A Free Radical Initiator, 2,2′-azobis (2-aminopropane) Dihydrochloride Enhances Hyperthermia-induced Apoptosis in Human Uterine Cervical Cancer Cell Lines

Hyperthermia-induced apoptosis and its enhancement in the presence of a temperature-dependent free radical initiator, 2,2′-azobis (2-aminopropane) dihydrochloride (AAPH) were examined in human uterine cervical cancer cell lines, CaSki and HeLa. When both cell lines were treated with hyperthermia at 44°C for 60?min, minimal apoptosis was observed. When combined with nontoxic AAPH (50?mM), significant enhancement of apoptosis was observed, where the initial rate of free radical formation was about twice as high than that at 37°C. Augmentation of the growth delay, lipid peroxidation (LPO), activation of caspase-3 and increase in [Ca²⁺]i were also observed after the combined treatment. A water-soluble vitamin E, Trolox, blocked the increase in [Ca²⁺]i and an intracellular Ca²⁺ chelator, BAPTA-AM, prevented the DNA fragmentation induced by the combination. Cytochrome c release was also revealed by fluorescence microscopy. However, no significant change in mitochondrial membrane potential and expression of Bax and Bcl-2 was observed. A slight increase in Fas expression was observed only in CaSki cells after the combined treatment. These results indicate that hyperthermia and AAPH induce enhanced apoptosis and subsequent cell killing via two pathways; a pathway dependent on increase in LPO and [Ca²⁺]i, and a pathway associated with cytochrome c release and subsequent caspase activation without changes of mitochondrial membrane potential and Bax/Bcl-2 expression in these cell lines. Since it is known that cancer cells are generally resistant to physical and chemical stress-induced apoptosis, free radical generators like AAPH appear to be a useful thermosensitizer for hyperthermic cancer therapy.     

Enhancement of hyperthermia-induced apoptosis by a free radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride, in human histiocytic lymphoma U937 cells

To elucidate the mechanism how a free radical initiator, 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH), induces cell death at hyperthermic temperatures, apoptosis in a human histiocytic lymphoma cell line, U937, was investigated. Free radical formation deriving from the thermal decomposition of AAPH was examined by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An assay for DNA fragmentation, observation of nuclear morphological changes, and flow cytometry for phosphatidylserine (PS) externalization were used to detect apoptosis and revealed enhancement of 44.0°C hyperthermia-induced apoptosis by free radicals due to AAPH. However, free radicals alone derived from AAPH did not induce apoptosis. Hyperthermia induced the production of lipid peroxidation (LPO), an increase in intracellular Ca²⁺ concentration ([Ca²⁺]i) and enhanced expression of the type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1). The effects of hyperthermia on LPO and [Ca²⁺]i were enhanced markedly by the combination with AAPH. A significant decrease in Bcl-2 expression, increase in Bax expression, a loss of mitochondrial membrane potential (ΔΨm) and a marked increase in cytochrome c expression were found only in cells treated with hyperthermia and AAPH. Although an intracellular Ca²⁺ ion chelator, BAPTA-AM, completely inhibited DNA fragmentation, water-soluble vitamine E, Trolox, only partially suppressed DNA fragmentation and the increase in [Ca²⁺]i. In contrast, LPO was inhibited completely by Trolox, but no inhibition by BAPTA-AM was found. These results suggest that apoptosis induced by hyperthermia alone is due to the increase in [Ca²⁺]i arising from increased expression of IP₃R1 and LPO. Additional increase in [Ca²⁺]i due to increased LPO and the activation of mitochondria-caspase dependent pathway play a major role in the enhancement of apoptosis by the combination with hyperthermia and AAPH.     

