Hsp90 inhibitor geldanamycin and its derivatives as novel cancer chemotherapeutic agents

Geldanamycin, an ansamycin-derivative benzoquinone compound, was originally isolated as a natural product with anti-fungal activity. Later, geldanamycin was found to have anti-proliferative activity on tumor cells transformed by oncogene kinases such as v-Src. Geldanamycin neither bind nor inhibit oncogene kinases directly, but specifically binds and inhibits a major molecular chaperone, Hsp90. Hsp90 is a highly abundant and essential cytosolic protein and the expression level of Hsp90 increases by environmental stress. Hsp90 functions as a molecular chaperone by binding to various cellular proteins and supporting the proper folding, stability, and function of target proteins. The Hsp90 client proteins include a wide variety of signal-transducing proteins that regulate cell growth and differentiation, such as protein kinases and steroid hormone receptors. Hsp90 functions in an ATP-dependent manner in cooperation with other molecular chaperones such as Cdc37 and FKBP52. Geldanamycin specifically inhibits the essential ATPase activity of Hsp90. Thus, treatment of cells with geldanamycin results in inactivation, destabilization, and degradation of Hsp90 client proteins. Because Hsp90 client proteins play important roles in the regulation of the cell cycle, cell growth, cell survival, apoptosis, and oncogenesis, geldanamycin obstructs the proliferation of cancer cells and shows anti-cancer activity in experimental animals. Although difficulties with solubility and toxicity should be overcome, Hsp90 inhibitors will be potential and effective cancer chemotherapeutic drugs with a unique profile. In fact, a modified geldanamycin with lower toxicity, 17-allylaminogeldanamycin (17-AAG), has been examined in phase I clinical trials with encouraging results.     

Lipopolysaccharide induces apoptotic insults to human alveolar epithelial A549 cells through reactive oxygen species-mediated activation of an intrinsic mitochondrion-dependent pathway

Alveolar type II epithelial cells can regulate immune responses to sepsis-induced acute lung injury. Lipopolysaccharide (LPS), an outer membrane component of Gram-negative bacteria, can cause septic shock. This study was designed to evaluate the cytotoxic effects of LPS on human alveolar epithelial A549 cells and its possible molecular mechanisms. Exposure of A549 cells to LPS decreased cell viability in concentration- and time-dependent manners. In parallel, LPS concentration- and time-dependently induced apoptosis of A549 cells. Meanwhile, LPS only at a high concentration of 10 μg/ml caused mildly necrotic insults to A549 cells. In terms of the mechanism, exposure of A549 cells to LPS increased the levels of cellular nitric oxide and reactive oxygen species (ROS). Pretreatment with N-acetylcysteine (NAC), an antioxidant, significantly lowered LPS-caused enhancement of intracellular ROS in A549 cells and simultaneously attenuated the apoptotic insults. Sequentially, treatment of A549 cells with LPS caused significant decreases in the mitochondrial membrane potential and biosynthesis of adenosine triphosphate. In succession, LPS triggered the release of cytochrome c from the mitochondria to the cytoplasm. Activities of caspase-9 and caspase-6 were subsequently augmented following LPS administration. Consequently, exposure of A549 cells induced DNA fragmentation in a time-dependent manner. Pretreatment of A549 cells with NAC significantly ameliorated LPS-caused alterations in caspase-9 activation and DNA damage. Therefore, this study shows that LPS specifically induces apoptotic insults to human alveolar epithelial cells through ROS-mediated activation of the intrinsic mitochondrion–cytochrome c-caspase protease mechanism.     

Sulforaphane induces reactive oxygen species-mediated mitotic arrest and subsequent apoptosis in human bladder cancer 5637 cells

The present study was undertaken to determine whether sulforaphane-derived reactive oxygen species (ROS) might cause growth arrest and apoptosis in human bladder cancer 5637 cells. Our results show that the reduced viability of 5637 cells by sulforaphane is due to mitotic arrest, but not the G2 phase. The sulforaphane-induced mitotic arrest correlated with an induction of cyclin B1 and phosphorylation of Cdk1, as well as a concomitant increased complex between cyclin B1 and Cdk1. Sulforaphane-induced apoptosis was associated with the activation of caspase-8 and -9, the initiators caspases of the extrinsic and intrinsic apoptotic pathways, respectively, and activation of effector caspase-3 and cleavage of poly (ADP-ribose) polymerase. However, blockage of caspase activation inhibited apoptosis and abrogated growth inhibition in sulforaphane-treated 5637 cells. This study further investigated the roles of ROS with respect to mitotic arrest and the apoptotic effect of sulforaphane, and the maximum level of ROS accumulation was observed 3 h after sulforaphane treatment. However, a ROS scavenger, N-acetyl-L-cysteine, notably attenuated sulforaphane-mediated apoptosis as well as mitotic arrest. Overall, these results suggest that sulforaphane induces mitotic arrest and apoptosis of 5637 cells via a ROS-dependent pathway.     

