A novel radiolytic rotenone derivative,rotenoisin A,displays potent anticarcinogenic activity in breast cancer cells

Chemotherapy for cancer treatment has therapeutic limitations, such as drug resistance, excessive toxic effects and undesirable adverse effects. Therefore, efforts to improve the safety and efficacy of chemotherapeutic agents are essential. Ionizing radiation can improve physiological and pharmacological properties by transforming structural modifications of the drug. In this study, in order to reduce the adverse effects of rotenone and increase anticancer activity, a new radiolytic rotenone derivative called rotenoisin A was generated through radiolytic transformation. Our findings showed that rotenoisin A inhibited the proliferation of breast cancer cells and increased the rate of apoptosis, whereas it had no inhibitory effect on primary epidermal keratinocytes compared with rotenone. Moreover, rotenoisin A-induced DNA damage by increasing reactive oxygen species (ROS) accumulation. It was also confirmed not only to alter the composition ratio of mitochondrial proteins, but also to result in structural and functional changes. The anticancer effect and molecular signalling mechanisms of rotenoisin A were consistent with those of rotenone, as previously reported. Our study suggests that radiolytic transformation of highly toxic compounds may be an alternative strategy for maintaining anticancer effects and reducing the toxicity of the parent compound.     

Bee Venom Protects against Rotenone-Induced Cell Death in NSC34 Motor Neuron Cells

Rotenone, an inhibitor of mitochondrial complex I of the mitochondrial respiratory chain, is known to elevate mitochondrial reactive oxygen species and induce apoptosis via activation of the caspase-3 pathway. Bee venom (BV) extracted from honey bees has been widely used in oriental medicine and contains melittin, apamin, adolapin, mast cell-degranulating peptide, and phospholipase A₂. In this study, we tested the effects of BV on neuronal cell death by examining rotenone-induced mitochondrial dysfunction. NSC34 motor neuron cells were pretreated with 2.5 μg/mL BV and stimulated with 10 μM rotenone to induce cell toxicity. We assessed cell death by Western blotting using specific antibodies, such as phospho-ERK1/2, phospho-JNK, and cleaved capase-3 and performed an MTT assay for evaluation of cell death and mitochondria staining. Pretreatment with 2.5 μg/mL BV had a neuroprotective effect against 10 μM rotenone-induced cell death in NSC34 motor neuron cells. Pre-treatment with BV significantly enhanced cell viability and ameliorated mitochondrial impairment in rotenone-treated cellular model. Moreover, BV treatment inhibited the activation of JNK signaling and cleaved caspase-3 related to cell death and increased ERK phosphorylation involved in cell survival in rotenone-treated NSC34 motor neuron cells. Taken together, we suggest that BV treatment can be useful for protection of neurons against oxidative stress or neurotoxin-induced cell death.     

激活小胶质细胞对帕金森病模型大鼠黑质多巴胺能神经元的影响

目的 探讨脂多糖(LPS)激活小胶质细胞(MG)对黑质损伤多巴胺(DA)能神经元细胞存活的影响.方法 采用立体定向技术向大鼠单侧黑质(SNpc)内注入LPS激活MG后,隔48h于原位注射高、低剂量鱼藤酮(BT)分别建立重度及轻度损伤模型;采用免疫组化法观察黑质酪氨酸羟化酶(TH)阳性细胞和离子钙结合转接分子1(Ib α...     

Melatonin protects against rotenone-induced cell injury via inhibition of Omi and Bax-mediated autophagy in Hela cells

Parkinson's disease is the second most common neurodegenerative disease, and environmental toxins such as rotenone play an important role in causing degeneration of dopaminergic neurons. Melatonin, a major secretory product of pineal, is recently reported to protect against rotenone-induced cell death in animal models. Yet, the mechanism involved in this protection needs to be elucidated. Here, we report that rotenone treatment (0-100 μM) decreased cell survival of Hela cells in a dose-dependent manner. At concentrations ranging from 0.1 to 100 μM, rotenone induced a dose-dependent increase in the expression of microtubule-associated protein 1 light chain 3 (LC3)-II, a protein associated with the autophagosomal membrane. Knockdown of Bax or Omi using shRNA inhibited 1 μM rotenone-induced autophagy. To determine whether melatonin would protect cells against rotenone-induced cell death and autophagy, we pretreated Hela cells with 250 μM melatonin for 24 hr in the presence of rotenone. Melatonin inhibited Bax expression and the release of the omi/HtrA2 into the cytoplasm induced by 1 μM rotenone. Melatonin 250 μM treatment also suppressed cell death induced by 0.1-100 μM rotenone and protected against the formation of LC3-II in cells exposed to 1 μM rotenone. This work demonstrates a novel role for melatonin as a neuroprotective agent against rotenone.     