Enhancement of hyperthermia-induced apoptosis by a free radical initiator, 2,2′-azobis (2-amidinopropane) dihydrochloride,in human histiocytic lymphoma U937 cells

To elucidate the mechanism how a free radical initiator, 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH), induces cell death at hyperthermic temperatures, apoptosis in a human histiocytic lymphoma cell line, U937, was investigated. Free radical formation deriving from the thermal decomposition of AAPH was examined by spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). An assay for DNA fragmentation, observation of nuclear morphological changes, and flow cytometry for phosphatidylserine (PS) externalization were used to detect apoptosis and revealed enhancement of 44.0°C hyperthermia-induced apoptosis by free radicals due to AAPH. However, free radicals alone derived from AAPH did not induce apoptosis. Hyperthermia induced the production of lipid peroxidation (LPO), an increase in intracellular Ca²⁺ concentration ([Ca²⁺]i) and enhanced expression of the type 1 inositol 1,4,5-trisphosphate receptor (IP₃R1). The effects of hyperthermia on LPO and [Ca²⁺]i were enhanced markedly by the combination with AAPH. A significant decrease in Bcl-2 expression, increase in Bax expression, a loss of mitochondrial membrane potential (ΔΨm) and a marked increase in cytochrome c expression were found only in cells treated with hyperthermia and AAPH. Although an intracellular Ca²⁺ ion chelator, BAPTA-AM, completely inhibited DNA fragmentation, water-soluble vitamine E, Trolox, only partially suppressed DNA fragmentation and the increase in [Ca²⁺]i. In contrast, LPO was inhibited completely by Trolox, but no inhibition by BAPTA-AM was found. These results suggest that apoptosis induced by hyperthermia alone is due to the increase in [Ca²⁺]i arising from increased expression of IP₃R1 and LPO. Additional increase in [Ca²⁺]i due to increased LPO and the activation of mitochondria-caspase dependent pathway play a major role in the enhancement of apoptosis by the combination with hyperthermia and AAPH.     

Oxidative stress underlies the mechanism for Ca(2+)-induced permeability transition of mitochondria

Recent studies demonstrated that the generation of intracellular reactive oxygen species (ROS) was enhanced prior to the onset of mitochondrial membrane permeability transition (MPT), a critical step for the induction of DNA fragmentation and apoptosis. Although Ca₂₊ induces typical MPT that involves depolarization and swelling of mitochondria and finally releases cytochrome c into cytosol, the mechanism by which ROS induce MPT remains unclear. In the presence of inorganic phosphate, Ca₂₊ increased the oxygen consumption and ROS production by isolated mitochondria as determined by a chemiluminescence (CHL) method using L-012. Ca₂₊ increased the generation of H₂O₂ by some mechanism that was inhibited by cyclosporin A but not by superoxide dismutase (SOD) and trifluoperazine. Ca₂₊ decreased the content of free thiols in adenine nucleotide translocase (ANT) in mitochondrial membranes with concomitant increase in ROS generation. The presence of cyclosporin A, trifluoperazine, or SOD inhibited the Ca₂₊-induced increase of L-012 CHL and decrease in the free thiols of ANT. These results indicate that Ca₂₊ increases the generation of ROS which oxidize the free thiol groups in mitochondrial ANT, thereby inducing MPT to release cytochrome c.     

Apoptosis and Bcl-2 protein changes in L1210 leukaemic cells exposed to oxidative stress

