upregulates p53 expression and causes DNA damage as assessed by the comet assay. These findings yield new insight into the mechanisms by which HA exposure may trigger atherosclerosis through modulation of the macrophage‐mediated immune system. Environ. Mol. Mutagen. 55:741–750, 2014. © 2014 Wiley Periodicals, Inc.     

Supplementation With Quercetin Attenuates 4‐Nitrophenol‐Induced Testicular Toxicity in Adult Male Mice

The beneficial effects of quercetin on reproductive damage elicited by 4‐nitrophenol (PNP) were studied in adult male mice. A six‐week treatment of weekly intraperitoneal injections of PNP (50 mg/kg) resulted in severe damage to the seminiferous tubules, a remarkable increase in both hydroxyl radical and malondiadehyde production, and notably decreased glutathione peroxidase and superoxide dismutase activities. Moreover, PNP treatment induced germ cell apoptosis, inhibited Bcl‐xl expression, and then activated Bax expression and the caspase‐3 enzyme. Exposure to PNP also increased XBP‐1 and HO‐1 mRNAs levels. However, simultaneous supplementation with quercetin (75 mg/kg) attenuated the toxicity induced by PNP through renewal of the antioxidant enzyme's status, alleviating apoptosis by regulating the expressions of Bax and Bcl‐xl, XBP‐1 and HO‐1mRNAs, and the regulation of caspase‐3 activity. Taken together, these findings indicated that the antioxidant quercetin displays a potential preventive effect on PNP‐induced oxidative damage in mouse testes and may represent an efficient supplement to attenuate reproductive toxicity from environmental toxicants in order to ensure reproductive health and sperm production. Anat Rec, 296:1650–1657, 2013. © 2013 Wiley Periodicals, Inc.     

c‐Jun NH2‐terminal kinase is a critical node in the death of CD4+CD8+ thymocytes during Salmonella enterica serovar Typhimurium infection

Thymic atrophy, due to the depletion of CD4⁺CD8⁺ thymocytes, is observed during infections with numerous pathogens. Several mechanisms, such as glucocorticoids and inflammatory cytokines, are known to be involved in this process; however, the roles of intracellular signaling molecules have not been investigated. In this study, the functional role of c‐Jun NH₂‐terminal kinase (JNK) during infection‐induced thymic atrophy was addressed. The levels of phosphorylated JNK in immature CD4⁺CD8⁺ thymocytes from C57BL/6 (Nramp‐deficient) and 129/SvJ (Nramp‐sufficient) mice were increased upon oral infection of mice with Salmonella enterica serovar Typhimurium (S. typhimurium). Furthermore, inhibition of JNK signaling, but not ERK or p38 MAPK, prevented the in vitro death of infected thymocytes. Importantly, the in vivo inhibition of JNK signaling with SP600125 protected C57BL/6 CD4⁺CD8⁺ thymocytes from depletion via multiple mechanisms as follows: lower intracellular ROS, inflammatory cytokines, Bax and caspase 3 activity, increase in Bcl‐xL amounts, and prevention of the loss in mitochondrial membrane potential. Notably, thymic architecture was preserved in infected mice treated with SP600125. Overall, this study identifies a novel role for JNK as a crucial regulator of the death of CD4⁺CD8⁺ thymocytes during S. typhimurium infection.     

Proteinase activated receptor‐2‐mediated dual oxidase‐2 up‐regulation is involved in enhanced airway reactivity and inflammation in a mouse model of allergic asthma

Airway epithelial cells (AECs) express a variety of receptors, which sense danger signals from various aeroallergens/pathogens being inhaled constantly. Proteinase‐activated receptor 2 (PAR‐2) is one such receptor and is activated by cockroach allergens, which have intrinsic serine proteinase activity. Recently, dual oxidases (DUOX), especially DUOX‐2, have been shown to be involved in airway inflammation in response to Toll‐like receptor activation. However, the association between PAR‐2 and DUOX‐2 has not been explored in airways of allergic mice. Therefore, this study investigated the contribution of DUOX‐2/reactive oxygen species (ROS) signalling in airway reactivity and inflammation after PAR‐2 activation. Mice were sensitized intraperitoneally with intact cockroach allergen extract (CE) in the presence of aluminium hydroxide followed by intranasal challenge with CE. Mice were then assessed for airway reactivity, inflammation, oxidative stress (DUOX‐2, ROS, inducible nitric oxide synthase, nitrite, nitrotyrosine and protein carbonyls) and apoptosis (Bax, Bcl‐2, caspase‐3). Challenge with CE led to up‐regulation of DUOX‐2 and ROS in AECs with concomitant increases in airway reactivity/inflammation and parameters of oxidative stress, and apoptosis. All of these changes were significantly inhibited by intranasal administration of ENMD‐1068, a small molecule antagonist of PAR‐2 in allergic mice. Administration of diphenyliodonium to allergic mice also led to improvement of allergic airway responses via inhibition of the DUOX‐2/ROS pathway; however, these effects were less pronounced than PAR‐2 antagonism. The current study suggests that PAR‐2 activation leads to up‐regulation of the DUOX‐2/ROS pathway in AECs, which is involved in regulation of airway reactivity and inflammation via oxidative stress and apoptosis.     

