Silymarin augments human cervical cancer HeLa cell apoptosis via P38/JNK MAPK pathways in serum-free medium

Silymarin was proved to have a protective effect of UV-induced A375-S2 cell apoptosis in our previous research. In this study, its pro-apoptotic and anti-apoptotic activities on human cervical cancer (HeLa) cells in vitro were investigated. Silymarin induced HeLa cell death through both apoptotic and necrotic pathways. At low doses (below 80 micromol l-1), it induced cell apoptosis, but caused necrosis at high dose (160 micromol l-1). Silymarin induced typical chromatin condensation and nuclear fragmentation as a hallmark of apoptosis. In this case, mitochondrial Bcl-2 family, Bcl-2 and Bax, were not involved in apoptotic effects; however, silymarin-induced cell death was regulated by the activation of p38 and JNK MAPKs. We also found that pan-caspase inhibitor and caspase-3 inhibitor could not antagonise silymarin-induced apoptosis. Therefore, silymarin induced and augmented HeLa cell apoptosis through p38/JNK MAPKs in the serum-free medium.     

Antiapoptotic Effect of Paricalcitol in Gentamicin-induced Kidney Injury

While the anti-apoptotic effect of paricalcitol has been demonstrated in various animal models, it is not yet clear whether paricalcitol attenuates the apoptosis in gentamicin (GM)-induced kidney injury. We investigated the effect of paricalcitol on apoptotic pathways in rat kidneys damaged by GM. Rats were randomly divided into three groups: 1) Control group (n=8), where only vehicle was delivered, 2) GM group (n=10), where rats were treated with GM (150 mg/kg/day) for 7 days, 3) PARI group (n=10), where rats were co-treated with paricalcitol (0.2 µg/kg/day) and GM for 7 days. Paricalcitol attenuated renal dysfunction by GM administration in biochemical profiles. In terminal deoxynucleotidyl transferase dUTP nick end labeling staining, increased apoptosis was observed in GM group, which was reversed by paricalcitol co-treatment. Immunoblotting using protein samples from rat cortex/outer stripe of outer medulla showed increased Bax/Bcl-2 ratio and cleaved form of caspase-3 in GM group, both of which were reversed by paricalcitol. The phosphorylated Jun-N-terminal kinase (JNK) expression was increase in GM, which was counteracted by paricalcitol. The protein expression of p-Akt and nitro-tyrosine was also enhanced in GM-treated rats compared with control rats, which was reversed by paricalcitol co-treatment. Paricalcitol protects GM-induced renal injury by antiapoptotic mechanisms, including inhibition of intrinsic apoptosis pathway and JNK.     

Silymarin augments human cervical cancer HeLa cell apoptosis via P38/JNK MAPK pathways in serum-free medium

Silymarin was proved to have a protective effect of UV-induced A375-S2 cell apoptosis in our previous research. In this study, its pro-apoptotic and anti-apoptotic activities on human cervical cancer (HeLa) cells in vitro were investigated. Silymarin induced HeLa cell death through both apoptotic and necrotic pathways. At low doses (below 80 μmol l^? 1), it induced cell apoptosis, but caused necrosis at high dose (160 μmol l^? 1). Silymarin induced typical chromatin condensation and nuclear fragmentation as a hallmark of apoptosis. In this case, mitochondrial Bcl-2 family, Bcl-2 and Bax, were not involved in apoptotic effects; however, silymarin-induced cell death was regulated by the activation of p38 and JNK MAPKs. We also found that pan-caspase inhibitor and caspase-3 inhibitor could not antagonise silymarin-induced apoptosis. Therefore, silymarin induced and augmented HeLa cell apoptosis through p38/JNK MAPKs in the serum-free medium.     