The aim of this study was to explore the dose- and time-dependent effects of hydrophilic peroxyl radical initiator 2,2'azobis(2amidinopropane)dihydrochloride (AAPH) on apoptosis, and on expression of Bcl-2 in L1210 leukaemic cells. We observed a progressive increase of intracellular concentration of oxygen free radicals (OFR), manifested by the rise of 6-carboxy-2', 7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) oxidation, during 24 h of cells exposure to AAPH. Oxidative stress was associated with peroxidation of cellular lipids, which was demonstrated by the measurement of thiobarbituric acid-reactive substances and conjugated dienes. Analysis of cell viability by the use of trypan blue exclusion method revealed that AAPH reduced the ability of L1210 cells to multiply or survive. AAPH increased the number of leukaemic cells with typical features of apoptosis like condensation of chromatin, pyknosis and fragmentation of nucleus, followed by secondary necrosis. A characteristic internucleosomal DNA cleavage, visualized as a DNA ‘ladder’ consisting of fragments that are multiples of 180-200 bp was also observed. The intensity of apoptosis was dependent on AAPH concentration, time of cell exposure and the availability of growth factors and nutrients in extracellular environment (FCS concentration). The novel observation is the increase of Bcl-2 level in L1210 leukaemic cells surviving an oxidative stress. The level of Bcl-2 protein significantly rose with increasing AAPH concentration, and time of cell exposure to this oxidant. This phenomenon could be the result of: (1) negative selection of cells with the lowest expression of bcl-2, being more susceptible to oxidative stress and (2) increased synthesis and/or decreased degradation of Bcl-2 protein as an adaptation to continuous OFR loading. In contrast to growth-promoting medium (10% FCS/RPMI), the maintenance medium (2% FCS/RPMI) did not cover cell requirements for progressive Bcl-2 increase at the highest AAPH concentration (2 mM) applied in this study. Several observations indicate that the increased Bcl-2 level in surviving L1210 leukaemic cells exposed to oxidative stress is a symptom of their natural defence against cellular lipids peroxidation and apoptosis. This revised version was published online in June 2006 with corrections to the Cover Date.     

Histone Deacetylase Inhibitors Sensitize Lung Cancer Cells to Hyperthermia: Involvement of Ku70/SirT-1 in Thermo-Protection

This study describes the sensitization mechanism to thermal stress by histone deacetylase inhibitors (HDACIs) in lung cancer cells and shows that Ku70, based on its acetylation status, mediates the protection of lung cancer from hyperthermia (42.5°C, 1-6 hrs). Ku70 regulates apoptosis by sequestering pro-apoptotic Bax. However, its role in thermal stress is not fully understood. The findings showed that, pre-treating lung cancer cells with HDACIs, nicotinamide (NM) or Trichostatin A (TsA) or both significantly enhanced hyperthermia-induced Bax-dependent apoptosis in PC-10 cells. We found that hyperthermia induces SirT-1, Sirtuin, upregulation but not HDAC6 or SirT-3, therefore transfection with dominant negative SirT-1 (Y/H) also eliminated the protection and resulted in more cell death by hyperthermia, in H1299 cells through Bax activation. Hyperthermia alone primed lung cancer cells to apoptosis without prominent death. After hyperthermia Bax was upregulated, Bcl-2 was downregulated, the Bax/Bcl-2 ratio was inversed and Bax/Bcl-2 heterodimer was dissociated. Although hyperthermia did not affect total Ku70 expression level, it stimulated Ku70 deacetylation, which in turn could bind more Bax in the PC-10 cells. These findings suggest an escape mechanism from hyperthermia-induced Bax activation. To verify the role of Ku70 in this protection mechanism, Ku70 was silenced by siRNA. Ku70 silencing significantly sensitized the lung cancer cells to hyperthermia. The Ku70 KD cells underwent cytotoxic G1 arrest and caspase-dependant apoptosis when compared to scrambled transfectants which showed only G2/M cytostatic arrest in the cell lines investigated, suggesting an additional cell cycle-dependent, novel, role of Ku70 in protection from hyperthermia. Taken together, our data show a Ku70-dependent protection mechanism from hyperthermia. Targeting Ku70 and/or its acetylation during hyperthermia may represent a promising therapeutic approach for lung cancer.     

