15-脱氧-D12,14-前列腺素 J2 的体外抗骨髓瘤活性与内质网应激相关,Blood

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15-脱氧-D12,14-前列腺素 J2 的体外抗骨髓瘤活性与内质网应激相关
Blood ( IF 21.0 ) Pub Date : 2013-11-15 , DOI: 10.1182/blood.v122.21.4913.4913
Ana Paula Demasi ₁ , Marcelo Henrique Napimoga ₂ , Elizabeth Ferreira Martinez ₁ , Adriana Silva Santos Duarte ₃ , Fernando V Pericole ₃ , Sara T O Saad ₄ , Ney Soares Araujo ₁ , Vera Cavalcanti Araujo ₁

Affiliation

多发性骨髓瘤 (MM) 是一种无法治愈的恶性肿瘤，其特征是终末分化的浆细胞在骨髓中积聚，通常伴有持续的单克隆免疫球蛋白产生。这种生产需要免疫球蛋白链的正确折叠和组装，通过内质网 (ER) 中的二硫键形成进行。二硫键是通过蛋白质中继系统中硫醇基团的电子转移形成的，从蛋白质二硫键异构酶 (PDI) 到内质网氧化还原酶 1 (ERO1)，最后到分子氧，生成一分子过氧化氢 (H 2 O 2 ) 作为副产物对于每个形成的二硫键。通常对 MM 细胞的高 ER 蛋白折叠需求使它们易于积累未折叠蛋白，从而导致 ER 应激和氧化应激，两者都能够诱导细胞死亡。针对这些特定压力形式的细胞防御系统，称为未折叠蛋白反应 (UPR) 和氧化应激反应，已被探索作为 MM 的治疗靶点。15-Deoxy-D 12,14-前列腺素 J 2 (15d-PGJ2) 是 J 2 系列 PG 的成员，其特征在于环戊烯酮环中存在亲电羰基，这赋予它们不同于PG 系列的其他组件。这些包括抗炎、抗增殖和促凋亡活性，具体取决于浓度和细胞类型。从观察到 15d-PGJ2 正在成为 J 2 系列 PG 中最有效的抗肿瘤剂开始，在本研究中，我们研究了它对 MM 细胞的影响。我们发现 15d-PGJ2 在 MM 细胞系中显示出优于地塞米松的细胞毒性。使用流式细胞术，我们证明了这些细胞在 15d-PGJ2 处理后细胞内活性氧 (ROS) 的形成，这与氧化谷胱甘肽 (GSH) 的比例增加有关，表明细胞处于氧化应激状态。由于其亲电性，有人认为 15d-PGJ2 可以直接与 GSH 和/或其他硫醇化合物反应。这种反应会消耗必需的细胞抗氧化剂，从而导致 MM 细胞的氧化应激。qPCR 分析的结果表明，15d-PGJ2 诱导 UPR 基因的转录，包括 GRP78、GRP94、EDEM1 和 PRDX4。GRP78/BiP 和 GRP94 是内质网驻留分子伴侣，是热休克蛋白 (HSP) 70 家族和 HSP 90 家族的成员，分别。这些分子伴侣被描述为与免疫球蛋白链结合，帮助它们组装。ER 降解促进剂甘露糖苷酶样蛋白 1 (Edem1) 参与错误折叠蛋白的降解，以防止其聚集和细胞毒性。PRDX4 编码 ER 定位的过氧化物氧还蛋白 (Prx) IV，这是一种尚未被识别为 UPR 组分的酶，但最近证明使用 ERO1 在二硫键形成过程中产生的 H 2 O 2 生成另一对二硫键，具有额外的好处将 H 2 O 2 还原为水。这些基因的转录增加表明尝试保留 ER 处理功能和 MM 细胞对积累的错误折叠蛋白的反应。在这方面，15d-PGJ2 诱导的氧化应激，通过引发蛋白质的氧化修饰，可能会加剧错误折叠蛋白的形成，从而激活 UPR 信号。