三氧化二砷对非急性早幼粒细胞白血病和实体瘤的实验研究进展

在急性早幼粒细胞白血病(APL)中,三氧化二砷(As₂O₃)有效诱导了APL细胞的凋亡,如果能避免早期死亡,多数患者可达到完全缓解和治愈。近几年,人们也研究了它对非APL白血病和多种实体瘤治疗的可能性。同时,越来越多的研究发现,它在体外或体内也抑制非APL白血病和几种实体瘤如肝癌、食管癌和胃癌等细胞的增殖。人们对非APL白血病和实体瘤中As₂O₃诱导细胞凋亡的机制也进行了研究。并且为取得更满意的疗效和减轻As₂O₃的细胞毒等副作用,人们对新型As₂O₃靶向制剂的研发也进行了探索。本文就上述有关研究进展进行综述。     

Arsenic trioxide therapy in acute promyelocytic leukemia and beyond: from bench to bedside

Arsenic trioxide (As₂O₃) has a long history of use in medicine. However, it was almost forgotten in Western medicine in the recent centuries. Prompted by reports from China about successful treatment of acute promyelocytic leukemia (APL) with As₂O₃, there was again increasing interest in this drug in the 1990s. This review summarizes the considerable knowledge about the mechanisms of action of As₂O₃ that was gained during the last 5 - 10 years. It is focused in particular on the effects of As₂O₃ in non-APL cells. Since As₂O₃ seems to induce apoptosis and inhibits growth in a large variety of cellular targets, it might become an alternative or adjunct drug to conventional chemotherapy. As₂O₃ can even be effective in cells resistant to conventional cytostatic agents. Insight into the cellular mechanisms, in particular the impact of the redox state on sensitivity towards As₂O₃ opens the possibility to enhance As₂O₃ effects by appropriate combination therapies.     

Phenylarsine oxide (PAO) more intensely induces apoptosis in acute promyelocytic leukemia and As2O3-resistant APL cell lines than As2O3 by activating the mitochondrial pathway

We studied the cytotoxic effect of an organic arsenical compound, phenylarsine oxide (PAO) on an acute promyelocytic leukemia (APL) cell line (NB4) and an As₂O₃-resistant NB4 subline (NB4/As). Cell growth was inhibited by 50% (IC₅₀) upon 2-day treatment with As₂O₃ or PAO at 0.54 and 0.06 μM, respectively in NB4 cells (P = 0.025), and 2.80 and 0.08 μM, respectively in NB4/As (P = 0.030). 0.1 μM PAO increased the proportion of hypodiploid cells (50.3%) by a greater degree than the same dose of As₂O₃ (3.8%) in NB4 cells. In NB4 cells, 0.1 μM PAO reduced the mitochondrial transmembrane potential (20.5% in a PI^negative-Rhodamine123^low fraction) by a greater degree than 1 μM As₂O₃ (7.1%). Western blotting showed that 0.1 μM PAO downregulated the expression of both Bcl-2 and Bcl-X_L proteins, whereas I μM As₂O₃ downregulated only Bcl-2 expression. These results suggest that the cytotoxic effect of PAO on an APL cell line and As₂O₃-resistant subline is significantly higher than that of As₂O₃. PAO-induced apoptosis seems to be related to the activation of the mitochondrial pathway and downregulation of both Bcl-2 and Bcl-X_L. PAO is a considerable agent for relapsed/refractory APL and for purging APL cells following stem cell transplantation.     

受体介导的c-myc反义核酸提高肝癌细胞对不同化疗药物的敏感性

目的: 探讨半乳糖(Galactose,Gal)-聚乙烯亚胺(Polyethyleneimine,PEI)-c-myc反义核酸(Antisense-Oligonucleotides,ASODN)复合物联合化疗药物三氧化二砷(Arsenic Trioxide As₂O₃),5-氟脲嘧啶(5-Fluorouracil,5-FU),表阿霉素(Pharmorbincin,PHA)对人肝癌Bel-7402细胞增殖的抑制作用。 方法: 选用不同浓度的As₂O₃、5-FU、PHA单独使用、联合c-myc ASODN、联合Gal-PEI-ASODN,作用于Bel-7402细胞,采用WST-8法检测细胞增殖的抑制率,并计算药物作用的IC₅₀。 结果: 不同浓度的化疗药物(As₂O₃,5-FU,PHA)联合0.25μmol/LGal-PEI-ASODN均明显降低了化疗药物的IC₅₀,提高了Bel-7402细胞对化疗药物的敏感性(分别提高药效3.38,1.58,2.05倍)。 结论: 半乳糖受体介导的c-myc反义核酸能提高人肝癌Bel-7402细胞对As₂O₃,5-FU,PHA的敏感性,与As2O3联合作用效果最好。     

