Establishment of a retinoic acid-resistant human acute promyelocytic leukaemia (APL) model in human granulocyte-macrophage colony-stimulating factor (hGM-CSF) transgenic severe combined immunodeficiency (SCID) mice.

To understand the mechanisms and identify novel approaches to overcoming retinoic acid (RA) resistance in acute promyelocytic leukaemia (APL), we established the first human RA-resistant APL model in severe combined immunodeficiency (SCID) mice. UF-1 cells, an RA-resistant APL cell line established in our laboratory, were transplanted into human granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing SCID (hGMTg SCID) mice and inoculated cells formed subcutaneous tumours in all hGMTg SCID mice, but not in the non-transgenic control SCID mice. Single-cell suspensions (UF-1/GMTg SCID cells) were similar in morphological, immunological, cytogenetic and molecular genetic features to parental UF-1 cells. All-trans RA did not change the morphological features of cells or their expression of CD11b. RA did not alter the growth curve of cells as determined by MTT assay, suggesting that UF-1/GMTg SCID cells are resistant to RA. These results demonstrate that this is the first RA-resistant APL animal model that may be useful for investigating the biology of this myeloid leukaemia in vivo, as well as for evaluating novel therapeutic approaches including patients with RA-resistant APL.     

亚砷酸联合全反式维甲酸治疗急性早幼粒细胞白血病16例疗效观察

目的:观察亚砷酸(As2O3)与全反式维甲酸(ATRA)联合治疗初发急性早幼粒细胞白血病(APL)的疗效和不良反应.方法:As2O3联合ATRA治疗初治APL患者16例,As2O30.1%注射液10ml加入5%葡萄糖溶液500ml静脉点滴,持续4h,1次/d;ATRA40-60mg/d,分2次口服,观察完全缓解(CR)率,获得CR所需时间、不良反应.结果:15例患者获得CR,CR率93.8%,获得缓解时间(27.3±3.6)d,没有发现明显的不良反应.结论:As2O3,联合ATRA治疗初发APL患者疗效好,能缩短CR的时间,长期CR时间需要进一步观察.     

Arsenic trioxide (ATO) and MEK1 inhibition synergize to induce apoptosis in acute promyelocytic leukemia cells.

Recent studies suggest that components of the prosurvival signal transduction pathways involving the Ras-mitogen-activated protein kinase (MAPK) can confer an aggressive, apoptosis-resistant phenotype to leukemia cells. In this study, we report that acute promyelocytic leukemia (APL) cells exploit the Ras-MAPK activation pathway to phosphorylate at Ser112 and to inactivate the proapoptotic protein Bad, delaying arsenic trioxide (ATO)-induced apoptosis. Both in APL cell line NB4 and in APL primary blasts, the inhibition of extracellular signal-regulated kinases 1/2 (ERK1/2) and Bad phosphorylation by MEK1 inhibitors enhanced apoptosis in ATO-treated cells. We isolated an arsenic-resistant NB4 subline (NB4-As(R)), which showed stronger ERK1/2 activity (2.7-fold increase) and Bad phosphorylation (2.4-fold increase) compared to parental NB4 cells in response to ATO treatment. Upon ATO exposure, both NB4 and NB4-As(R) cell lines doubled protein levels of the death antagonist Bcl-xL, but the amount of free Bcl-xL that did not heterodimerize with Bad was 1.8-fold greater in NB4-As(R) than in the parental line. MEK1 inhibitors dephosphorylated Bad and inhibited the ATO-induced increase of Bcl-xL, overcoming ATO resistance in NB4-As(R). These results may provide a rationale to develop combined or sequential MEK1 inhibitors plus ATO therapy in this clinical setting.     

Arsenic trioxide (As(2)O(3)) induces apoptosis through activation of Bax in hematopoietic cells