Quinalizarin induces cycle arrest and apoptosis via reactive oxygen species-mediated signaling pathways in human melanoma A375 cells

Quinalizarin, a bioactive and highly selective compound, is known to promote apoptosis in colon and lung cancer cells. However, studies evaluating quinalizarin-induced apoptosis in melanoma cells have not been conducted. In the present study, we investigated the underlying mechanisms of antimelanoma activity of quinalizarin in human melanoma A375 cells. The MTT assay and Trypan blue staining were used to evaluate the cell viability. The flow cytometry was used to detect cell cycle, apoptosis and reactive oxygen species (ROS). Western blot was used to detect the expression of cell cycle and apoptosis-related proteins, MAPK, and STAT3. The results revealed a significant dose and time dependent effect of quinalizarin on inhibiting proliferation in three kinds of human melanoma cells, and had no significant toxic effects on normal cells. Moreover, quinalizarin triggered G2/M phase cell arrest by modulating the protein expression levels of CDK 1/2, cyclin A, cyclin B, p21 and p27, and induced apoptosis by down-regulating the antiapoptotic protein Bcl-2 and upregulating the proapoptotic protein BAD, leading to the activation of caspase-3 and PARP in the caspase cascade in A375 cells. Quinalizarin treatment led to apoptosis of A375 cells via activation of MAPK and inhibition of STAT3 signaling pathways. In addition, quinalizarin increased the level of ROS, but ROS scavenger NAC inhibited quinalizarin-induced apoptosis by regulating MAPK and STAT3 signaling pathways. In summary, quinalizarin induces cell cycle arrest and apoptosis via ROS-mediated MAPK and STAT3 signaling pathways in human melanoma A375 cells, and quinalizarin may be used as a novel and effective antimelanoma therapeutic.     

Hsp90抑制剂FS-108抑制癌基因依赖肿瘤细胞增殖及运动的机制研究

目的探讨Hsp90抑制剂FS-108抑制癌基因依赖的肿瘤细胞EBC-1和A375的增殖及运动能力的作用机制。方法采用磺酰罗丹明B法检测FS-108对细胞增殖的影响;采用蛋白免疫印迹法检测FS-108对Hsp90客户蛋白、周期调控蛋白及凋亡调控蛋白的变化;采用流式细胞术检测FS-108作用后细胞周期分布及凋亡情况;采用Transwell小室方法检测FS-108对细胞运动能力的影响。结果 FS-108对EBC-1和A375均有明显的增殖抑制作用,IC_(50)分别为25.53 nmol·L~(-1)和30.02 nmol·L~(-1)。FS-108能有效降解肿瘤细胞中的客户蛋白c-Met和B-Raf并进而抑制下游AKT及ERK信号通路。FS-108能诱导细胞产生G_2/M期阻滞及凋亡。同时,FS-108还能明显抑制EBC-1和A375细胞的运动能力。结论 Hsp90抑制剂FS-108主要通过诱导肿瘤细胞产生G_2/M期阻滞和细胞凋亡,发挥其抑制增殖的细胞生物学效应,同时,FS-108也具有一定抗转移潜能。     

Guanylate cyclase activator YC-1 potentiates apoptotic effect of licochalcone A on human epithelial ovarian carcinoma cells via activation of death receptor and mitochondrial pathways

Natural phenol licorice compounds have been shown to induce apoptosis in cancer cells. 3-(5'-Hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) may enhance the sensitivity of cancer cells to anticancer drugs. However, the combined effect of licochalcone A and YC-1 on cell death in ovarian cancer cells has not been studied. We assessed the combined effect of licochalcone A and YC-1 on apoptosis in human epithelial ovarian carcinoma cell lines in relation to the cell death process. In the OVCAR-3 and SK-OV-3 cell lines, licochalocone A induced a decrease in Bid, Bcl-2, Bcl-xL and survivin protein levels; an increase in Bax levels; loss of the mitochondrial transmembrane potential; cytochrome c release; activation of caspases (-8, -9 and -3); cleavage of PARP-1; and an increase in the tumor suppressor p53 levels. YC-1 enhanced licochalcone A-induced apoptosis-related protein activation, nuclear damage and cell death. These results suggest that YC-1 may potentiate the apoptotic effect of licochalcone A on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-dependent pathway and the mitochondria-mediated apoptotic pathway, leading to caspase activation. The combination of licochalcone A and YC-1 may confer a benefit in the treatment of human epithelial ovarian adenocarcinoma.     