MAPK inhibitors and pifithrin-alpha block cinnamaldehyde-induced apoptosis in human PLC/PRF/5 cells

Cinnamaldehyde (Cin) has been shown to be effective in inducing apoptotic cell death in a number of human cancer cells. The aim of this study was to investigate the effect of pifithrin-alpha (PFTalpha; a specific p53 inhibitor) and mitogen-activated protein kinases (MAPKs) inhibitors [namely SP600125 (a specific JNK inhibitor), SB203580 (a specific p38 inhibitor) and PD98059 (a specific ERK inhibitor)] on apoptotic signaling transduction mechanism induced by Cin in human hepatoma PLC/PRF/5 (CD95-negative) cells. Using XTT assay, Cin exhibited a powerful cytotoxic effect and apoptotic induction in PLC/PRF/5 cells. Apoptosis was elicited when cells were treated with 1 microM Cin as characterized by morphological changes and the appearance of phosphatidylserine on the outer surface of the plasma membrane. Cin down-regulated the expression of Bcl-(XL), up-regulated mutant p53 and Bax proteins and promoted caspase-3 to active forms, as well as cleaving poly (ADP-ribose) polymerase (PARP) in a time-dependent pattern. This could be supported by the activation and phosphorylation of MAPKs, including JNK, ERK and p38 kinases. Pre-incubation with PFTalpha and specific MAPK inhibitors significantly diminished the effect of Cin-induced apoptosis. The activities of anti-apoptotic (Bcl-(XL)) and pro-apoptotic (Bax) proteins were remarkably affected by PFTalpha and PD98059 pre-treatment. PFTalpha effectively blocked PARP cleavage in cells treated with Cin, and also markedly prevented the phosphorylation of JNK, p38 and ERK proteins. These results suggest that p53 induction and MAPK signaling pathways are required for Cin-mediated apoptosis in PLC/PRF/5 cells.     

Sildenafil attenuates renal injury in an experimental model of rat cisplatin-induced nephrotoxicity

Sildenafil is the first commercially available selective inhibitor of phosphodiesterase-5 (PDE5) and is widely used for the treatment of erectile dysfunction. In recent years, investigations of the role of sildenafil in cardioprotection in animal models have received considerable interest. We evaluated whether sildenafil can attenuate cisplatin-induced nephrotoxicity in a rat experimental model. Male Sprague–Dawley rats were divided into five groups: control rats, sildenafil-control rats, cisplatin-injected rats (5 mg kg⁻¹ IP, single dose), sildenafil-treated cisplatin-injected rats (0.4 mg kg⁻¹, daily), and sildenafil + NG-nitro-l-arginine methyl ester hydrochloride (l-NAME)-treated rats. The molecular, functional, and structural parameters of the kidney were measured. At 96 h after cisplatin injection, serum levels of creatinine were lower in rats treated with both sildenafil + cisplatin compared with rats treated with cisplatin alone, and renal iNOS and eNOS expression was significantly higher in sildenafil + cisplatin-treated rats compared with rats treated with cisplatin alone (all P < 0.05). Renal Bax gene and protein expression was significantly higher in cisplatin-treated rats compared with control rats, and sildenafil treatment significantly reduced the levels of Bax and increased the renal Bax/Bcl-2 ratio (P < 0.05). Sildenafil treatment also reduced renal caspase-3 activation and TUNEL-positive apoptotic cells. These data suggest that sildenafil attenuates experimental cisplatin-induced nephrotoxicity by preventing apoptosis.     

Cisplatin-induced apoptosis by translocation of endogenous Bax in mouse collecting duct cells