热疗联合奥沙利铂对SW480细胞增殖和凋亡的影响

目的:观察奥沙利铂联合热疗对人结肠癌细胞SW480增殖及凋亡的影响,确定联合用药的效果,为临床方案提供参考。方法:采用MTT(四唑盐)法检测热疗、奥沙利铂及联合用药对细胞增殖的影响;瑞士吉姆萨染色法观察细胞形态;流式细胞仪检测细胞凋亡和周期;Western blot检测Bax、Bcl-2以及Caspase8蛋白表达量变化;q PCR检测Bax、Bcl-2以及Caspase8 m RNA的积累。结果:热疗联合奥沙利铂可以显著抑制细胞增殖,与对照组相比,热疗组、化疗组、联合组细胞凋亡率分别为16.2%、20.5%和36.1%,具有显著性差异(P0.01);细胞学形态中,热疗组细胞发生皱缩,化疗组细胞膜破裂;化疗将细胞阻滞在G2/M期,热疗和联合组将细胞阻滞S期;Western blot和qPCR显示Bax/Bcl-2比值上升,Caspase8表达量增加,联合组三种蛋白的表达量均与对照组具有显著性差异(P0.01)。结论:热疗联合奥沙利铂可以显著促进细胞凋亡,提高治疗效果,为结肠癌的治疗提供参考。     

Adriamycin (ADM) induced apoptosis in Transitional Cell Cancer (TCC) cell lines accompanied by p21 WAF1/CIP1 induction

Genotoxic stimuli, including anticancer drugs, induce apoptosis in cancer cells through increase of p53, p21WAF1/CIP1 , at least in part. Bcl-2 and Bax modify this pathway or directly regulated by p53. Here we studied Adriamycin (ADM)-induced apoptosis in four human bladder cancer cell lines in respect of p53, p21WAF1/CIP1 and Bcl-2 family proteins. After ADM, treatment bladder cancer cells underwent dose-dependent cell death with typical morphologic features of apoptosis. Among four cell lines RT4 with wt p53, low ratio of Bcl-2 to Bax and induction of p21WAF1/CIP1 after ADM treatment, was the most sensitive to induction of apoptosis. Thus, p53, p21WAF1/CIP1 , Bcl-2 and Bax status might determine susceptibility of bladder cancer cells to ADM induced apoptosis.     

Enhancement of radiation-induced apoptosis by 6-formylpterin

Radiation-induced apoptosis and its possible enhancement in the presence of 6-formylpterin (6-FP), a metabolite of folic acid, were examined in human myelomonocytic lymphoma U937 cells. When cells were treated with 6-FP at a nontoxic concentration of 300 μM, and then exposed to X-rays at a dose of 10 Gy, significant enhancement of radiation-induced apoptosis as determined by nuclear morphological change, phosphatidylserine (PS) externalization and DNA fragmentation were observed. Flow cytometry for the detection of intracellular hydrogen peroxide (H₂O₂) revealed that 6-FP increased the formation of intracellular H₂O₂, which further increased when the cells were irradiated. Decrease of mitochondria trans-membrane potential (MMP), release of cytochrome c from mitochondria, and activation of caspase-3 were enhanced after the combined treatment. Remarkable activation of protein kinase C δ (PKC δ) and its translocation from cytosol to mitochondria were detected in combined treatment. Increase of intracellular Ca₂₊ concentrations ([Ca₂₊]i) was also observed, however, neither calpain I nor calpain II could inhibit the apoptosis. In addition, c-Jun NH₂-terminal kinase ( JNK) activation was not enhanced in the combined treatment. A protein involved in a caspase-independent apoptosis pathway, apoptosis inducing factor (AIF), remained unchanged even 3 h after treatment. These results indicate that intracellular H₂O₂ generated by 6-FP enhances radiation-induced apoptosis via the mitochondria-mediated caspase-dependent pathway, with the active involvement of PKC δ.     