Diarylheptanoid hirsutenone enhances apoptotic effect of TRAIL on epithelial ovarian carcinoma cell lines via activation of death receptor and mitochondrial pathway

Tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL) induces apoptosis in various cancer cells. Diarylheptanoids such as hirsutenone and oregonin have been shown to have anti-inflammatory and anti-tumor effects. However, it is still unknown by which mechanism diarylheptanoids induce cell death. In addition, the effect of hirsutenone on TRAIL-induced apoptosis in the human epithelial ovarian carcinoma cell lines is unknown. To assess the apoptosis promoting effect of hirsutenone, we investigated the effect of hirsutenone on the apoptotic effect of TRAIL using the human epithelial carcinoma cell lines OVCAR-3 and SK-OV-3. TRAIL induced nuclear damage, decrease in Bid, Bcl-2 and Bcl-xL protein levels, increase in Bax levels, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases (8, 9 and 3) and increase in tumor suppressor p53 levels. Hirsutenone enhanced the TRAIL-induced apoptosis-related protein activation, nuclear damage and cell death. The results suggest that hirsutenone may enhance the apoptotic effect of TRAIL on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-dependent pathways and the mitochondria-mediated apoptotic pathway, leading to caspase activation. Hirsutenone may confer a benefit in the TRAIL treatment of epithelial ovarian adenocarcinoma.     

进口紫杉醇与国产紫杉醇治疗上皮性卵巢癌的临床疗效分析

目的 分析并对比进口紫杉醇与国产紫杉醇治疗上皮性卵巢癌的近期疗效与副作用.方法 回顾性分析我院2006年1月至2010年12月收治的59例上皮性卵巢癌患者的临床资料,其中经进口紫杉醇(泰素)治疗的患者18例(进口紫杉醇组),经国产紫杉醇治疗的患者41例(国产紫杉醇组),对2组患者的年龄、病理类型、临床分期、是否合并腹水及肠管或肝脾转移情况、治疗方法(常规治疗或是先期化疗),及治疗总有效率和无瘤生存期以及患者的骨髓抑制情况进行对比.结果 2组患者年龄、病理类型、临床分期、合并转移情况及治疗方法差异均无统计学意义(P＞0.05).进口紫杉醇组治疗后总有效率为94.4％(17/18),其中完全缓解(CR) 13例,部分缓解(PR)4例,进展(PD)1例;国产紫杉醇组总有效率95.1％(39/41),其中CR 29例,PR 10例,PD 2例;2组总有效率比较差异无统计学意义(P＞0.05).进口紫杉醇组和国产紫杉醇组无瘤生存期差异无统计学意义(P＞0.05).进口紫杉醇组发生0度骨髓抑制为5例,Ⅰ度0例,Ⅱ度7例,Ⅲ度3例,Ⅳ度3例;国产紫杉醇组0度骨髓抑制为10例,Ⅰ度为3例,Ⅱ度14例,Ⅲ度11例,Ⅳ度3例.2组骨髓抑制发生情况差异无统计学意义(P＞0.05).结论 进口紫杉醇与国产紫杉醇在治疗上皮性卵巢癌的近期疗效与不良反应方面无差异.     