Induction of heat shock protein 70 reduces the alteration of striatal electrical activity caused by mitochondrial impairment

Since mild thermal stress seems to exert neuroprotection via induction of heat-shock protein 70 kDa (hsp70), we tested whether hsp70 would preserve striatal bioelectrical activity under conditions of mitochondrial impairment. Corticostriatal slices from rats that had undergone mild thermal stress were exposed to either rotenone or 3-nitropropionic acid (3-NP), that selectively inhibits mitochondrial complex I and complex II, respectively. Rotenone is utilized to obtain an experimental model of Parkinson's disease while 3-NP replicates Huntington's disease phenotype in experimental animals. The cerebral hsp70 increase did not alter field potential amplitude of the slices but partially protected them against rotenone-induced neurotoxicity. Similarly, induction of hsp70 had also a partial neuroprotective effect on the neurotoxicity caused by 3-NP on striatal field potential. Since rotenone and 3-NP treatments mimic the mitochondrial impairment and oxidative stress that contribute to development of Parkinson's disease and Huntington's disease, these data suggest that induction of hsp70 might represent a possible neuroprotective mechanism against the pathophysiological chain of events implicated in these neurodegenerative disorders.     

Two pathways for iron uptake by guinea pig reticulocytes

We have demonstrated that the intracellular processing of transferrin to effect iron removal involves two pathways, one sensitive to rotenone and the other not. We have also found that the effect of the rotenone is dependent on the transferrin concentration: iron uptake was suppressed with concentrations of transferrin in the micromolar range, and was not suppressed at physiologic concentrations of transferrin. Rotenone does not disturb transferrin's interaction with its extracellular receptor, indicating that its action must be intracellular. The following model is suggested: that separate pathways are entered by transferrin in the cell. The first pathway is preferentially utilized when transferrin is in short supply. It begins with an intracellular site which has a high affinity (and low capacity) for either iron or transferrin. The second pathway begins with an intracellular site which has a high capacity (but low affinity) for either iron or transferrin and is utilized when transferrin is in physiologic concentration (and the low-capacity, high-affinity site is saturated); the pathway it initiates is dominant when transferrin is abundant. We speculate that the high-affinity low-capacity pathway may serve to direct intracellular iron to sites which would be critically injured by iron excess.     

Rapamycin protects against rotenone-induced apoptosis through autophagy induction

Ubiquitin proteasome system (UPS) and autophagy lysosome pathway (ALP) are the two most important routes for degradation of aggregated/misfolded proteins. Additionally, ALP is so far the only known route to clear entire organelles, such as mitochondria. We proposed that enhancement of ALP may be beneficial for some neurodegenerative disorders, such as Parkinson's disease (PD), in which the accumulation of aggregated/misfolded proteins and the dysfunction of mitochondria are the two major pathogenesis. Mitochondrial complex I inhibitor rotenone, which causes dysfunction mitochondria and UPS, has been considered as one of the neurotoxins related to PD. In this study, rotenone-exposed human neuronal SH-SY5Y cells were used as an in vitro model for us to determine whether autophagy enhancer rapamycin could protect against rotenone-induced injury and its underlying mechanisms. The observed results showed that rapamycin alleviated rotenone-induced apoptosis, whose effects were partially blocked when autophagy related gene 5 (Atg5) was suppressed by Atg5 small interference RNA (siRNA) transfection. Additionally, the results showed that rapamycin pretreatment diminished rotenone-induced accumulation of high molecular weight ubiquitinated bands, and reduced rotenone-induced increase of cytochrome c in cytosolic fraction and decreased mitochondrial marker cytochrome oxidase subunit IV (COX IV) in mitochondrial fraction. The changes in cytochrome c and COX IV indicated that the decreased translocation of cytochrome c from mitochondria to cytosol was probably due to the turn over of entire injured mitochondria. The results that lysosome and mitochondria were colocolized within the cells pretreated with rapamycin and that the mitochondria could be found within autophagy double membrane structures further supported that the damaged mitochondria might be cleared through autophagy, which process has been termed as “mitophagy.” Our studies suggested that autophagy enhancer rapamycin is neuroprotective against rotenone-induced apoptosis through autophagy enhancement. We concluded that pharmacologically induction of autophagy by rapamycin may represent a useful therapeutic strategy as disease-modifiers in PD.     