香蕉皮抗氧化和抑制人肝癌HepG2细胞活性研究

对香蕉皮提取物（banana peel extraction,BPE）的体外抗氧化作用和抗肿瘤活性进行了研究。使用福林酚和硝酸铝法测定BPE中总多酚和总黄酮含量;通过检测DPPH和O₂^-自由基清除能力测定BPE的体外抗氧化活性;采用Alamar Blue法检测BPE对HepG2细胞活性的影响;荧光染色及流式技术检测HepG2细胞凋亡;免疫印迹技术检测凋亡蛋白的变化;Caspase活性检测试剂盒测定Caspase-3和Caspase-9的活性;Caspase-9和Caspase-3抑制剂验证BPE抗肿瘤相关的信号转导通路。结果表明,BPE中总多酚和总黄酮含量分别为39.23 ± 2.35 mg·L^-1和26.53 ± 1.97 mg·L^-1;BPE显示出一定的DPPH和O₂^-自由基清除能力（65.31%±3.82%和51.29%±4.23%）,可明显抑制HepG2细胞的增殖（IC₅₀=54.32 mL·L^-1）;BPE通过上调Caspase-3、Bax、Bid、Apaf-1、Bak、p53、Caspase-9的表达,下调Bcl-2、Bcl-xl的表达诱导HepG2细胞凋亡;Caspase-9和Caspase-3抑制剂（Z-LEHD-FMK、Ac-DEVD-CHO）在一定程度上逆转了BPE的增殖抑制活性。BPE具有一定的自由基清除能力,对HepG2细胞具有明显增殖抑制作用,这些结果为香蕉皮的进一步开发利用提供了数据支撑。     

Activation of Bak,Bax, and BH3-only proteins in the apoptotic response to doxorubicin

The anthracyclin doxorubicin (DXR) is a major antitumor agent known to cause cellular damage via a number of mechanisms including free radical formation and inhibition of topoisomerase II. It is not clear, however, how the subsequent lesions may lead to the apoptotic death of the cell. We have here examined the effects of DXR on activation of pro-apoptotic members of the Bcl-2 family, all of which are connected to the mitochondrial events of apoptosis. In two human cell lines (lymphoma and myeloma), clinically relevant concentrations of DXR were found to induce apoptosis, first observed after 24 h of treatment. Apoptosis correlated with modulation of Bak and Bax to their active conformations. bax- as well as bak-deficient mouse embryo fibroblasts were resistant to DXR compared with wild-type mouse embryo fibroblasts further supporting a role for these proteins as main DXR-induced apoptosis regulators. Furthermore, using immunocytochemistry as well as chemical blocking of putative apical pathways we could demonstrate that Bak is activated prior to Bax. In the human cell lines, DXR was furthermore found to induce high protein levels of Bik, another BH3-only protein. DXR-induced apoptosis was completely blocked in Bcl-2-overexpressing U266 cells. Interestingly, in Bcl-2-transfected cells Bak activation was also blocked, while Bax was still partially active in agreement with differential regulation of these two proteins. Furthermore, co-incubation of the phosphatidylinositol 3-kinase (PI3K)-inhibitor LY294002 potentiated the apoptotic response to DXR. This enhanced apoptosis was preceded by enhanced Bak and Bax activation, and both responses as well as apoptosis were blocked in transfectants overexpressing Bcl-2. In summary, several pieces of evidence suggest that DXR induces apoptosis through a sequential and differential activation of Bak and Bax.     

Bax signaling regulates palmitate-mediated apoptosis in C(2)C(12) myotubes

Insulin resistance is a primary characteristic of type 2 diabetes. Several lines of evidence suggest that accumulation of free fatty acids in skeletal muscle may at least in part contribute to insulin resistance and may be linked to mitochondrial dysfunction, leading to apoptosis. Palmitate treatment of several cell lines in vitro results in apoptosis and inhibits protein kinase B (Akt) activity in response to insulin. However, the role of Bax and Bcl-2 in regulating palmitate-induced apoptosis has not been well studied. Therefore, the purpose of this study was to determine whether palmitate-induced apoptosis in C(2)C(12) myotubes is dependent on Bax to Bcl-2 binding. An additional purpose of this study was to determine whether the changes in Bax to Bcl-2 binding corresponded to decreases in Akt signaling in palmitate-treated myoblasts. Apoptotic signaling proteins were examined in C(2)C(12) myotubes treated overnight with palmitate. Bax to Bcl-2 binding was determined through a coimmunoprecipitation assay that was performed in myotubes after 2 h of serum starvation, followed by 10 min of serum reintroduction. This experiment evaluated whether temporal Akt activity coincided with Bax to Bcl-2 binding. Last, the contribution of Bax to palmitate-induced apoptosis was determined by treatment with Bax siRNA. Palmitate treatment increased apoptosis in C(2)C(12) myotubes as shown by a twofold increase in DNA fragmentation, an approximately fivefold increase in caspase-3 activity, and a 2.5-fold increase in caspase-9 activity. Palmitate treatment significantly reduced Akt protein expression and Akt activity. In addition, there was a fourfold reduction in Bax to Bcl-2 binding with palmitate treatment, which mirrored the reduction in Akt(Ser473) phosphorylation. Furthermore, treatment of the C(2)C(12) myotubes with Bax siRNA attenuated the apoptotic effects of palmitate treatment. These data show that palmitate induces Bax-mediated apoptosis in C(2)C(12) myotubes and that this effect corresponds to reductions in Akt(Ser473) phosphorylation.     