新生霉素对STI571耐药K562/G01细胞的作用

目的研究热休克蛋白90(Hsp90)抑制剂新生霉素(novobiocin,NB)对STI571耐药K562/G01细胞生长的抑制作用,并研究NB和STI571联合应用对STI571敏感和耐药细胞的作用,并进一步探讨该作用与Bcr-Abl蛋白水平和Bcr-Abl激酶水平的关系。方法用MTT法检测NB对K562和K562/G01细胞生长的抑制作用,用金氏公式评价药物合用体外是否有协同作用,用蛋白免疫印迹法检测Bcr-Abl蛋白水平和p-Tyrosine水平。结果NB能抑制K562和K562/G01细胞增殖,IC50分别是0.4353mmol.L-1和0.3490mmol.L-1;NB和STI571联合应用对STI571敏感和耐药细胞均有协同作用;NB能使K562和K562/G01细胞内Bcr-Abl和p-Tyrosine蛋白含量减少。结论NB通过减少Bcr-Abl和p-Tyrosine蛋白含量,抑制STI571耐药细胞K562/G01的增殖,NB和STI571有协同效应。     

Hirsutanol A induces apoptosis and autophagy via reactive oxygen species accumulation in breast cancer MCF-7 cells

Hirsutanol A is a novel sesquiterpene compound purified from the marine fungus Chondrostereum sp in the coral Sarcophyton tortuosum. Our previous studies had demonstrated that hirsutanol A exerted potent cytotoxic effect in many kinds of cancer cell lines. Here, the anticancer molecular mechanisms of hirsutanol A were investigated in breast cancer MCF-7 cells. The results showed that hirsutanol A could inhibit cell proliferation, elevate reactive oxygen species (ROS) level, and induce apoptosis and autophagy. Co-treatment with the potent antioxidant agent N-acetyl-L-cysteine could effectively reverse the effect of enhanced ROS production, which in turn, reduces growth inhibition, apoptosis, and autophagy mediated by hirsutanol A. In addition, blocking autophagy by bafilomycin A1 or Atg7-siRNA could synergistically enhance the antiproliferative effect and apoptosis induced by hirsutanol A. These data suggested that hirsutanol A could induce apoptosis and autophagy via accumulation of ROS and co-treatment with an autophagy inhibitor could sensitize MCF-7 cells to hirsutanol A.     

The role of reactive oxygen species in WP 631-induced death of human ovarian cancer cells: A comparison with the effect of doxorubicin

In the present study, we investigated the anticancer activity of WP 631, a new anthracycline analog, in weakly doxorubicin-resistant SKOV-3 ovarian cancer cells. We studied the time-course of apoptotic and necrotic events: the production of reactive oxygen species (ROS) and changes in the mitochondrial membrane potential in human ovarian cancer cells exposed to WP 631 in the presence and absence of an antioxidant, N-acetylcysteine (NAC). The effect of WP 631 was compared with the activity of doxorubicin (DOX), the best known first-generation anthracycline.Cytotoxic activity was determined by the MTT assay. The morphological changes characteristic of apoptosis and necrosis in drug-treated cells were analyzed by double staining with Hoechst 33258 and propidium iodide (PI) using fluorescence microscopy. The production of reactive oxygen species and changes in mitochondrial membrane potential were studied using specific fluorescence probes: DCFH₂-DA and JC-1, respectively.The experiments showed that WP 631 was three times more cytotoxic than DOX in the tested cell line. It was found that the new anthracycline analog induced mainly apoptosis and, marginally, necrosis. Apoptotic cell death was associated with morphological changes and a decrease in mitochondrial membrane potential. In comparison to DOX, the novel bisanthracycline induced a significantly higher level of ROS and a greater drop in the membrane potential.The results provide direct evidence that the novel anthracycline WP 631 is considerably more cytotoxic to human SKOV-3 ovarian cancer cells than doxorubicin. The drug can produce ROS, which are immediately involved in the induction of apoptotic cell death.     

Synergistic apoptotic effect of crocin and cisplatin on osteosarcoma cells via caspase induced apoptosis

Crocin is well-known traditional Chinese medicine which is extracted from saffron. However, its role in osteosarcoma has not been well understood. Therefore, we used crocin and cisplatin individually or jointly on MG63 and OS732 cells so as to explore whether crocin could induce cellular apoptosis and suppress the ability of invasion of osteosarcoma cells. Cell survival rates, changes of cellular shape, cell apoptosis and cell invasion were analyzed, respectively, by 3-(4,5)-dimethylthiahiazo (-z-y1)-2,5-di- phenytetrazoliumromide (MTT) assay, inverted phase contrast microscope and fluorescence microscope, ?ow cytometry, and Transwell invasion chamber methods. The expressions of caspase-3 and caspase-8 were detected by Western blot. The survival rate of combined application was significantly lower than that of the individual application. Apoptosis-inducing effect of combined application was much stronger than that of individual application. The invasion ability of MG63 and OS732 cells was restrained significantly in the combined group compared with the individual group and control group. Combined group has the effect of up-regulating the expressions of cleaved-caspase-3 and caspase-8. The results suggested that combination of crocin and cisplatin has a strong killing effect on osteosarcoma cells and suppresses the ability of invasion of MG63 and OS732 cells which might be related to up-regulate the expression of caspase-3 and caspase-8.     