Inhibition of JNK3 Promotes Apoptosis Induced by BH3 Mimetic S1 in Chemoresistant Human Ovarian Cancer Cells

Previous studies have suggested that the novel BH3 mimetic S1 could induce apoptosis in diverse tumor cell lines through endoplasmic reticulum (ER) stress or mitochondrial cell death pathways. The activation of c‐Jun N‐terminal kinase (JNK) through inositol requiring enzyme‐1 (IRE1) is closely connected to ER stress‐induced apoptosis. However, the role of JNK is complex, as there are different JNK subtypes and the function of each subtype is still not entirely clear. Here we found that the mRNA expression of JNK3 was continuously high in S1‐treated human ovarian cancer SKOV3/DDP cells using a human unfolded protein response (UPR) pathway PCR array. Pharmacological inhibition of JNK3 increased cell sensitivity to apoptosis induced by S1. Furthermore, inhibition of JNK3 induced accumulation of both acidic compartment and p62, and upregulated ROS production. Our results suggest that JNK3 plays a pro‐survival role during ER stress through preventing the block of autophagic flux and reducing oxidative stress in SKOV3/DDP cells. Inhibition of JNK3 may be a potential method to enhance the killing effect of the Bcl‐2 inhibitor S1. Anat Rec, 298:386–395, 2015. © 2014 Wiley Periodicals, Inc.     

2‐Chlorodeoxyadenosine (cladribine) induces apoptosis in human monocyte‐derived dendritic cells

2‐Chlorodeoxyadenosine (cladribine, CdA) is an immunosuppressive drug that is licensed to treat hairy cell leukaemia, and has been shown recently to have beneficial effects in patients with multiple sclerosis (MS). The therapeutic effects of CdA have been suggested to be mediated partly through its potent toxicity towards lymphocytes. However, the effects of CdA on other immune cells are poorly understood. In the present study, we investigated the effects of CdA on the induction of apoptosis in human monocytes, monocyte‐derived immature (ImDC) and mature (mDC) dendritic cells. Treatment of monocytes with CdA strongly induced apoptosis after 24 h, while apoptosis induction in DC was evident after 72 h. Furthermore, CdA treatment strongly induced caspase‐3 and caspase‐9 in monocytes, whereas activation of caspases was undetected in DC. The mitochondrial membrane potential in DC was reduced significantly after CdA treatment. DNA hypodiploid assessment showed fragmented nuclei in DC after CdA treatment together with activation of p53 protein. These results revealed that CdA induces caspase‐independent apoptosis in DC and suggest cell type specific effects of CdA. This mechanism may contribute to the effect of CdA in autoimmune diseases.     

Bcl‐2 protects TK6 cells against hydroquinone‐induced apoptosis through PARP‐1 cytoplasm translocation and stabilizing mitochondrial membrane potential