三氧化二砷与传统及新型药物协同抑制皮肤T细胞淋巴瘤细胞系增殖

  目的  探讨三氧化二砷（arsenic trioxide,As₂O₃）与传统药物地塞米松（Dexamethasone,DXM）、依托泊苷（Etoposide,VP-16）、甲氨蝶呤（Methotrexate,MTX）和新型药物硼替佐米（Bortezomib,BTZ）、组蛋白去乙酰化酶抑制剂-辛二酰苯胺异羟肟酸（suberoylanilide hydroxamic acid,SAHA）联合对人皮肤T细胞淋巴瘤（CTCL）细胞系Hut-78、Hut-102细胞的增殖抑制作用。  方法  不同浓度的As₂O₃单药或与DXM/VP-16/MTX/BTZ/SAHA联合作用于Hut-78、Hut-102细胞48 h后,采用MTT法检测细胞增殖抑制率,应用联合指数分析两药是否具有协同效应。  结果  As₂O₃、DXM、VP-16、MTX、BTZ、SAHA单药对Hut-78、Hut-102细胞的增殖均具有显著的抑制作用,呈剂量依赖性,培养48h的半数抑制浓度分别为5 μmol/L、500 μg/L、2.5 μg/L、1 μg/L、10 μmol/L、2.5 μmol/L。As₂O₃与DXM/VP-16/MTX/BTZ/SAHA联合时具有协同效应,抗肿瘤作用更为显著。  结论  As₂O₃单药及其与DXM/VP-16/MTX/ BTZ/SAHA联合在体外可有效抑制CTCL细胞增殖。As₂O₃是一种很有前景的治疗CTCL的药物,As₂O₃与DXM/VP-16/MTX/BTZ/ SAHA等传统或新型药物联合可为CTCL临床的治疗提供实验依据。      

葛根素与三氧化二砷协同作用通过Akt/p38途径促进人胶质瘤细胞凋亡

  目的  研究葛根素（PRN）是否通过Akt/p38途径协同三氧化二砷（As₂O₃）促进人胶质瘤细胞的凋亡。  方法  MTT检测细胞的存活率,流式细胞仪（FCM）技术检测细胞的凋亡状态,蛋白免疫印迹（Immunoblotting）检测细胞phosphorylated Akt和p38-MAPK,Cleaved Caspase-3蛋白的表达,PCR检测Caspase-3的mRNA的表达。  结果  PRN协同As₂O₃降低人胶质瘤细胞U87的存活。与对照组相比,PRN（16 μM）组,As₂O₃（2μM）组显著增加细胞内钙水平（1.13±0.015）,（1.18±0.33）。此外,PRN能够协同As₂O₃增加细胞内钙（1.34 ± 0.72）,下调蛋白phosphorylated Akt,上调phosphorylated p38-MAPK和Cleaved Caspase-3蛋白及Cleaved Caspase-3 mRNA表达水平。  结论  PRN协同As₂O₃增加胶质瘤细胞内钙水平,抑制细胞存活。此外,PRN联合As₂O₃下调phosphorylated Akt,增强phosphorylated p38-MAPK和Cleaved Caspase-3的表达,进而促进肿瘤细胞凋亡。PRN可能成为临床上辅助As₂O₃治疗肿瘤中的潜在的辅助治疗药物。      