A novel class of geldanamycin derivatives as HCV replication inhibitors targeting on Hsp90: synthesis, structure-activity relationships and anti-HCV activity in GS4.3 replicon cells

A novel class of geldanamycin (GA) derivatives as hepatitis C virus (HCV) replication inhibitors has been synthesized and their anti-HCV activities were evaluated in GS4.3 HCV replicon cells. Most of the synthesized compounds demonstrated potential activities against HCV in vitro. Substitution with an aliphatic cyclic group (2b) and polar phosphate group (2f) at the 17 position of GA resulted in more potent inhibitory activity. The configurations of the tetrahydrofurfurylamino (THFM) substituents obviously affected their antiviral activities. The 2b with a 2'-(R)-THFM group at the 17 position showed much potent activity and higher selectivity than its 2'-(S) and 2'-(R, S) epimers. In the tested GA derivatives, 2b and 2f show the most potential leading compounds for development of novel anti-HCV agents.     

Growth inhibition and apoptotic effect of alpha-eleostearic acid on human breast cancer cells

Alpha-eleostearic acid (α-ESA) is a natural and biologically active compound which possesses potent antioxidant and anti-tumor activity. The purpose of this study was to confirm the anticancer activity of α-ESA against human breast cancer cells and to further elucidate its mechanism of activity. Human breast cancer cells and normal liver cells were used for in-vitro tests of the anticancer activity of α-ESA, including cytotoxicity, colony formation inhibition, EdU incorporation, AO/EB staining of apoptotic cells, cell cycle distribution through flow cytometry, and PPARγ, p21, Bax, p53, and caspase-3 mRNA expressions through RT-PCR. After α-ESA treatment, the proliferation, colony formation, and EdU labeling indices of cancer cells decreased (p < 0.05), while the AO/EB-stained apoptotic cells increased (p < 0.05). By FCM analysis, the apoptotic indices increased (p < 0.01), and the cell population decreased in S phase (p < 0.01) and increased in G₂/M phase (p < 0.05) in α-ESA treated cancer cells. RT-RCR showed that α-ESA significantly increased the expression levels of PPARγ, p21, Bax, p53, and caspase-3 mRNA. The findings in these studies suggested that α-ESA exhibited a potential cytotoxicity and apoptosis induction effect on human breast cancer cells, with little effect on normal cells at certain concentrations. The mechanism for such effects might be associated with the inhibition of DNA synthesis, induction of apoptosis, and cell cycle arrest of cancer cells through up-regulation of PPARγ, p21, Bax, p53, and caspase-3 expressions.     

DCZ5248, a novel dual inhibitor of Hsp90 and autophagy,exerts antitumor activity against colon cancer

Hsp90 is a potential therapeutic target for tumor,as it maintains the stability of a variety of proteins related to tumor development and progression.Autophagy is a self-degradation process to maintain cellular homeostasis and autophagy inhibitors can suppress tumor growth.In this study,we identified DCZ5248,a triazine derivative,was a dual inhibitor of both Hsp90 and late-autophagy with potent antitumor activity against colon cancer cells in vitro and in vivo.We showed that DCZ5248(0.1–10μM)induced dose-dependent degradation of Hsp90 client proteins(AKT,CDK4,CDK6 and RAF-1)in HCT 116 colon cancer cells through a proteasome-dependent pathway.Meanwhile,DCZ5248(0.3μM)induced cytoplasmic vacuole formation,LC3 II conversion,p62 protein upregulation,and inhibited autophagy at the late stage in the colon cancer cell lines tested.We further revealed that the inhibition of autophagy was achieved by impairing lysosomal functions through induction of lysosomal acidification and attenuation of lysosomal cathepsin activity.The modulation of autophagy by DCZ5248 was independent of Hsp90 inhibition as the autophagy inhibition was not blocked by Hsp90 knockdown.Importantly,inhibition of both Hsp90 function and autophagy by DCZ5248 induced G1-phase cell cycle arrest,apoptosis,and exerted potent antitumor activity against colon cancer cells both in vitro and in vivo.These findings demonstrate that DCZ5248 is a novel dual inhibitor of Hsp90 and autophagy with potential for colon cancer therapy.     