cis-platinum(II) (cis-diammine dichloroplatinum; cisplatin) is a potent antitumor compound that is widely used for the treatment of many malignancies. An important side-effect of cisplatin is nephrotoxicity, which results from injury to renal tubular epithelial cells and can be manifested as either acute renal failure or a chronic syndrome characterized by renal electrolyte wasting. Recently, apoptosis has been recognized as an important mechanism of cell death mediating the antitumor effect of cisplatin. This study was undertaken to examine the mechanisms of cell death induced by cisplatin in M-1 cells, which were derived from the outer cortical collecting duct cells of SV40 transgenic mice. Treatment of M-1 cells with high concentrations of cisplatin (0.5 and 1 mM) for 2 hr led to necrotic cell death, whereas a 24-hr treatment with 5-20 microM cisplatin led to apoptosis. Antioxidants protected against cisplatin-induced necrosis, but not apoptosis, indicating that reactive oxygen species play a role in mediating necrosis but not apoptosis induced by cisplatin and that the mechanism of cell death induced by cisplatin is concentration dependent. The low concentrations of cisplatin, which induced apoptosis in M-1 cells, did not affect the expression levels of Bcl-2-related proteins and did not activate c-Jun NH2-terminal kinase (SAPK/JNK). Cisplatin induced the translocation of endogenous Bax from the cytosolic to the membrane fractions and, subsequently, the release of cytochrome c. Overexpression of Bcl-2 blocked cisplatin-induced apoptosis and Bax translocation. These observations suggest that the subcellular redistribution of Bax is a critical event in the apoptosis induced by cisplatin.     

癌痛消方对DEN诱癌大鼠不同时间点凋亡相关蛋白的影响

目的 通过分析癌痛消方(AP)干预二乙基亚硝胺(DEN)诱癌大鼠模型在不同时间点的肝脏组织病理变化、细胞凋亡的相关蛋白表达变化,来探讨癌痛消方治疗原发性肝癌的机制.方法 将50只♂ Wistar大鼠随机分为预防组、模型组(每组20只)和空白对照组(10只),预防组和模型组的大鼠以64 ppm DEN饮水饲养,连续16周.同时,自诱癌之日起,预防组大鼠予以AP灌胃0.2 g·(100 g)-1,qd,每周连续用药6d,给药周期为18周;模型与空白对照组正常饮食,饮水(自来水).分别在4、8、12、16、18周时,随机各取预防组和模型组的4只大鼠、2只空白对照组的,断颈处死,取肝组织(阳性部位)免疫组化法检测Bax、Bcl-2、survivin、Fas、Fasl、caspase-3蛋白的表达情况.结果 空白对照组未见survivin、casepase-3、Fas/Fasl、Bax、Bcl-2蛋白的表达;模型组各时间点均可见凋亡相关蛋白的表达,以凋亡抑制蛋白Bcl-2、survivin表达量较多;AP干预过程可以全程下调survivin、Bcl-2蛋白的表达,上调Fas、casepase、Bax蛋白的表达.结论 AP可以促进受损肝细胞或癌细胞凋亡,其预防和治疗肝癌机理是上调促凋亡蛋白、下调抑凋亡蛋白的表达,促进炎症细胞或肿瘤细胞凋亡.     

Caspase-3 activation in rat frontal cortex following treatment with typical and atypical antipsychotics.

In schizophrenia, studies indicate that apoptotic susceptibility in cortex may be increased. A role for apoptosis in schizophrenia could potentially contribute to post-mortem evidence of reduced cortical neuropil and neuroimaging studies showing progressive cortical gray matter loss. Interestingly, antipsychotic treatment has been associated with higher cortical levels of anti-apoptotic Bcl-2 protein in rat cortex and preliminary data has suggested a similar association in schizophrenia and bipolar disorder. To better understand the effects of antipsychotics on apoptotic regulation, rats were administered haloperidol, clozapine, quetiapine, or saline daily for 4 weeks. Multiple apoptotic markers, including Bcl-2, pro-apoptotic Bax, anti-apoptotic XIAP, and the downstream protease caspase-3 were measured in frontal cortex using Western blot. Caspase-3 activity, activated caspase-3-positive cell number, and DNA/histone fragmentation levels were also determined. Western blot showed that immunoreactivity of Bax and Bcl-2 bands were unchanged with treatment. However, mean density of the 19 kD activated caspase-3 band was 55% higher with haloperidol (p<0.001), 40% higher with clozapine (p<0.05), and 48% higher with quetiapine (p<0.01) compared to saline control. Specific activity of caspase-3 was also increased across all treatments (p<0.0001), while DNA fragmentation rates remained unchanged. These data suggest that sub-chronic antipsychotic treatment is associated with non-lethal caspase-3 activity. The findings do not support a prominent Bcl-2-mediated neuroprotective role for antipsychotics. Although the association between antipsychotic treatment and increased pro-apoptotic caspase-3 is intriguing, further study is needed to understand its potential effects.     