三氧化二砷联合环腺苷酸拟似物诱导骨髓瘤细胞凋亡的实验研究

背景与目的：近年来随着新型药物和免疫疗法的成功应用，多发性骨髓瘤（multiple myeloma，MM）患者的缓解率、缓解深度和无病生存率较以往显著提高，然而仍有相当一部分MM患者属于复发/难治性病例。我们的前期研究发现，环腺苷酸（cyclic adenosine monophosphate，cAMP）拟似物和三氧化二砷（arsenic trioxide，As₂O₃）可以抑制MM细胞增殖并诱导其凋亡，成为MM治疗的新途径。该研究进一步探究As₂O₃单独和联合环腺苷酸拟似物8-对氯苯硫基环腺苷酸［8-(4-chlorophenylthio) adenosine 3’, 5’-cyclic monophosphate，8-CPT-cAMP］对MM细胞的影响及可能作用机制。方法：以不同浓度As₂O₃和8-CPT-cAMP单独和联合处理MM细胞系U266细胞，采用细胞计数试剂盒（cell counting kit-8，CCK-8）检测其增殖，应用药物联合指数（combination index，CI）分析两药是否存在协同效应，同时采用流式细胞术检测细胞周期和凋亡率，进一步利用蛋白质印迹法（Western blot）检测细胞凋亡相关蛋白caspase-3和Bcl-2表达的变化。结果：U266细胞经As₂O₃和8-CPT-cAMP单独或联合处理72和120 h后，联合用药组细胞增殖抑制率分别为（18.01±0.13）%和（28.01±0.14）%，明显高于单独用药组［As₂O₃组为（11.35±0.01）%和（16.01±0.14）%，8-CPTcAMP组为（12.26±0.30）%和（15.43±0.23）%，P均<0.05］；但两药联合处理U266细胞的CI值均大于1。联合用药组72和120 h后U266细胞凋亡率分别达到（22.26±0.13）%和（31.03±0.14）%，显著高于单药组［As₂O₃组为（10.06±0.01）%和（12.35±0.14）%，8-CPT-cAMP组为（13.26±0.30）%和（18.76±0.23）%，P均<0.05］。同时联合用药组U266细胞内caspase-3蛋白剪切活化和Bcl-2表达下降。结论：As₂O₃联合8-CPT-cAMP对MM细胞凋亡诱导效应强于单药，但无协同效应。     

Ras和磷酸化ERK信号通路参与三氧化二砷逆转耐药作用机制的研究

  目的   体外实验研究三氧化二砷(As₂O₃)对人胃癌阿霉素耐药细胞株(SGC7901/ADM)的逆转耐药机制。   方法   MTT法检测磷酸化ERK(p-ERK)激动剂G-CSF作用前后As₂O₃的逆转耐药倍数; 免疫细胞化学法测定As₂O₃及G-CSF作用前后SGC7901/ADM细胞内Ras和p-ERK的变化; 流式细胞仪测定G-CSF和As₂O₃干预后SGC7901/ADM的细胞周期和凋亡率。   结果   SGC7901/ADM对ADM耐药, As₂O₃可逆转耐药, 其中0.5μmol/L As₂O₃作用48 h后逆转耐药倍数约为6.29。G-CSF干预后, 逆转耐药倍数降至4.72;SGC7901/ADM中Ras表达高于亲本细胞株SGC7901/S, 而p-ERK无明显差异, As₂O₃可下调Ras及p-ERK的表达。G-CSF干预后, As₂O₃下调Ras及p-ERK表达的能力较干预前显著降低。0.1μmol/L和0.5μmol/L As₂O₃组的G₀~G₁期细胞比例和凋亡率均显著高于各个对照组; G-CSF干预后同一剂量的As₂O₃组G₀~G₁期细胞及凋亡率较干预前均显著降低。   结论   As₂O₃可逆转人胃癌耐药细胞株SGC7901/ADM对ADM的耐药作用, 其机制与下调Ras/p-ERK信号传导通路中Ras、磷酸化ERK的表达有关。      

三氧化二砷在胃癌治疗中的应用

三氧化二砷(As2O3)是中药砒霜的主要成分,最初被用于治疗血液系统疾病.近年研究发现,其对多种实体瘤也有良好疗效,其中对胃癌疗效显著.研究证实,As2O3可以抑制胃癌细胞增殖及转移,诱导胃癌细胞凋亡和保护性自噬,抑制胃癌淋巴管和新生血管形成,影响胃癌细胞内端粒相关蛋白的表达.     

三氧化二砷对人胃癌耐药细胞系SGC7901/ADM耐药逆转及凋亡诱导作用的探讨

  目的   体外实验研究三氧化二砷(As₂O₃)对人胃癌阿霉素耐药细胞株(SGC7901/ADM)的逆转耐药机制。   方法   MTT法检测磷酸化ERK(p-ERK)激动剂G-CSF作用前后As₂O₃的逆转耐药倍数; 免疫细胞化学法测定As₂O₃及G-CSF作用前后SGC7901/ADM细胞内Ras和p-ERK的变化; 流式细胞仪测定G-CSF和As₂O₃干预后SGC7901/ADM的细胞周期和凋亡率。   结果   SGC7901/ADM对ADM耐药, As₂O₃可逆转耐药, 其中0.5μmol/L As₂O₃作用48 h后逆转耐药倍数约为6.29。G-CSF干预后, 逆转耐药倍数降至4.72;SGC7901/ADM中Ras表达高于亲本细胞株SGC7901/S, 而p-ERK无明显差异, As₂O₃可下调Ras及p-ERK的表达。G-CSF干预后, As₂O₃下调Ras及p-ERK表达的能力较干预前显著降低。0.1μmol/L和0.5μmol/L As₂O₃组的G₀~G₁期细胞比例和凋亡率均显著高于各个对照组; G-CSF干预后同一剂量的As₂O₃组G₀~G₁期细胞及凋亡率较干预前均显著降低。   结论   As₂O₃可逆转人胃癌耐药细胞株SGC7901/ADM对ADM的耐药作用, 其机制与下调Ras/p-ERK信号传导通路中Ras、磷酸化ERK的表达有关。     