This study explores the roles of Bax and other Bcl-2 family members play in arsenic trioxide (As(2)O(3))-induced apoptosis. We showed that As(2)O(3) treatment triggered Bax conformational change and subsequent translocation from cytosol to mitochondria to form various multimeric homo-oligomers in IM-9 cells. On the other hand, human leukemic Jurkat cells deficient in Bax showed dramatically reduced apoptosis in response to As(2)O(3). Stable overexpression of Bcl-2 in IM-9 cells (IM-9/Bcl-2) inhibited As(2)O(3)-mediated Bax activation and apoptosis, and this inhibition could be partially averted by cell-permeable Bid-Bcl-2 homology (BH)3 peptide. Meanwhile, Bax conformational change and oligomerization induced by As(2)O(3) were not inhibited by the pancaspase inhibitor z-VAD-fmk, although Bid cleavage could be completely abolished. Bax activation by As(2)O(3) seemed to require stress-induced intracellular reactive oxygen species (ROS), since the ROS scavengers (N-acetyl-L-cysteine and lipoic acid) could completely block the conformational change and translocation of Bax from cytosol to mitochondria. These data suggest that As(2)O(3) might exert the cell killing in part by inducing Bax activation through a Bcl-2-suppressible pathway in hematopoietic cells that is caspase independent and intracellular ROS regulated.     

Involvement of mitochondrial aggregation in arsenic trioxide (As2O3)-induced apoptosis in human glioblastoma cells

Arsenic trioxide (As(2)O(3)) is effective against acute promyelocytic leukemia and has potential as a novel treatment against malignant solid tumors. As(2)O(3) induces differentiation and inhibits growth. It also causes mitochondrial damage mediated by the production of reactive oxygen species (ROS) and the dissipation of mitochondrial transmembrane potential (DeltaPsi(m)), leading to apoptosis. Mitochondria might be the key target of antitumor activity by As(2)O(3); however, its mechanisms have not been completely elucidated. Using two human glioblastoma cell lines, A172 and T98G, we found that As(2)O(3) induced apoptosis in A172 cells but not in T98G cells. As(2)O(3)-induced ROS production was observed in both cell lines; however, the dissipation of DeltaPsi(m), Bax oligomerization and caspase activation occurred only in As(2)O(3)-sensitive A172 cells. To determine the mechanisms of As(2)O(3)-induced apoptosis after ROS generation, we examined the change of mitochondrial morphology. As we reported previously, mitochondrial aggregation occurs before cytochrome c release during apoptosis, thus playing a role in cell death progression. We observed mitochondrial aggregation in As(2)O(3)-sensitive A172 cells but not in T98G cells treated with As(2)O(3). Using laser scanning cytometry, we quantitatively confirmed the results, which indicate that mitochondrial aggregation plays an important role in regulating sensitivity to As(2)O(3)-induced apoptosis. We propose a sequential process involving ROS generation, mitochondrial aggregation, Bax oligomerization and DeltaPsi(m) dissipation, and caspase activation during As(2)O(3)-induced apoptosis.     

Use of arsenic trioxide (As2O3) in the treatment of patients with acute promyelocytic leukemia: the M. D. Anderson experience

BACKGROUND: Approximately 20-30% of patients with acute promyelocytic leukemia (APL) who are treated with all-trans retinoic acid (ATRA) and an anthracycline develop recurrent disease. It has been reported that arsenic trioxide (As(2)O(3)) is effective in this setting. The authors report the experience of The M. D. Anderson Cancer Center with As(2)O(3) in the treatment of patients with recurrent APL. METHODS: Twelve patients who developed recurrent APL after treatment with ATRA were included. Patients received intravenous As(2)O(3) 0.15 mg/kg per day until they achieved a complete remission (CR) or up to a maximum of 60 days. Their median age was 44 years (range, 26-72 years), and the median duration of first remission was 52 weeks (range, 23-292 weeks). RESULTS: All 12 patients achieved a CR. The median time to achieve CR was 52 days (range, 27-75 days). Seven of 10 evaluable patients achieved a molecular remission (i.e., polymerase chain reaction [PCR] analysis was negative for the gene encoding fusion of the nuclear receptor for retinoic acid to the PML gene at the time of CR; 70% of patients; 95% confidence interval, 0.35-0.93), and all other patients had negative PCR results after they received post-remission therapy. All patients received subsequent therapy: Four patients received As(2)O(3) alone, six patients received As(2)O(3) with other chemotherapeutic agents, and two patients received idarubicin plus ATRA without As(2)O(3). Eight patients continued in CR after a median follow-up of 24 months (range, 9-45 months). Side effects were mild, except for two patients who developed Grade 2 and 3 peripheral neuropathy, respectively; one of those patients required discontinuation of therapy. CONCLUSIONS: As(2)O(3) is effective and well tolerated therapy for patients with recurrent APL. Molecular remission may be achieved at the time of CR in the majority of patients, and remissions are durable.     

Arsenic trioxide-induced apoptosis and differentiation are associated respectively with mitochondrial transmembrane potential collapse and retinoic acid signaling pathways in acute promyelocytic leukemia.