Pharmaceutical development of IPI‐504, an Hsp90 inhibitor and clinical candidate for the treatment of cancer

IPI‐504 (retaspimycin hydrochloride) is an Hsp90 inhibitor that is the subject of multiple clinical trials for the treatment of cancer. IPI‐504 is an aqueous soluble (>200 mg/ml) hydroquinone hydrochloride salt of 17‐(allylamino)‐17‐demethoxygeldanamycin (17‐AAG), a quinone derivative also undergoing clinical evaluation, albeit with suboptimal formulations that address its inferior aqueous solubility (～50 µg/ml). IPI‐504 interconverts with 17‐AAG in vivo through oxidation‐reduction reactions that result in a dynamic redox equilibrium. The development challenges associated with redox active molecules are significant due to the pH, oxygen, and temperature sensitivities associated with such chemotypes. The API and sterile drug product manufacturing processes thus warrant the monitoring and control of these key variables. Furthermore, the pharmaceutical development challenges associated with cancer agents that are often fast‐tracked due to unmet medical needs mandate a rapid development cycle with associated regulatory hurdles. Drug Dev Res 71: 429–438, 2010. © 2010 Wiley‐Liss, Inc.     

活性氧自由基对膀胱癌患者淋巴因子激活的杀伤细胞的作用

目的:探讨活性氧自由基对膀胱癌患者淋巴因子激活的杀伤(LAK)细胞的增殖和抗膀胱癌细胞系活性的作用.方法:分别用细胞计数和MTT法测定LAK细胞的增殖和细胞毒作用.结果:羟自由基浓度依赖性地抑制IL-2所诱导的LAK细胞增殖.用抗坏血酸400μmol·L~(-1)和硫酸亚铁40μmol·L~(-1)培养细胞96h,细胞增殖被抑制34.5％.这种抑制可被一定浓度的甘露醇和依地酸(edetic acid)扭转.一定浓度的超氧阴离子或一氧化氮释放剂硝普钠可刺激由IL-2所诱导的LAK细胞的增殖.超氧化物歧化酶(SOD)可抵消超氧阴离子的刺激作用.外源性超氧阴离子可加强LAK细胞对BIU-87和EJ细胞的杀伤.羟自由基和SOD对LAK细胞杀伤作用则影响不明显.结论:超氧阴离子和一氧化氮可增强膀胱癌患者LAK细胞的增殖、激活和抗肿瘤的细胞毒,而羟自由基对此起抑制作用.这两种活性氧自由基对IL-2诱导的LAK细胞增殖的作用是不同的.     

Nonylphenol‐induced apoptotic cell death in mouse TM4 Sertoli cells via the generation of reactive oxygen species and activation of the ERK signaling pathway

Nonylphenol (NP), a representative endocrine disruptor, interferes with reproductive function in aquatic organisms and animals. Although many previous studies have focused on apoptotic cell death by NP, the fundamental mechanism of NP on apoptosis remains poorly understood. Here, we investigated the molecular mechanism on NP‐induced apoptotic cell death in mouse TM4 Sertoli cells. To evaluate NP treatment on cell viability, formazan and lactate dehydrogenase (LDH) assays were performed. Results indicate that NP reduced cell viability and increased the release of LDH in dose‐ and time‐dependent manners. The reduction of cell viability by NP treatment appeared to involve necrosis as well as apoptosis based on nuclear fragmentation, an increase in the sub G1 population, and the detection of poly(ADP ribose) polymerase and caspase‐3 cleavage. Additionally, the anti‐apoptotic protein Bcl‐2 diminished, whereas the pro‐apoptotic protein Bax increased in a time‐dependent manner. Note that NP‐induced apoptotic cell death was enhanced by the generation of reactive oxygen species (ROS) and activation of extracellular signal‐regulated kinase (ERK) signaling. Pretreatment with N‐acetylcysteine, an antioxidant, attenuated NP‐induced apoptotic cell death. Moreover, NP caused a transient activation of the MAPK pathway. In particular, NP‐induced cell death was significantly suppressed by U0126, a specific inhibitor of ERK. Taken together, our results suggest that NP induces apoptosis in mouse TM4 Sertoli cells via ROS generation and ERK activation. Copyright © 2013 John Wiley & Sons, Ltd.