Skp2在三氧化二砷诱导的肝癌SMMC-7721细胞周期阻滞中的表达变化和意义

目的: 探讨三氧化二砷(As₂O₃)对肝癌SMMC-7721细胞周期的影响,及其与细胞周期素依赖性激酶抑制因子(CDKI)p27^kip1和p27^kip1相关蛋白S期激酶相关蛋白2(S-phase kinase-associated protein2,Skp2)的关系,为临床应用提供依据。 方法: 体外培养人肝癌细胞株SMMC-7721,用2μmol/LAs₂O₃处理72h,流式细胞仪检测细胞周期变化,采用核浆分离、Western Blot技术及细胞免疫荧光技术检测该过程中p27^kip1、Skp2在SMMC-7721细胞中的表达变化及亚细胞定位情况。 结果: 与对照组比较,As₂O₃使SMMC-7721细胞周期阻滞在G₂/M期。经As₂O₃作用的人肝癌细胞p27^kip1蛋白水平增加,Skp2蛋白水平降低,同时27^kip1发生从胞浆到胞核的易位。 结论: As₂O₃可下调Skp2的表达,从而促进SMMC-7721细胞中p27^kip1的积聚,干扰细胞周期的进程,抑制SMMC-7721细胞的增殖。     

维生素C联合三氧化二砷对膀胱癌T-24细胞株的作用

 目的 探讨维生素C联合三氧化二砷（As2O3）对膀胱癌T-24细胞的影响及其机制。方法 体外培养T-24细胞,分为6组,对照组不做处理,实验组分别加入不同浓度的As₂O₃ （0.4、4、40 μg/ml）组,维生素C（400 μg/ml）组、维生素C（400 μg/ml）+ As₂O₃ （0.4 μg/ml）组（联合组）,采用细胞增殖曲线检测T-24细胞生长差异情况,用流式细胞术检测细胞周期及凋亡率变化,RT-PCR、Western blot技术检测分析caspase-3、survivin mRNA及蛋白的表达情况。结果 联合组、维生素C（400 μg/ml）组、As₂O₃ （4 μg/ml）组及As₂O₃ （40 μg/ml）组均对膀胱癌T-24细胞增殖有显著抑制作用;联合组将细胞阻止在G₀/G₁期,且凋亡率显著高于其他五组（P<0.05）;RT-PCR及Western blot结果显示,维生素C（400 μg/ml）组、联合组的caspase-3 mRNA和蛋白表达升高,survivin mRNA和蛋白表达降低。结论 维生素C联合As₂O₃ 可抑制膀胱癌T-24细胞增殖,促进细胞凋亡,其机制可能与上调caspase-3 mRNA、蛋白,下调survivin mRNA、蛋白表达有关。     

Arsenic trioxide: new clinical experience with an old medication in hematologic malignancies.

Arsenic trioxide has shown great promise in the treatment of patients with relapsed or refractory acute promyelocytic leukemia (APL). In clinical trials, arsenic trioxide induces complete remission in 87% of patients and molecular remission in 83% of patients. Two-year overall and relapse-free survival estimates are 63% and 49%, respectively. Treatment with arsenic trioxide may be associated with the APL differentiation syndrome, leukocytosis, and electrocardiographic abnormalities. The expanded use of arsenic trioxide in APL for postremission therapy, in conjunction with transplantation, and in patients with newly diagnosed APL is under investigation. The multiple mechanisms of action of arsenic trioxide suggest that it may have antitumor activity in malignancies other than APL and that it may be used in combination with other agents to expand its potential use. This article reviews the clinical use of arsenic trioxide to date and discusses new therapeutic strategies evolving from its diverse biologic activities.     