Diallyl trisulfide-induced apoptosis of bladder cancer cells is caspase-dependent and regulated by PI3K/Akt and JNK pathways

Diallyl trisulfide (DATS) is one of the major organosulfur components of garlic (Allium sativum L.), which inhibits the proliferation of various cancer cells, but the exact mechanisms of this action in human bladder cancer cells still remain largely unresolved. In this study, we investigated how DATS induces apoptosis in T24 human bladder cancer cells in vitro. Treatment of T24 cells with DATS resulted in potent anti-proliferative activity. Additionally, some typical apoptotic characteristics, such as chromatin condensation and an increase in the population of sub-G1 hypodiploid cells, were observed. With respect to the mechanism underlying the induction of apoptosis, DATS reduced the expression of anti-apoptotic Bcl-2 and Bcl-xL, and inhibitor of apoptosis protein family proteins, but the expression of pro-apoptotic Bax and death receptor-related proteins was increased compared with the controls. DATS also activated caspase-8 and -9, the respective initiator caspases of the extrinsic and the intrinsic apoptotic pathways. The increase in mitochondrial membrane depolarization was correlated with activation of effector caspase-3 and cleavage of poly-ADP-ribose polymerase, a vital substrate of activated caspase-3. Blockage of caspase activation through treatment with a pan-caspase inhibitor consistently inhibited apoptosis and abrogated growth inhibition in DATS-treated T24 cells. The study further investigated the roles of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinases (MAPKs) pathways with respect to the apoptotic effect of DATS, and showed that DATS deactivates Akt. Additionally, DATS activates extracellular signal-regulated kinase (ERK) and c-Jun N-terminal protein kinase (JNK), but not p38 MAPK, in T24 cells. Unlike ERK, JNK inhibitors reversed DATS-induced apoptosis and growth inhibition; however, inhibition of PI3K/Akt notably enhanced the apoptotic action of DATS. The results suggest that the pro-apoptotic activity of DATS is probably regulated by a caspase-dependent cascade through the activation of both intrinsic and extrinsic signaling pathways, which is mediated through the blocking of PI3K/Akt and the activation of the JNK pathway.     

Induction of apoptosis by streptochlorin isolated from Streptomyces sp. in human leukemic U937 cells

Streptochlorin is a small molecule that produced by marine Streptomyces sp. that is known to have anti-angiogenic and anti-cancer properties. However, the mechanism by which streptochlorin functions is not well understood. In this study, we investigated the pro-apoptotic effect of streptochlorin in human leukemic U937 cells. Streptochlorin treatment resulted in concentration- and time-dependent growth inhibition by inducing apoptosis. The increase in apoptosis that was induced by streptochlorin was correlated with down-regulation of anti-apoptotic Bcl-2 expression, up-regulation of pro-apoptotic Bax and FasL, a decrease in the mitochondrial membrane potential (MMP), activation of caspases and degradation of poly-(ADP-ribose)polymerase and phospholipase C-gamma1 protein. In addition, the cytotoxic effects and apoptotic characteristics induced by streptochlorin were significantly inhibited by z-DEVD-fmk, a caspase-3 inhibitor, which demonstrates the important role that caspase-3 played in the process. Furthermore, Bcl-2 overexpression significantly reversed the streptochlorin-induced growth inhibitory effects via inhibition of the MMP collapse and caspases activation and effectively attenuated the apoptotic response to streptochlorin. However, the elevated levels of FasL expression induced by streptochlorin were not reduced by Bcl-2 overexpression. Taken together, these findings demonstrate that the pro-apoptotic effect of streptochlorin is mediated through activation of caspases and mitochondria in U937 cells.     