Free Radical Formation from the Antineoplastic Agent VP 16-213

Free radical formation from VP 16-213 was studied by ESR spectroscopy. Incubation of VP 16-213 with the one-electron oxidators persulphate-ferrous, myeloperoxidase (MPO)/hydrogen peroxide and horseradish peroxidase (HRP)/hydrogen peroxide readily led to the formation of a free radical. The ESR spectra obtained in the last two cases, were in perfect accord with that of a product obtained by electrochemical oxidation of VP 16-213 at +550 mV. The half-life of the free radical in 1 mM Tris (pH 7.4), 0.1 MNaClat 20°C, was 257 ± 4 s. The signal recorded on incubation with HRP/H₂O₂ or MPO/H₂O₂ did not disappear on addition of 0.3 - 1.2 mg/ml microsomal protein. From incubations with rat liver microsomes in the presence of NADPH, no ESR signals were obtained.     

Synthesis of Bcl-2 in response to anthracycline treatment may contribute to an apoptosis-resistant phenotype in leukemic cell lines.

BACKGROUND: Some forms of chemoresistance in leukemia may start from failure of tumour cells to successfully undergo apoptosis and Bcl-2 may play a role in this defect. Therefore, we evaluated the Bcl-2 content and synthesis in relation with the apoptotic potential in leukemic cell lines after anthracycline treatment. METHODS: U937, HL60, and K562 cells and their drug resistant (DR) variants were treated with varying concentrations of Idarubicin (IDA). Apoptosis was evaluated by fluorescence microscopy after acridine orange staining. Bcl-2 and Bax content were evaluated either by flow cytometry after indirect immunolabelling or by Western blot. RESULTS: High Bcl-2 contents were not related to a poor ability to undergo apoptosis in U937, HL60, K562 and their DR variants. IDA induced a concentration-dependent increase in Bcl-2 content in all cell lines as long as they do not perform apoptosis. Enhanced Bcl-2 expression was inhibited by cycloheximide, actinomycin D, or antisense oligonucleotide directed against bcl-2 mRNA. Bcl-2 expression was also increased in the resistant U937 variant after serum deprivation or C2-ceramide treatment. The synthesis of Bcl-2 led to an increased Bcl-2/Bax ratio solely in the cells with an apoptosis-resistance phenotype. CONCLUSIONS: These data suggest that exposure to IDA induces Bcl-2 expression in leukemic cell lines, and that this mechanism could contribute to apoptosis resistance and participate in the acquisition of chemoresistance. They also confirm that the evolution of the Bcl-2/Bax ratio reflects apoptotic ability better than the steady state level of Bcl-2 expression.     