与 15d-PGJ2 介导的错误折叠蛋白的积累一致，该积累被阻止沿着分泌途径进一步发展，我们的 ELISA 结果显示，在 15d-存在下，MM 细胞分泌的轻链免疫球蛋白浓度大幅降低。 PGJ2，但不是地塞米松。这些数据表明 15d-PGJ2 有利于 ROS 积累和蛋白质错误折叠，特别是威胁具有高蛋白质折叠负荷和/或易受氧化应激影响的细胞的存活。从这个意义上说，多发性骨髓瘤和相关疾病可能受益于 15d-PGJ2 治疗。披露：Demasi：FAPESP：研究资金。Napimoga：FAPESP：研究资金。与 15d-PGJ2 介导的错误折叠蛋白的积累一致，该积累被阻止沿着分泌途径进一步发展，我们的 ELISA 结果显示，在 15d-存在下，MM 细胞分泌的轻链免疫球蛋白浓度大幅降低。 PGJ2，但不是地塞米松。这些数据表明 15d-PGJ2 有利于 ROS 积累和蛋白质错误折叠，特别是威胁具有高蛋白质折叠负荷和/或易受氧化应激影响的细胞的存活。从这个意义上说，多发性骨髓瘤和相关疾病可能受益于 15d-PGJ2 治疗。披露：Demasi：FAPESP：研究资金。Napimoga：FAPESP：研究资金。与 15d-PGJ2 介导的错误折叠蛋白的积累一致，该积累被阻止沿着分泌途径进一步发展，我们的 ELISA 结果显示，在 15d-存在下，MM 细胞分泌的轻链免疫球蛋白浓度大幅降低。 PGJ2，但不是地塞米松。这些数据表明 15d-PGJ2 有利于 ROS 积累和蛋白质错误折叠，特别是威胁具有高蛋白质折叠负荷和/或易受氧化应激影响的细胞的存活。从这个意义上说，多发性骨髓瘤和相关疾病可能受益于 15d-PGJ2 治疗。披露：Demasi：FAPESP：研究资金。Napimoga：FAPESP：研究资金。我们的 ELISA 结果显示，在 15d-PGJ2 存在下，MM 细胞分泌的轻链免疫球蛋白浓度大幅降低，但地塞米松不存在。这些数据表明 15d-PGJ2 有利于 ROS 积累和蛋白质错误折叠，特别是威胁具有高蛋白质折叠负荷和/或易受氧化应激影响的细胞的存活。从这个意义上说，多发性骨髓瘤和相关疾病可能受益于 15d-PGJ2 治疗。披露：Demasi：FAPESP：研究资金。Napimoga：FAPESP：研究资金。我们的 ELISA 结果显示，在 15d-PGJ2 存在下，MM 细胞分泌的轻链免疫球蛋白浓度大幅降低，但地塞米松不存在。这些数据表明 15d-PGJ2 有利于 ROS 积累和蛋白质错误折叠，特别是威胁具有高蛋白质折叠负荷和/或易受氧化应激影响的细胞的存活。从这个意义上说，多发性骨髓瘤和相关疾病可能受益于 15d-PGJ2 治疗。披露：Demasi：FAPESP：研究资金。Napimoga：FAPESP：研究资金。尤其威胁具有高蛋白质折叠负荷和/或易受氧化应激影响的细胞的存活。从这个意义上说，多发性骨髓瘤和相关疾病可能受益于 15d-PGJ2 治疗。披露：Demasi：FAPESP：研究资金。Napimoga：FAPESP：研究资金。尤其威胁具有高蛋白质折叠负荷和/或易受氧化应激影响的细胞的存活。从这个意义上说，多发性骨髓瘤和相关疾病可能受益于 15d-PGJ2 治疗。披露：Demasi：FAPESP：研究资金。Napimoga：FAPESP：研究资金。

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Antimyeloma In Vitro Activity Of 15-Deoxy-D12,14-Prostaglandin J2 Is Associated With Endoplasmic Reticulum Stress In a Reactive Oxygen Species-Dependent Fashion