Paricalcitol prevents cisplatin-induced renal injury by suppressing apoptosis and proliferation

The present study was performed to examine whether paricalcitol may prevent the cisplatin-induced kidney injury. Furthermore, potential molecular mechanisms underlying the protective effect of paricalcitol were explored. Male Sprague-Dawley rats were treated with vehicle (n=12), cisplatin (n=12, 6 mg/kg/day, i.p.), or cisplatin+paricalcitol (n=12, 0.2 μg/kg/day, s.c.) for 4 days. In another series of experiment, HK-2 cells were treated with cisplatin (50 μM), with or without paricalcitol (0.2 ng/ml). Paricalcitol counteracted the cisplatin-induced decline in renal function. Paricalcitol also suppressed the expression of TGF-β1, Smad signaling, and the subsequent epithelial-to-mesenchymal process in cisplatin-treated rats. The expression of P-p53 and p21 was increased in cisplatin-induced nephropathy. These changes were completely prevented or significantly attenuated with paricalcitol co-treatment. The expression of p27(kip1) was increased in cisplatin-treated rats, which was, however, further augmented by the paricalcitol co-treatment. In HK-2 cells, cisplatin increased the expression of p-ERK1/2 and P-p38. Cisplatin also increased the expression of fibronectin and CTGF. Cisplatin increased the expression of pro-apoptotic markers. The expression of CDK2 and Cyclin E as well as that of PCNA was increased. These changes were completely prevented or significantly attenuated by the paricalcitol pretreatment. In contrast, cisplatin increased the expression of p27(kip1), which was further augmented by the paricalcitol-pretreatment. These results suggest that paricalcitol may ameliorate cisplatin-induced renal injury by suppressing the fibrotic, apoptotic and proliferative factors. Its underlying mechanisms may include inhibition of TGF-β1, mitogen-activated protein kinase signaling, p53-induced apoptosis, and augmentation of p27(kip1).     

维生素D对过氧化氢诱导MIN6细胞凋亡的保护作用

目的探讨维生素D(vitamin D,VitD)对过氧化氢(hydrogen peroxide,H 2O 2)诱导小鼠胰岛β细胞株MIN6细胞凋亡的保护作用及机制。方法VitD预处理后,用H 2O 2处理MIN6细胞,分别运用CCK-8法、Hoechst 33258荧光染色法、流式细胞术检测MIN6细胞增殖、形态及凋亡百分率。Western blot检测增殖与凋亡相关基因Bax、Bcl-2、Caspase-3以及Cleaved caspase-3的表达。结果H 2O 2呈时间和剂量依赖性抑制MIN6细胞增殖,诱导细胞凋亡,降低Bcl-2表达、增加Bax及Cleaved caspase-3表达,Bcl-2/Bax比值降低,Cleaved caspase-3/caspase-3比值增加;当用VitD预处理后,Bcl-2表达增加,Bax、Cleaved caspase-3表达降低,Bcl-2/Bax比值增加,Cleaved caspase-3/caspase-3比值降低,MIN6细胞活力增加。结论VitD预处理后,通过增加抗凋亡Bcl-2表达,降低促凋亡Bax和Cleaved caspase-3表达,减少由H 2O 2诱导的小鼠胰岛β细胞株MIN6细胞凋亡。     

大鼠脑缺血再灌注后Bcl-2和Bax的表达

目的探讨大鼠脑缺血再灌注（CIR）后早期凋亡相关基因Bcl-2和Bax在脑皮质神经元中的表达变化。方法将56只大鼠随机分为假手术组（对照组）及缺血再灌注组（观察组）,采用免疫组织化学法测定Bcl-2和Bax的表达。结果随着CIR时间的延长,凋亡细胞增多,缺血组Bcl-2、Bax表达增多,与对照组相比,差异有统计学意义（P〈0．01）,Bcl-2的表达在12h达到最高,Bax的增加一直持续到48h。结论CIR后Bcl-2表达的增加早于Bax,CIR后神经细胞凋亡可能与Bcl-2／Bax失调有关。     

Protective effect of Bojungbangdocktang on cisplatin-induced cytotoxicity and apoptosis in MCF-10A breast endothelial cells