Recent studies showed that arsenic trioxide (As2O3) could induce apoptosis and partial differentiation of leukemic promyelocytes. Here, we addressed the possible mechanisms underlying these two different effects. 1.0 microM As2O3-induced apoptosis was associated with condensation of the mitochondrial matrix, disruption of mitochondrial transmembrane potentials (DeltaPsim) and activation of caspase-3 in acute promyelocytic leukemia (APL) cells regardless of their sensitivity to all-trans retinoic acid (ATRA). All these effects were inhibited by dithiothreitol (DTT) and enhanced by buthionine sulfoximine (BSO). Furthermore, BSO could also render HL60 and U937 cells, which had the higher cellular catalase activity, sensitive to As2O3-induced apoptosis. Surprisingly, 1.0 microM As2O3 did not induce the DeltaPsim collapse and apoptosis, while 0.1 microM As2O3 induced partial differentiation of fresh BM cells from a de novo APL patient. In this study, we also showed that 0.2 mM DTT did not block low-dose As2O3-induced NB4 cell differentiation, and 0. 10.5 microM As2O3 did not induce differentiation of ATRA-resistant NB4-derived sublines, which were confirmed by cytomorphology, expression of CD11b, CD33 and CD14 as well as NBT reduction. Another interesting finding was that 0.10.5 microM As2O3 could also induce differentiation-related changes in ATRA-sensitive HL60 cells. However, the differentiation-inducing effect could not be seen in ATRA-resistant HL60 sublines with RARalpha mutation. Moreover, low-dose As2O3 and ATRA yielded similar gene expression profiles in APL cells. These results encouraged us to hypothesize that As2O3 induces APL cell differentiation through direct or indirect activation of retinoic acid receptor-related signaling pathway(s), while DeltaPsim collapse is the common mechanism of As2O3-induced apoptosis.     

Induction of differentiation of retinoic acid-resistant acute promyelocytic leukemia cells by the combination of all-trans retinoic acid and granulocyte colony-stimulating factor

An acute promyelocytic leukemia (APL) cell line with natural resistance to all-trans retinoic acid (ATRA), UF-1, was induced to differentiate into mature granulocyte when treated with the combination of ATRA and granulocyte colony-stimulating factor (G-CSF), while neither of them alone was capable of inducing the differentiation effectively. Continuous presence of both agents was required for the maximal differentiation-inductive effect. Neither proliferation arrest nor induction of apoptosis preceded the differentiation. Differentiated phenotype was accompanied by growth arrest, however, not by increased apoptosis. It was assumed that cellular events at the downstream of the signaling pathways of ATRA and G-CSF cooperatively played pivotal roles in the differentiation-induction.     

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.     

Arsenic trioxide-induced mitotic arrest and apoptosis in acute promyelocytic leukemia cells.

Arsenic trioxide (As(2)O(3)), an effective drug for the treatment of acute promyelocytic leukemia (APL), can induce apoptosis and partial differentiation in APL cells in vitro and in vivo. However, As(2)O(3) also induces apoptosis in cancer cells other than APL with complex mechanisms, which seem to be cell type dependent. In this study, we report that APL cells (NB4 cell line) are arrested at early mitotic phase before the collapse of mitochondrial transmembrane potential (Deltavarphi(m)) and apoptosis after treatment with pharmacological concentrations (1.0-2.0 micro M) of As(2)O(3). We have also made the following new discoveries: (1) 0.5 micro M As(2)O(3) that fails to induce apoptosis has no effects on cell cycle distribution. (2) With inhibition of As(2)O(3)-induced Deltavarphi(m) collapse and apoptosis, dithiothreitol also effectively inhibits As(2)O(3)-induced mitotic arrest, suggesting that both As(2)O(3)-induced apoptosis and mitotic arrest involve proteins with thiol groups. (3) 1.5 mM caffeine that relieves cells from G(2)/M arrest also inhibits As(2)O(3)-induced Deltavarphi(m) collapse and apoptosis, (4) 1.0 micro M As(2)O(3) increases the expression of both cyclin B(1) and hCDC20 whereas it inhibits Tyr15 phosphorylation of p34(cdc2). In conclusion, our results strongly support that there is a tight link between As(2)O(3)-induced apoptosis and mitotic arrest, the latter being one of common mechanisms for As(2)O(3)-induced apoptosis in cancer cells.     