B cell leukemia/lymphoma‐2 (Bcl‐2) suppresses apoptosis by binding the BH3 domain of proapoptotic factors and thereby regulating mitochondrial membrane potential (MMP). This study aimed to investigate the role of Bcl‐2 in controlling the mitochondrial pathway of apoptosis during hydroquinone (HQ)‐induced TK6 cytotoxicity. In this study, HQ, one metabolite of benzene, decreased the MMP in a concentration‐dependent manner and induced the generation of reactive oxygen species (ROS), the activation of the DNA damage marker γ‐H2AX, and production of the DNA damage‐responsive enzyme poly(ADP‐ribose)polymerase‐1 (PARP‐1). Exposure of TK6 cells to HQ leads to an increase in Bcl‐2 and co‐localization with PARP‐1 in the cytoplasm. Inhibition of Bcl‐2 using the BH3 mimetic, ABT‐737, suppressed the PARP‐1 nuclear to cytoplasm translocation and sensitized TK6 cells to HQ‐induced apoptosis through depolarization of the MMP. Western blot analysis indicated that ABT‐737 combined with HQ increased the levels of cleaved PARP and γ‐H2AX, but significantly decreased the level of P53. Thus, ABT‐737 can influence PARP‐1 translocation and induce apoptosis via mitochondria‐mediated apoptotic pathway, independently of P53. In addition, we found that knockdown of PARP‐1 attenuated the HQ‐induced production of cleaved PARP and P53. These results identify Bcl‐2 as a protective mediator of HQ‐induced apoptosis and show that upregulation of Bcl‐2 helps to localize PARP‐1 to the cytoplasm and stabilize MMP. Environ. Mol. Mutagen. 59:49–59, 2018. © 2017 Wiley Periodicals, Inc.     

Protective Effect of Quercetin on Cadmium‐Induced Oxidative Toxicity on Germ Cells in Male Mice

Cadmium is a toxic heavy metal that is widely distributed in the environment. As a critical process, oxidative toxicity mediates the morphological and functional damages in germ cells after cadmium exposure. In this study, the protective effect of quercetin on cadmium‐induced oxidative toxicity was investigated in mouse testicular germ cells. After oral administration of cadmium chloride at 4 mg/kg body weight for 2 weeks, damages in spermatozoa occurred in the early stage of spermatogenesis. Cadmium treatment significantly decreased the testicular antioxidant system, including decreases in the glutathione (GSH) level, superoxide dismutase (SOD), and GSH peroxidase (GSH‐Px) activities. Moreover, exposure to cadmium resulted in an increase of hydrogen peroxide production and lipid peroxidation in testes. In addition, cadmium provoked germ cell apoptosis by upregulating expression of the proapoptotic proteins Bax and caspase‐3 and downregulating expression of the antiapoptotic protein Bcl‐XL. However, combined administration of a common flavonoid quercetin at 75 mg/kg body weight significantly attenuated cadmium‐induced germ cell apoptosis by suppressing the hydrogen peroxide production and lipid peroxidation in testicular tissue. Simultaneous supplementation of quercetin markedly restored the decrease in GSH level and SOD and GSH‐Px activities elicited by cadmium treatment. Additionally, quercetin protected germ cells from cadmium‐induced apoptosis by downregulating the expression of Bax and caspase‐3 and upregulating Bcl‐XL expression. These results indicate that quercetin, due to its antioxidative and antiapoptotic characters, may manifest effective protective action against cadmium‐induced oxidative toxicity in mouse testicular germ cells. Anat Rec, 2011. © 2010 Wiley‐Liss, Inc.     

Angiostatic Effects of NK Cell‐Derived IFN‐γ Counteracted by Tumour Cell Bcl‐xL Expression

Anti‐apoptotic proteins that block death receptor‐mediated apoptosis favour tumour evasion of the immune system, leading to enhanced tumour progression. However, it is unclear whether blocking the mitochondrial pathway of apoptosis will protect tumours from immune cell attack. Here, we report that the anti‐apoptotic protein Bcl‐x_L, known for its ability to block the mitochondrial pathway of apoptosis, exerted tumour‐progressive activity in a murine lymphoma model. Bcl‐x_L overexpressing tumours exhibited a more aggressive development than control tumours. Surprisingly, Bcl‐x_L protection of tumours from NK cell‐mediated attack did not involve protection from NK cell‐mediated cytotoxicity. Instead, Bcl‐x_L‐blocked apoptosis resulting from hypoxia and/or nutrient loss associated with the inhibition of angiogenesis caused by NK cell‐secreted IFN‐γ. These results support the notion that NK cells may inhibit tumour growth also by mechanisms other than direct cytotoxicity. Hence, the present results unravel a pathway by which tumours with a block in the mitochondrial pathway of apoptosis can evade the immune system.     