Multiple myeloma (MM) is an incurable malignancy characterized by the accumulation of terminally differentiated plasma cells in the bone marrow, usually accompanied by continuous monoclonal immunoglobulin production. This production requires proper folding and assembly of immunoglobulin chains, conducted by disulfide bonds formation in the endoplasmic reticulum (ER). Disulfide bonds are formed via electron transfer from thiol groups in a protein relay system, from protein disulfide isomerase (PDI) to ER oxidoreductase1 (ERO1) and finally to molecular oxygen, generating one molecule of hydrogen peroxide (H 2 O 2 ) as secondary product for each disulfide bond formed. The high ER protein-folding demand usually placed on MM cells predisposes them to accumulation of unfolded proteins, which results in ER stress and also oxidative stress, both capable to induce cell death. Cellular defense systems for these specific forms of stress, known as the unfolded protein response (UPR) and oxidative stress response, have been explored as therapeutic targets for MM. 15-Deoxy-D 12,14 -prostaglandin J 2 (15d-PGJ2) is a member of J 2 series of PGs, characterized by the presence of an electrophilic carbonyl group in the cyclopentenone ring, which confers them biological properties that are different from those of other components of the PG family. These include anti-inflammatory, anti-proliferative and pro-apoptotic activities, depending on the concentration and cell type. Starting from the observation that15d-PGJ2 is emerging as the most potent antineoplastic agent of the J 2 series of PGs, in the present study we investigated its effect on MM cells. We found that 15d-PGJ2 displayed superior cytotoxicity than dexamethasone in MM cell lines. Using ﬂow cytometry, we demonstrated intracellular reactive oxygen species (ROS) formation in these cells after 15d-PGJ2 treatment, which was associated with an augmented ratio of oxidized to reduced glutathione (GSH), indicating that the cells were under oxidative stress. Due to its electrophilic property, it has been suggested that 15d-PGJ2 may directly react with GSH and/or other thiol compounds. Such reactions would deplete essential cellular antioxidants thereby leading to oxidative stress in MM cells. Results from qPCR analysis showed that 15d-PGJ2 induces transcription of UPR genes, including GRP78 , GRP94 , EDEM1 and also PRDX4 . GRP78/BiP and GRP94 are ER-resident chaperones, members of the heat shock protein (HSP) 70 family and HSP 90 family, respectively. These chaperones were described to bind to immunoglobulin chains assisting their assembly. The ER degradation-enhancer mannosidase-like protein 1 (Edem1) is involved in the degradation of misfolded proteins to prevent their aggregation and cytotoxicity. PRDX4 encodes the ER-localized peroxiredoxin (Prx) IV, an enzyme not yet recognized as an UPR component, but recently demonstrated to use the H 2 O 2 produced by ERO1 during disulfide bond formation to generate another pair of disulfide, with the additional benefit of reducing H 2 O 2 to water. Increased transcription of these genes indicates an attempt to preserve ER-processing function and MM cells survival in response to accumulated misfolded proteins. In this regard, 15d-PGJ2-induced oxidative stress, by provoking oxidative modification of proteins, could have intensified the formation of misfolded proteins, thereby activating UPR signaling. In agreement with 15d-PGJ2-mediated accumulation of misfolded proteins, which are prevented from progressing further along the secretory pathway, our results from ELISA have shown a massive decrease in the concentration of light chain immunoglobulin secreted by MM cells in the presence of 15d-PGJ2, but not dexamethasone. These data suggest that 15d-PGJ2 favors ROS accumulation and protein misfolding, especially threatening the survival of cells that have a high protein-folding load and/or are susceptible to oxidative stress. In this sense, multiple myeloma and related disorders may benefit from 15d-PGJ2 therapy. Disclosures: Demasi: FAPESP: Research Funding. Napimoga: FAPESP: Research Funding.

更新日期：2013-11-15

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