Opposing actions of the progesterone metabolites, 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αHP) on mitosis,apoptosis, and expression of Bcl-2, Bax and p21 in human breast cell lines

Previous studies have shown that breast tissues and breast cell lines convert progesterone (P) to 5α-dihydroprogesterone (5αP) and 3α-dihydroprogesterone (3αHP) and that 3αHP suppresses, whereas 5αP promotes, cell proliferation and detachment. The objectives of the current studies were to determine if the 5αP- and 3αHP-induced changes in cell numbers are due to altered rates of mitosis and/or apoptosis, and if 3αHP and 5αP act on tumorigenic and non-tumorigenic cells, regardless of estrogen (E) and P receptor status. The studies were conducted on tumorigenic (MCF-7, MDA-MB-231, T47D) and non-tumorigenic (MCF-10A) human breast cell lines, employing several methods to assess the effects of the hormones on cell proliferation, mitosis, apoptosis and expression of Bcl-2, Bax and p21. In all four cell lines, 5αP increased, whereas 3αHP decreased cell numbers, [³H]thymidine uptake and mitotic index. Apoptosis was stimulated by 3αHP and suppressed by 5αP. 5αP resulted in increases in Bcl-2/Bax ratio, indicating decreased apoptosis; 3αHP resulted in decreases in Bcl-2/Bax ratio, indicating increased apoptosis. The effects of either 3αHP or 5αP on cell numbers, [³H]thymidine uptake, mitosis, apoptosis, and Bcl-2/Bax ratio, were abrogated when cells were treated simultaneously with both hormones. The expression of p21 was increased by 3αHP, and was unaffected by 5αP. The results provide the first evidence that 5αP stimulates mitosis and suppresses apoptosis, whereas 3αHP inhibits mitosis and stimulates apoptosis. The opposing effects of 5αP and 3αHP were observed in all four breast cell lines examined and the data suggest that all breast cancers (estrogen-responsive and unresponsive) might be suppressed by blocking 5αP formation and/or increasing 3αHP. The findings further support the hypothesis that progesterone metabolites are key regulatory hormones and that changes in their relative concentrations in the breast microenvironment determine whether breast tissues remain normal or become cancerous.     

SIRT2抑制对MPP+诱导的帕金森病细胞模型凋亡和线粒体动态平衡的影响*

探究siRNA敲减沉默信息调节因子2(SIRT2)对1-甲基-4-苯基吡啶离子(MPP⁺)诱导的帕金森病细胞模型细胞损伤的影响和机制。CCK-8法检测不同浓度MPP⁺处理对体外培养小鼠海马神经元HT-22细胞生存率的影响。将细胞分为对照组、MPP⁺最佳浓度处理组(1 mmol/L MPP⁺处理组)、阴性转染组(对照组基础上转染SIRT2阴性序列)、SIRT2 siRNA处理组(损伤组基础上转染SIRT2 siRNA)。观察各组细胞凋亡情况,检测凋亡相关蛋白(Bcl-2、Bax、Caspase-9)、线粒体分裂及融合相关蛋白(Drp1、Fis1、OPA1、Mfn1、Mfn2)。与对照组相比,MPP⁺处理组细胞抑制率均升高,细胞抑制率随MPP⁺浓度增加而逐渐增加(P<0.05)。与SIRT2 siRNA转染组相比,损伤组Bax、Caspase-9、Drp1、Fis1蛋白表达和细胞凋亡率升高,Bcl-2、Mfn1、Mfn2蛋白表达降低(P<0.05)。SIRT2在MPP⁺诱导帕金森病细胞模型中表达升高,抑制SIRT2可减轻MPP⁺诱导帕金森病细胞模型中细胞凋亡并促进线粒体融合,从而对神经元具有一定的保护作用。     

Modulation of Ion Gradients and Glutamate Release in Cultured Cerebellar Granule Cells by Ouabain