Exogenous High‐Mobility Group Box 1 Inhibits Apoptosis and Promotes the Proliferation of Lewis Cells via RAGE/TLR4‐Dependent Signal Pathways

Upregulated high‐mobility group box 1 (HMGB1) has been found in many diseases. Nevertheless, the function of HMGB1 on modulating the proliferation of lung cancer cells (Lewis cells) and inhibiting apoptosis is poorly understood, as well as the involved intracellular signalling. In the present study, we firstly found the apoptosis of Lewis was increased following Hanks’ balanced salt solution (HBSS)‐induced starvation, while it was rescued after exogenous HMGB1 protein was added; furthermore, the receptor for advanced glycation end products (RAGE) and Toll‐like receptor (TLR4) could coordinately improve the proliferation of tumour cells in vitro, and HMGB1 could enhance the phosphorylation of PI3K/Akt and Erk1/2, inhibit the expression of pro‐apoptosis protein Bax and promote the expression of anti‐apoptosis protein Bcl‐2. These findings clearly demonstrated that HMGB1–RAGE/TLR4‐ PI3K‐Akt/Erk1/2 pathway contributed to the proliferation of Lewis. Moreover, our observations provide experimental and theoretical basis for clinical biological therapy for cancers; it also may be a new target for intervention and treatment of lung cancer.     

Upregulation of LAPTM4B‐35 Promotes Malignant Transformation and Tumorigenesis in L02 Human Liver Cell Line

Hepatocellular carcinoma (HCC) is one of the most frequent malignant neoplasms worldwide and is the second leading cause of cancer death in China. We have previously demonstrated that LAPTM4B‐35, encoded by lysosomal protein transmembrane 4 beta gene, is overexpressed in over 80% of HCCs and is a novel‐independent prognostic factor for metastasis, recurrence, and postoperative survival in HCC. In this study, we investigated the role of LAPTM4B‐35 in malignant transformation and tumorigenesis using L02 cells, a cell line originated from human normal liver cells. Our data show that replication‐deficient adenovirus vector‐mediated upregulation of LAPTM4B‐35 promotes anchorage‐independent proliferation and resistance to adriamycin‐induced apoptosis. Study of the underlying mechanisms demonstrated alterations of molecular events involved in these processes, which included the activation of phosphoinositide 3‐kinases (PI3K)/serine/threonine protein kinase B (PKB/AKT)/bcl‐xL/bcl‐2‐associated death promoter homolog (Bad) signaling pathway, inhibition of caspase‐3 activation, upregulation of Bcl‐2, and downregulation of Bax. In addition, upregulation of LAPTM4B‐35 in L02 cells resulted in tumorigenesis in 100% (6/6) of inoculated nude mice and accelerated the death of mice with xenografts in vivo. In conclusion, LAPTM4B‐35 promotes malignant transformation and tumorigenesis in human liver L02 cell line through promotion of deregulated proliferation and inhibition of apoptosis. These findings suggest that overexpression of LAPTM4B‐35 may play a critical role in hepatocarcinogenesis and therefore, may be a therapeutic target for HCC. Anat Rec, 2011. © 2011 Wiley‐Liss, Inc.     

Pannexin‐1‐dependent caspase‐1 activation and secretion of IL‐1β is regulated by zinc

Inflammatory processes induced by IL‐1β are critical for host defence responses, but are also implicated in disease. Zinc deficiency is a common consequence of, or contributor to, human inflammatory disease. However, the molecular mechanisms through which zinc contributes to inflammatory disease remain largely unknown. We report here that zinc metabolism regulates caspase‐1 activation and IL‐1β secretion. One of the endogenous mediators of IL‐1β secretion is adenosine triphosphate, acting via the P2X7‐receptor and caspase‐1 activation in cells primed with an inflammatory stimulus such as LPS. We show that this process is selectively abolished by a brief pre‐treatment with the zinc chelator N,N,N′,N′‐tetrakis‐(2‐pyridylmethyl) ethylene diamine (TPEN). These effects on IL‐1β secretion were independent of rapid changes in free zinc within the cell, not a direct effect on caspase‐1 activity, and upstream of caspase‐1 activation. TPEN did however inhibit the activity of pannexin‐1, a hemi‐channel critical for adenosine triphosphate and nigericin‐induced IL‐1β release. These data provide new insights into the mechanisms of caspase‐1 activation and how zinc metabolism contributes to inflammatory mechanisms.     