Phase II clinical trial of arsenic trioxide with liposomal doxorubicin, vincristine, and dexamethasone in newly diagnosed multiple myeloma

In patients with multiple myeloma, there is preclinical justification to combine arsenic trioxide (ATO and As₂O₃) with DVd (Doxil™, vincristine, and dexamethasone) for newly diagnosed patients. Eleven patients on this phase II trial received 0.15 mg/kg of ATO for five consecutive days followed by four cycles of DVd plus ATO with the ATO at 0.25 mg/kg IV twice per week. The most common grade 3 toxicities were hyperglycemia, hyponatremia, and hypocalcemia. There were four partial and no complete responses. We could not demonstrate that the addition of ATO with this schedule improved the response rate of MM to DVd.     

三氧化二砷联合吉西他滨及多西他赛治疗晚期转移性骨肉瘤26例临床分析

  目的   探讨三氧化二砷（arsenic trioxide,As₂O₃）联合吉西他滨及多西他赛治疗骨肉瘤肺转移的临床疗效。   方法   收集26例对一线化疗药物耐药的骨肉瘤肺转移患者,男性14例,女性12例,年龄11~62岁,平均31.2岁,所有患者均接受过传统规范化疗后出现肺转移。应用As₂O₃（剂量为10 mg/d,d1~28）联合吉西他滨（剂量为800 mg/m2,d1、d8）及多西他赛（剂量为75 mg/m2,d8）每3周重复给药,每2个化疗疗程复查疗效。   结果   接受化疗的患者总体有效率（CR+PR）为34.6%（9/26）。中位随访时间28.2（1~48）个月,中位总生存期（OS）为16.7个月（95%CI:7.561~18.058）和中位无进展生存期（PFS）为10.3个月（95%CI:6.541~ 8.754）,1、2和4年生存率分别是61.5%、38.4%和15.4%。治疗后最常见的不良反应为骨髓抑制,而常见的非血液学不良反应包括心脏毒性、消化道反应及肝肾功能异常,对症处理后均明显缓解。   结论   As₂O₃联合吉西他滨及多西他赛作为二线化疗药物治疗骨肉瘤肺转移的近期临床疗效较好,且有较好的耐受性。      

Efficacy and safety results with the combination therapy of arsenic trioxide, dexamethasone, and ascorbic acid in multiple myeloma patients

BACKGROUND: Single-agent arsenic trioxide has shown promising results in patients with relapsed or refractory multiple myeloma (MM). Because preclinical data suggested greater activity with dexamethasone and ascorbic acid, a phase 2 trial of the combination of arsenic trioxide, dexamethasone, and ascorbic acid in patients with relapsed or refractory MM was conducted. METHODS: Twenty patients in whom no more than two previous therapies had failed were enrolled. The mean age was 62 yr, and 55% of the patients had refractory disease. The regimen consisted of 14- or 15-wk cycles, with the first cycle considered induction, followed by one or two consolidation cycles with a reduced steroid schedule and then a maintenance cycle in responding patients. RESULTS: The overall response rate was 30%, with at least stable disease in 80% of patients. Median progression- free survival was 316 d in all patients and 584 d in those with a response. The regimen was well tolerated, with most adverse events being mild or moderate. CONCLUSIONS: This study showed the clinical efficacy and tolerability of the combination of arsenic trioxide, dexamethasone, and ascorbic acid. Further study is warranted.     

Clinical experience of arsenic trioxide in relapsed acute promyelocytic leukemia

Acute promyelocytic leukemia (APL) has unique clinical, cytogenetic, and molecular features and is one of the most potentially curable human malignancies. The current standard treatment given to patients with newly diagnosed APL consists of all-trans retinoic acid and anthracycline-based cytotoxic chemotherapy, which is highly effective for remission induction. However, despite the potential for cure with existing treatments, approximately 20%-30% of patients relapse and require salvage therapy. Reports of the safety and efficacy of arsenic trioxide from centers in China led to a pivotal trial of this agent in the United States for patients with relapsed APL. In an initial pilot study, 11 of 12 patients experienced a complete response, and a subsequent multicenter trial confirmed the efficacy and safety of arsenic trioxide for remission induction in this patient population. Additional trials are under way to evaluate the use of this agent alone or as part of a chemotherapy regimen for consolidation and maintenance of patients with APL.     

三氧化二砷(As_2O_3)治疗2例儿童难治性急性早幼粒细胞白血病

为探讨三氧化二砷治疗儿童白血病的效果。对2例难治性儿童患者进行了治疗。1例达CR。使用过程无明显毒副反应，且与全反式维甲酸（ATRA）无交叉耐药。细胞形态学检查显示：AS2O3有诱导分化及促凋亡双重作用。