Abstract: Upon addition of the cardiac glycoside ouabain to cultured cerebellar granule cells, an immediate increase in intracellular free sodium is evoked mediated by two pathways, a voltage-sensitive channel blocked by tetrodotoxin and a channel sensitive to flunarizine. Ouabain induces a steady plasma membrane depolarization in low Ca²⁺ medium; whereas in the presence of Ca²⁺, a distinct discontinuity is observed always preceded by a large increase in intracellular free Ca²⁺ ([Ca²⁺]_c). The plateau component of the increase can be inhibited additively by the L-type Ca²⁺ channel antagonist nifedipine, the spider toxin Aga-Gl, and the NMDA receptor antagonist MK-801. Single-cell imaging reveals that the [Ca²⁺]_c increase occurs asynchronously in the cell population and is not dependent on a critical level of extracellular glutamate or synaptic transmission between the cells. A prolonged release of glutamate is also observed that is predominantly Ca²⁺ dependent for the first 6–10 min after the evoked increase in [Ca²⁺]_c. This release is four times as large as that observed with 50 m M KCl and is predominantly exocytotic because release was inhibited by tetanus toxin, the V-type ATPase inhibitor bafilomycin, and Aga-Gl. It is proposed, therefore, that ouabain induces a period of membrane excitability culminating in a sustained exocytosis above that observed upon permanent depolarization with KCl.     

High Bcl-2/Bax ratio in Walker tumor cells protects mitochondria but does not prevent H<Subscript>2</Subscript>O<Subscript>2</Subscript>-induced apoptosis via calcineurin pathways

It has been previously shown that Walker 256 tumor cells express a high content of the anti-apoptotic protein Bcl-2 which protects mitochondria against the damaging effects of Ca²⁺. In the present study, we analyze H₂O₂-induced apoptotic death in two different types of tumor cells: Walker 256 and SCC-25. Treatment with H₂O₂ (4mM) increased reactive oxygen species generation and the concentration of cytosolic free Ca²⁺. These alterations preceded apoptosis in both cell lines. In Walker cells, which show a high Bcl-2/Bax ratio, apoptosis was dependent on calcineurin activation and independent of changes in mitochondrial membrane potential (Δ < eqid1 > _m), as well as cytochrome c release. In contrast, in SCC-25 cells, which show a lower Bcl-2/Bax ratio, apoptosis was preceded by a decrease in Δ < eqid2 > _m, mitochondrial permeability transition, and cytochrome c release. Caspase-3 activation occurred in both cell lines. The data suggest that although the high Bcl-2/Bax ratio protected the mitochondria of Walker cells from oxidative stress, it was not sufficient to prevent apoptosis through calcineurin pathways.     

Mitochondrial function, apoptosis and cell cycle delay in the WEHI-3B leukaemia cell line and its variant Ciprofloxacin-resistant WEHI-3B/CPX

OBJECTIVE: The susceptibility of two cell lines, WEHI-3B myelomonocytic leukaemia and its variant Ciprofloxacin-resistant WEHI-3B/CPX to undergo apoptosis induced by Ciprofloxacin was studied and compared. MATERIALS AND METHODS: Apoptosis was checked by measuring the DNA fragmentation and determining the ratio of apoptotic/necrotic cells. The relationship between the induction of apoptosis and G(1), S or G(2) block in the cell cycle has also been investigated and cytogenetical evaluation of chromosomal aberrations in both cell lines has been carried out. The regulation of expression of Bax and Bcl-2 was also checked by western blotting after Ciprofloxacin treatment. RESULTS: We observed that the resistance of the subline was caused by a small percentage of cells that underwent apoptosis during continuous exposure to Ciprofloxacin in comparison with the parental cell line, whereas the percentage of necrotic cells remained unchanged. The WEHI-3B cells showed a G(2) block and a higher degree of cytogenetic damage after drug exposure. The two cell lines expressed the same level of Bax and Bcl-2 following stimulation by Ciprofloxacin. Only in the resistant subclone, the ratio Bcl-2/Bax reversed in the anti-apoptotic gene expression. CONCLUSION: The resistance to ciprofloxacin observed is not related to mitochondrial function and although Bcl-2/Bax ratio behaviour does not fully explain the resistance of the WEHI3B/CPX subclone it is consistent with phenotypic character of resistance to CPX. The toxic effect on sensitive cells could be mediated by the cell cycle arrest whereas in the resistant clone, the prolonged G(2) phase could play a key role to favour cell cycle progression and proliferation.