The tobacco‐specific nitrosamine 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) induces mitochondrial and nuclear DNA damage in Caenorhabditis elegans

The metabolites of the tobacco‐specific nitrosamine 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) form DNA adducts in animal models. While there are many reports of formation of nuclear DNA adducts, one report also detected NNK‐induced damage to the mitochondrial genome in rats. Using a different DNA damage detection technology, we tested whether this finding could be repeated in the nematode Caenorhabditis elegans. We treated N2 strain (wild‐type) nematodes with NNK in liquid culture, and applied quantitative PCR to analyze NNK‐induced nuclear and mitochondrial DNA (mtDNA) damage. Our results confirm that NNK causes both nuclear and mtDNA damage. However, we did not detect a difference in the level of nuclear versus mtDNA damage in C. elegans. To test whether the mtDNA damage was associated with mitochondrial dysfunction, we used a transgenic nematode strain that permits in vivo measurement of ATP levels and found lower levels of ATP in NNK‐exposed animals when compared with the unexposed controls. To test whether the lower levels of ATP could be attributed to inhibition of respiratory chain components, we investigated oxygen consumption in whole C. elegans and found reduced oxygen consumption in exposed animals when compared with the unexposed controls. Our data suggest a model in which NNK exposure causes damage to both C. elegans nuclear and mitochondrial genomes, and support the hypothesis that the mitochondrial damage is functionally important in this model. These results also represent a first step in developing this genetically tractable organism as a model for assessing NNK toxicity. Environ. Mol. Mutagen. 55:43–50, 2014. © 2013 Wiley Periodicals, Inc.     

Silibinin inhibited autophagy and mitochondrial apoptosis in pancreatic carcinoma by activating JNK/SAPK signaling

BackgroundPrevious investigation have indicated Silibinin induces apoptosis and JNK/SAPK in human pancreatic cancer cells. This study aims to evaluate the further mechanism of Silibinin in pancreatic cancer treatment.Materials and methodsHuman pancreatic cancer cell lines SW1990 was treated with Silibinin and/or JNK/SAPK inhibitor SP600125 followed by measurement of cell viability, apoptosis, autophagy, ROS and ATP, and western blotting.ResultsSilibinin promoted cell viability and promoted cell apoptosis. The expression of ROS and ATP associated with mitochondrial function was also promoted by the treatment of silibinin. Silibinin also promoted autophagy in pancreatic cancer cells. All these biological effects of Silibinin can be reversed by JNK/SAPK inhibitor.ConclusionsThe biological effects regulated by Silibinin can be mediated by JNK/SAPK signaling. This provides a solid theoretical basis for the role of Silibinin in the treatment of pancreatic cancer.     

Gemcitabine sensitizes lung cancer cells to Fas/FasL system‐mediated killing

Gemcitabine is a chemotherapy agent commonly used in the treatment of non‐small cell lung cancer (NSCLC) that has been demonstrated to induce apoptosis in NSCLC cells by increasing functionally active Fas expression. The aim of this study was to evaluate the Fas/Fas ligand (FasL) system involvement in gemcitabine‐induced lung cancer cell killing. NSCLC H292 cells were cultured in the presence or absence of gemcitabine. FasL mRNA and protein were evaluated by real‐time PCR, and by Western blot and flow cytometry, respectively. Apoptosis of FasL‐expressing cells was evaluated by flow cytometry, and caspase‐8 and caspase‐3 activation by Western blot and a colorimetric assay. Cytotoxicity of lymphokine‐activated killer (LAK) cells and malignant pleural fluid lymphocytes against H292 cells was analysed in the presence or absence of the neutralizing anti‐Fas ZB4 antibody, by flow cytometry. Gemcitabine increased FasL mRNA and total protein expression, the percentage of H292 cells bearing membrane‐bound FasL (mFasL) and of mFasL‐positive apoptotic H292 cells, as well as caspase‐8 and caspase‐3 cleavage. Moreover, gemcitabine increased CH11‐induced caspase‐8 and caspase‐3 cleavage and proteolytic activity. Cytotoxicity of LAK cells and pleural fluid lymphocytes was increased against gemcitabine‐treated H292 cells and was partially inhibited by ZB4 antibody. These results demonstrate that gemcitabine: (i) induces up‐regulation of FasL in lung cancer cells triggering cell apoptosis via an autocrine/paracrine loop; (ii) induces a Fas‐dependent apoptosis mediated by caspase‐8 and caspase‐3 activation; (iii) enhances the sensitivity of lung cancer cells to cytotoxic activity of LAK cells and malignant pleural fluid lymphocytes, partially via Fas/FasL pathway. Our data strongly suggest an active involvement of the Fas/FasL system in gemcitabine‐induced lung cancer cell killing.