Arsenic trioxide and low-dose cytarabine in older patients with untreated acute myeloid leukemia, excluding acute promyelocytic leukemia

BACKGROUND: Acute myeloid leukemia (AML) carries a dismal prognosis in older patients. In this study, the authors evaluated the safety and efficacy of arsenic trioxide combined with low-dose cytarabine in untreated patients aged >or=60 years with AML. METHODS: In a phase 1/2 design, arsenic trioxide was administered intravenously at a dose of 0.25 mg/kg on Days 1 through 5 and on Days 8 through 12, and low-dose cytarabine was given subcutaneously twice daily on Days 1 through 14 in escalating doses to a target of 10 mg/m(2) per dose. Of 64 patients who had pathologically confirmed AML, excluding patients with acute promyelocytic leukemia and using World Health Organization criteria, the median age was 71 years, 10 patients (16%) had treatment-related AML, 40 patients (63%) had an antecedent myelodysplastic syndrome or myeloproliferative disorder, and 35 patients (55%) had unfavorable cytogenetics. Thirty-four patients (53%) had an Eastern Cooperative Oncology Group performance status of 2 or 3. RESULTS: Complete remission was achieved in 21 of 61 patients (34%), including 15 of 50 patients (30%) who had secondary or treatment-related AML, 10 of 33 patients (30%) who had unfavorable cytogenetics, and 6 of 34 patients (18%) who had a poor baseline performance status. The mortality rate within the first 4 weeks was 8%. Neutropenic fever was observed in >80% of patients, and 41% of patients had bacteremia. Nonhematologic toxicity generally was mild and reversible and included fatigue, nausea, diarrhea, rash, peripheral edema, and elevated transaminases. There were no clinically significant cardiac arrhythmias. CONCLUSIONS: The addition of arsenic trioxide to low-dose cytarabine appeared to improve responses in elderly patients who had AML compared with either agent alone, and a randomized trial of the combination versus single-agent low-dose cytarabine is ongoing.     

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.     

Arsenic trioxide (trisenox) therapy for acute promyelocytic leukemia in the setting of hematopoietic stem cell transplantation

The relapse-free survival of patients with acute promyelocytic leukemia (APL) has significantly increased during the last decade. The introduction of all-trans retinoic acid (ATRA) doubled the survival of patients with this disease. However, despite ATRA and anthracycline-based chemotherapy, 12%-30% of patients will still relapse. Arsenic trioxide (ATO) has demonstrated efficacy and safety in patients with first and subsequent relapsed or refractory APL, regardless of the disease-free interval. Treatment of relapsed and refractory patients with this novel therapy produces complete remission in 87% of patients and molecular remission in 83%. Studies have documented the efficacy of autologous and allogeneic transplantation as salvage therapy in relapsed and refractory APL. The introduction of ATO into the treatment regimen for APL has stimulated discussion on its role in the transplantation setting. Investigators recently met to discuss the issue and make recommendations regarding ATO therapy in patients who are in their second or subsequent complete remission and are candidates for transplantation. This article describes the pivotal studies of this novel agent, discusses risk factor stratification for relapse in patients with APL, and proposes protocols for treatment incorporating ATO therapy. In addition, it describes scientific issues in ongoing and proposed clinical trials of ATO therapy for this disease.     

Leukocytosis and the retinoic acid syndrome in patients with acute promyelocytic leukemia treated with arsenic trioxide.

PURPOSE: Arsenic trioxide, like all-trans-retinoic acid (RA), induces differentiation of acute promyelocytic leukemia (APL) cells in vivo. Treatment of APL patients with all-trans RA is commonly associated with leukocytosis, and approximately 50% of patients develop the RA syndrome. We reviewed our clinical experience with arsenic trioxide to determine the incidence of these two phenomena. PATIENTS AND METHODS: Twenty-six patients with relapsed or refractory APL were treated with arsenic trioxide for remission induction at daily doses that ranged from 0.06 to 0.17 mg/kg. RESULTS: Twenty-three patients (88%) achieved complete remission. Leukocytosis was observed in 15 patients (58%). The median baseline leukocyte count for patients with leukocytosis was 3,900 cells/microL (range, 1,200 to 72,300 cells/microL), which was higher than that for patients who did not develop leukocytosis (2,100 cells/microL; range, 500 to 5,400 cells/microL; P =.01). No other cytotoxic therapy was administered, and the leukocytosis resolved in all cases. The RA syndrome was observed in eight patients (31%). Patients who developed leukocytosis were significantly more likely to develop the RA syndrome (P <.001), and no patient without a peak leukocyte count greater than 10,000 cells/microL developed the syndrome. Among the patients with leukocytosis, there was no observed relation between the leukocyte peak and the probability of developing the syndrome (P =.37). CONCLUSION: Induction therapy of APL with all-trans RA and arsenic trioxide is associated with leukocytosis and the RA syndrome. These clinical effects seem to be intrinsically related to the biologic responsiveness and the differentiation process induced by these new agents.     