2,2,6,6-四甲基-3-烯哌啶氮氧自由基鱼藤酮肟酯体外抗肿瘤作用

目的 研究2,2,6,6-四甲基-3-烯哌啶氮氧自由基鱼藤酮肟酯(PNR)体外抗肿瘤作用.方法 用磺酰罗丹明B(SRB)法检测PNR对肿瘤细胞增殖能力的影响;用流式细胞术检测PNR对Tea8113细胞周期的影响;用Transwell迁移法观察PNR对Tca8113细胞迁移能力的影响;用4',6-二脒基-2-苯基吲哚(DAPI)荧光染色法观察Tca8113细胞凋亡形态.结果 PNR对Tca8113、A549、HepG 2、BCG 823和EJ等人癌细胞均有增殖抑制作用.PNR在0.8～ 12.8μg/mL剂量范围内浓度、时间依赖性地抑制A549、HepG 2和BCG 823细胞的生长.2～7 μg/mL明显抑制Tca8113和EJ细胞生长,量效关系明显,对Tca8113和EJ细胞抑制作用在24 h已达高峰.综合比较,PNR对Tca8113细胞的抑制作用最强.PNR还明显抑制Tca8113细胞的迁移活性,阻止Tca8113细胞于S期,减少G1期细胞比例,并能明显诱导Tca8113细胞凋亡.结论 PNR对多种肿瘤细胞的生长有明显抑制作用,对Tca8113细胞的作用最强,并能抑制其迁移,诱导其凋亡.     

Melatonin treatment potentiates neurodegeneration in a rat rotenone Parkinson's disease model

Parkinson's disease (PD) is characterized pathologically by progressive neurodegeneration of the nigrostriatal dopamine (DA) system. Currently, the cause of the disease is unknown, except for a small percentage of familial cases (<10% of total). The rat rotenone model reproduces many of the pathological features of the human disease, including apomorphine‐responsive behavioral deficits, DA depletion, loss of striatal DA terminals and nigral dopaminergic neurons, and α‐synuclein/polyubiquitin‐positive cytoplasmic inclusions reminiscent of Lewy bodies. Therefore, this model is well‐suited to examine potential neuroprotective agents. Melatonin is produced mainly by the pineal gland and is known primarily for regulating circadian rhythms. It also has potent free radical scavenging and antiinflammatory properties. Melatonin has been reported to be neuroprotective in the 6‐hydroxydopamine (6‐OHDA) and 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) models of PD. However, there are conflicting reports suggesting that melatonin does not provide neuroprotection in these models. Melatonin elicits significant functional changes in the nigrostriatal DA system that may affect 6‐OHDA and MPTP entry into cells. Therefore, rotenone is an ideal model for assessing protection, because it does not rely on the dopamine transporter uptake to exert neurotoxicity. In this study, the neuroprotective potential of melatonin in the rotenone PD model was assessed. Melatonin potentiated striatal catecholamine depletion, striatal terminal loss, and nigral DA cell loss. Indeed, melatonin alone elicited alterations in striatal catecholamine content. Our findings indicate that melatonin is not neuroprotective in the rotenone model of PD and may exacerbate neurodegeneration. © 2009 Wiley‐Liss, Inc.     

自噬参与DJ-1保护大鼠黑质多巴胺能神经元抵抗鱼藤酮损伤

目的 观察过表达DJ-1是否缓解鱼藤酮致SD大鼠中脑黑质多巴胺能神经元损伤及其机制。方法 在大鼠黑质致密部注射携带DJ-1、LacZ或者GFP-DJ-1、GFP的腺相关病毒颗粒,4周后注射鱼藤酮。通过免疫组织化学方法鉴定基因表达情况,并检测 TH阳性神经元数量,验证DJ-1保护作用。通过Western blotting方法检测自噬相关蛋白表达水平。结果 鱼藤酮损伤4周时,可见过表达DJ-1组大鼠损伤侧黑质的TH阳性神经元数目显著高于单纯鱼藤酮损伤组,差异有统计学意义(P<0.05)。蛋白印迹检测动物损伤侧黑质自噬相关蛋白Beclin1、p-mTOR的表达和LC3 Ⅱ/Ⅰ的比值,发现DJ-1过表达组中Beclin1与LC3 Ⅱ/Ⅰ的比值均较对照组增加,差异有统计学意义;而p-mTOR的表达较对照组降低,差异有统计学意义(P<0.05)。结论 DJ-1能抵抗鱼藤酮对大鼠黑质多巴胺能神经元的损伤,DJ-1能上调自噬水平。