Although cisplatin has been extensively used as a cancer chemotherapeutic agent for the treatment of various human cancers, it causes significant side effects such as nephrotoxicity and hepatotoxicity due to lethal bystander damage to normal cells. Thus, in the current study, we investigated the Oriental herbal medicine Bojungbangdocktang (BJBDT), as we reported previously its anti-angiogenic activity at nontoxic concentrations that could prevent cisplatin-induced toxicity and apoptosis in human normal breast epithelial cell MCF-10A, but not in MCF-7 and MDA MB-231 breast cancer cells. BJBDT protected cisplatin-induced cytotoxicity in MCF-10A cells and potentiated cytotoxicity and MMP loss in MCF-7 cells. Also, 4′,6-diamidino-2-phenylindole (DAPI) staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay revealed that BJBDT reduced cisplatin-induced apoptotic bodies in MCF-10A cells compared with cisplatin-treated control. Consistently, BJBDT attenuated the apoptotic portion sub-G1 DNA contents as well as blocked the activation of caspase-3 and -9 and poly(ADP-ribose)polymerase (PARP) cleavage in cisplatin-treated MCF-10A cells. Taken together, our findings suggest that BJBDT can protect cisplatin-induced cytotoxicity and apoptosis in normal MCF-10A breast cells as a cancer chemopreventive agent.     

Penta-O-galloyl-β-D-glucose attenuates cisplatin-induced nephrotoxicity via reactive oxygen species reduction in renal epithelial cells and enhances antitumor activity in Caki-2 renal cancer cells

Cisplatin shows limited therapeutic efficacy due to serious side effects such as nephrotoxicity and hepatotoxicity. In the present study, we demonstrate that 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG) has protective effects against cisplatin-induced cytotoxicity and apoptosis in normal human primary renal epithelial cells (HRCs) while showing synergistic effect against cisplatin-induced cell death in human Caki-2 renal cancer cells. PGG significantly blocked cisplatin-mediated cytotoxicity and reduced cisplatin-induced sub-G1 accumulation in HRCs. Consistently, PGG reduced the number of apoptotic cell populations by TdT-mediated dUTP nick end labeling (TUNEL) and Live/Dead assays in cisplatin-treated HRCs. Furthermore, PGG suppressed PARP cleavage and caspase-3 activation, cytochrome c release, up-regulation of bax and p53 in cisplatin-treated HRCs. Moreover, PGG attenuated reactive oxygen species (ROS) production mediated by cisplatin treatment, suggesting that PGG prevented cisplatin-induced apoptosis by inhibiting ROS generation in HRCs. Notably, PGG significantly enhanced cytotoxicity and PARP cleavage in cisplatin-treated Caki-2 renal cancer cells. Combination Index (CI) revealed synergism between PGG and cisplatin in Caki-2 cells. Taken together, our findings suggest the dual effects of PGG as a protective supplement against cisplatin-induced toxicity in normal renal cells and a combination chemotherapeutic drug with cisplatin in renal cancer cells.     

L-硝基精氨酸对大鼠内毒素性肺损伤治疗作用及其机制的研究

目的探讨L-硝基精氨酸（NG—nitro-L—arginine,L—NA）对内毒素性肺损伤大鼠肺表面活性物质（PS）和细胞凋亡的影响,探讨L—NA对肺损伤的保护作用及其机制。方法健康雄性SD大鼠采用舌下静脉注射脂多糖（LPS）复制肺损伤模型,分别于给予LPS 1h和6b后给予0．9％氯化钠溶液（对照纽及LPS组,静脉给药）和L-NA（20mg／kg,静脉给药）（L-NA治疗组）,治疗3h,取肺组织,原位杂交法（ISH）测定肺组织肺表面活性物质蛋白A（SP-A）mRNA;流式细胞术（FCM）检测肺细胞凋亡率;Western blot法检测Caspase-3蛋白的表达;免疫组化法测定Bcl-2和Bax蛋白的表达。结果与对照组比较,大鼠肺损伤后SP-A mRNA表达明显下降（P〈0．01）;细胞凋亡率、Caspase-3和Bax蛋白表达明显升高（P〈0．01）,Bcl-2蛋白表达和Bcl-2／Bax降低（P〈0．01）;肺损伤1h用L—NA治疗3h后,SP-A mRNA阳性细胞表达明显增强（P〈0．05）,细胞凋亡率降低（P〈0．05）;但Caspase-3、Bax、Bcl-2蛋白表达、Bcl-2／Bax与LPS组比较没有明显的变化（P〈0．05）,肺组织病理改变减轻,肺损伤6h用L—NA治疗3h对LPS引起的以上变化没有明显影响。结论肺损伤1h后给予L—NA可减轻内毒素性肺损伤,增强PS表达,减少细胞凋亡是其机制之一。     