全反式维甲酸联合三氧化二砷治疗急性早幼粒细胞白血病的疗效观察

背景与目的:三氧化二砷(arsenictrioxide,As2O3)治疗急性早幼粒细胞白血病作用机制与全反式维甲酸(all-transretinonicacid,ATRA)有所不同,前者主要是加速细胞的凋亡,后者是诱导细胞的分化。但两者均能抑制急性早幼粒细胞白血病组织因子(tissuefactor,TF)mRNA的转录,降低TF水平和促凝活性,从而改善患者的出凝血异常。本研究旨在探讨两药联合应用能否提高疗效及是否加重不良反应。方法:采用历史对照分组。2000年1月～2001年10月的22例急性早幼粒细胞白血病作为ATRA组,其中初治17例,复发5例;单用ATRA治疗,剂量为25mg/(m2·d),分两次口服,一个疗程最长不超过50天。2001年11月～2003年6月的急性早幼粒细胞白血病19例为联合治疗组,其中初治15例,复发4例,采用As2O3与ATRA联合治疗。治疗剂量为0.1%As2O3溶液10ml,加入5%葡萄糖溶液中静脉滴注,每天1次,持续4～6h,28天为一疗程,ATRA的剂量用法同前组。结果:ATRA治疗组22例中19例(初治16例,复发3例)获完全缓解,完全缓解率为86.4%。联合治疗组19例中17例(初治15例,复发2例)获完全缓解,缓解率为89.5%。病死率ATRA组为18.6%,共3例(1例初治,2例复发);联合治疗组为10.5%,共2例(均为复发者)。完全缓解率和病死率两组比较无统计学差异(P>0.05)。达到完全缓解的中位时间,AT     

三氧化二砷联合热疗抑制大鼠肝癌的体内实验研究

目的:研究热疗联合三氧化二砷(As2O3)对大鼠肝癌多药耐药性的影响,探讨其作为肝癌辅助治疗的可行性。方法:采用含有walker-256肿瘤细胞的大鼠腹水稀释后接种于大鼠腹股沟皮下,7天后取下瘤块并切成(1.0-2.0)mm3大小的小块,然后将小瘤块接种于50只大鼠的左肝叶上,10天后将大鼠分成4组,分别为对照组,热疗组,As2O3组及(热疗+As2O3)组,同时给予治疗,治疗结束后第2天处死大鼠。测量瘤重分析As2O3联合热疗的体内抑瘤作用;用H-E染色法观察肿瘤细胞病理象变化;免疫组化S-P法检测肿瘤组织内Pgp(P-glycoprotein,P-糖蛋白)的表达情况。结果:热疗组,As2O3组及(热疗+As2O3)组的平均瘤重均低于对照组(P<0.05);同时(热疗+As2O3)组与As2O3组及热疗组相比较,前者抑瘤率高于后两者(P<0.05)。在实验过程中,对照组荷瘤鼠活动减少,毛发稀疏,进食水少,而热疗组大鼠活动稍差,其余两组基本正常,饮食状况无明显改变,体重未见明显减轻。各组细胞均有坏死,大部分为凝固性坏死。对照组肿瘤细胞生长比较旺盛,坏死面积较小。其他三组坏死都比较彻底,并且呈现大片状坏死,尤以(热疗+As2O3)组明显,镜下呈一片荒凉景象,肿瘤细胞溶解,破碎,多见细胞核碎裂及核固缩表象。免疫组化法观察到多药耐药细胞呈片状分布,Pgp主要分布于耐药细胞的胞膜面,少数分布于胞浆当中;Pgp显色指数在对照组与各处理组之间差异显著(P<0.05),且(热疗+As2O3)组较其他处理组差异显著(P<0.05)。结论:As2O3和热疗在体内对大鼠肝癌均有明显的抑制作用,两者联合应用有协同作用,其作用机制可能与抑制多药耐药基因(MDR1)的表达有关。