Bcl-xL and Mcl-1 are involved in prevention of in vitro apoptosis in rat late-stage erythroblasts derived from bone marrow

Apoptosis controls erythroid homeostasis by balancing survival and death of erythroid cells. The mitochondrial pathway of apoptosis involves regulation of apoptotic events caused by the Bcl-2 family proteins, including the anti-apoptotic and pro-apoptotic members. However, little has been reported on the role of the anti-apoptotic Bcl-2 family members in rat late-stage erythroblasts that are no longer erythropoietin (EPO)-dependent. In the present study, to investigate this we analyzed changes in apoptosis-related factors that occurred in vitro. EPO stimulation resulted in reduced apoptotic cell death of the late-stage erythroblasts accompanied by decreased caspase-3 and caspase-9 activities, which is indicative of the induction of apoptosis through the mitochondrial pathway. Analysis of mRNA expression of the Bcl-2 family proteins demonstrated that EPO stimulation up-regulated the Bcl-xL mRNA, resulting in decreases in the mRNA ratios of Bak, Bax, and Bad to Bcl-xL. Also, the mRNA ratios of Bak and Noxa to Mcl-1 were decreased, mainly due to up-regulation of Mcl-1 mRNA. These results showed a close association between reduced apoptotic cell death and increased mRNA levels of Bcl-xL and Mcl-1 in the presence of EPO. Thus, the present study suggests that Bcl-xL may be an important anti-apoptotic factor of rat late-stage erythroblasts as has been reported in murine erythroblasts. Moreover, the results also indicate the possibility that Mcl-1 may act on the rat late-stage erythroblasts as an anti-apoptotic factor.     

Cisplatin-treated murine peritoneal macrophages induce apoptosis in L929 cells: role of Fas-Fas ligand and tumor necrosis factor-tumor necrosis factor receptor 1

Cisplatin [cis-diamminedichloroplatinum (II)]-treated murine peritoneal macrophages interact with L929 cells in vitro in a sequential manner, resulting in the formation of contact between the two cells. This interaction leads to the death of L929 cells by the process of apoptosis. The detailed investigations have suggested the involvement of two different pathways in macrophage-mediated L929 cell apoptosis. It is observed that the induction of apoptosis in L929 cells by cisplatin-treated macrophages is contact dependent and is mediated through Fas-Fas ligand and tumor necrosis factor-tumor necrosis factor receptor 1 pathways. This conclusion was based on the Western blot and immunoprecipitation analysis of Fas-Fas ligand, tumor necrosis factor-tumor necrosis factor receptor 1, Fas-associated death domain and tumor necrosis factor receptor-associated death domain. The Fas-Fas ligand interaction between macrophages and L929 cells increased the expression of Fas-associated death domain, and tumor necrosis factor-tumor necrosis factor receptor 1 interaction between macrophages and L929 cells increased the expression of tumor necrosis factor receptor-associated death domain in L929 cells. The induction of apoptosis in L929 cells was investigated by DNA fragmentation, Annexin V staining and Western blot analysis of Bax, Bcl-2, Bid, cytochrome c, poly(ADP ribose) polymerase, CAD, caspase-8 and caspase-3.