Death by arsenic: implications of PML sumoylation

PML is a multifunctional protein that plays an important role in programmed cell death, albeit by mechanisms that remain unclear. In this issue of Cancer Cell, Hayakawa and Privalsky associate a MAP kinase pathway that mediates As(2)O(3)-induced PML phosphorylation with sumoylation and increased apoptotic activity of PML. Thus, specific MAP kinases may potentiate apoptosis in response to As(2)O(3), a compound that has dramatic activity against acute promyelocytic leukemia (APL) cells. This novel mechanism may have important implications for use of As(2)O(3) as a chemotherapeutic agent, especially in malignancies less sensitive to As(2)O(3) than APL.     

Leukemia-associated translocation products able to activate RAS modify PML and render cells sensitive to arsenic-induced apoptosis

Since the 19th century, arsenic (As2O3) has been used in the treatment of chronic myelogenous leukemia (CML) characterized by the t(9;22) translocation. As2O3 induces complete remissions in patients with acute promyelocytic leukemia. The response to As2O3 is genetically determined by the t(15;17)-or the t(9;22)-specific fusion proteins PML/RARalpha or BCR/ABL. The PML portion of PML/RARalpha is crucial for the sensitivity to As2O3. PML is nearly entirely contained in PML/RARalpha. PML is upregulated by oncogenic RAS in primary fibroblasts. The aberrant kinase activity of BCR/ABL leads to constitutive activation of RAS. Therefore, we hypothesized that BCR/ABL could increase sensitivity to As2O2-induced apoptosis by modifying PML expression. To disclose the mechanism of As2O3-induced apoptosis in PML/RARalpha- and BCR/ABL-expressing cells, we focused on the role of PML for As2O3-induced cell death. Here we report that (i) sensitivity to As2O3-induced apoptosis of U937 cells can be increased either by overexpression of PML, or by conditional expression of activated RAS; (ii) also the expression of the t(8;21)-related AML-1/ETO increased sensitivity to As2O3-induced apoptosis; (iii) both BCR/ABL and AML-1/ETO activated RAS and modified the PML expression pattern; (iv) the expression of either BCR/ABL or AML-1/ETO rendered U937 cells sensitive to interferon alpha-induced apoptosis. In summary, these data suggest a crucial role of factors able to upregulate PML for As2O2-induced cell death.     

A novel differentiation-inducing therapy for acute promyelocytic leukemia with a combination of arsenic trioxide and GM-CSF.

Arsenic trioxide (As2O3) effectively induces clinical remission via apoptosis in relapsed acute promyelocytic leukemia (APL). However, because this new anti-leukemic drug is also considered to be a poison, its possible adverse effects are a highly important issue related to its clinical use. We here investigated, both in vitro and in vivo, the effects of a combination of As2O3 and GM-CSF as a novel therapeutic approach for the treatment of APL. Treatment of both retinoic acid (RA)-sensitive and -resistant APL cell lines (NB4 and UF-1 cells, respectively), as well as primary APL cells with a combination of As2O3 and GM-CSF for 4 days resulted in inducing differentiation, but not apoptosis, to mature granulocytes. In addition, a combination of both agents induced degradation of the PML/RARalpha protein. GM-CSF was found to be associated with increased tyrosine phosphorylation of Jak2 kinase in both NB4 and UF-1 cells, and a specific inhibitor of Jak2, AG490, completely blocked the ability of GM-CSF to prevent apoptosis and induce differentiation of As2O3-treated UF-1 cells. In in vivo analysis, As2O3 induced differentiation of APL cells in a RA-resistant APL model of human GM-CSF-producing transgenic SCID mice that had a high level of human GM-CSF in their sera. In contrast, As2O3 alone diminished tumors in UF-1 cells transplanted into NOD/SCID mice via induction of apoptosis. In conclusion, a combination of As2O3 and GM-CSF appears to be a novel differentiation-inducing therapy in patients with APL, including relapsed or RA-resistant cases.     

BCR-ABL mediates arsenic trioxide-induced apoptosis independently of its aberrant kinase activity

In the prechemotherapy era arsenic derivatives were used for treatment of chronic myelogenous leukemia, a myeloproliferative disorder characterized by the t(9;22) translocation, the Philadelphia chromosome (Ph+). In acute promyelocytic leukemia response to arsenic trioxide (As2O3) has been shown to be genetically determined by the acute promyelocytic leukemia-specific t(15;17) translocation product PML/RARalpha. Hence, we reasoned that As2O3 might have a selective inhibitory effect on proliferation of BCR-ABL-expressing cells. Here, we report that: (a) As2O3 induced apoptosis in Ph+ but not in Ph- lymphoblasts; (b) enforced expression of BCR-ABL in U937 cells dramatically increased the sensitivity to As2O3; (c) the effect of As2O3 was independent of BCR-ABL kinase activity; and (d) As2O3 reduced proliferation of chronic myelogenous leukemia blasts but not of peripheral CD34+ progenitors. In summary, these data establish As2O3 as a tumor cell-specific agent, making its clinical application in Ph+ leukemia feasible.     

Apoptosis and growth inhibition in malignant lymphocytes after treatment with arsenic trioxide at clinically achievable concentrations.

BACKGROUND: Arsenic trioxide (As2O3) can induce clinical remission in patients with acute promyelocytic leukemia via induction of differentiation and programmed cell death (apoptosis). We investigated the effects of As2O3 on a panel of malignant lymphocytes to determine whether growth-inhibitory and apoptotic effects of As2O3 can be observed in these cells at clinically achievable concentrations. METHODS: Eight malignant lymphocytic cell lines and primary cultures of lymphocytic leukemia and lymphoma cells were treated with As2O3, with or without dithiothreitol (DTT) or buthionine sulfoximine (BSO) (an inhibitor of glutathione synthesis). Apoptosis was assessed by cell morphology, flow cytometry, annexin V protein level, and terminal deoxynucleotidyl transferase labeling of DNA fragments. Cellular proliferation was determined by 5-bromo-2'-deoxyuridine incorporation into DNA and flow cytometry and by use of a mitotic arrest assay. Mitochondrial transmembrane potential (delta psi(m)) was measured by means of rhodamine 123 staining and flow cytometry. Protein expression was assessed by western blot analysis or immunofluorescence. RESULTS: Therapeutic concentrations of As2O3 (1-2 microM) had dual effects on malignant lymphocytes: 1) inhibition of growth through adenosine triphosphate (ATP) depletion and prolongation of cell cycle time and 2) induction of apoptosis. As2O3-induced apoptosis was preceded by delta psi(m) collapse. DTT antagonized and BSO enhanced As2O3-induced ATP depletion, delta psi(m) collapse, and apoptosis. Caspase-3 activation, usually resulting from delta psi(m) collapse, was not always associated with As2O3-induced apoptosis. As2O3 induced PML (promyelocytic leukemia) protein degradation but did not modulate expression of cell cycle-related proteins, including c-myc, retinoblastoma protein, cyclin-dependent kinase 4, cyclin D1, and p53, or expression of differentiation-related antigens. CONCLUSIONS: Substantial growth inhibition and apoptosis without evidence of differentiation were induced in most malignant lymphocytic cells treated with 1-2 microM As2O3. As2O3 may prove useful in the treatment of malignant lymphoproliferative disorders.     

砷诱导肝母细胞瘤HepG2细胞凋亡机理研究

目的 研究三氧化二砷(As203)诱导肝母细胞瘤细胞系HePG2细胞凋亡的机理及其对细胞核基质相关蛋白——前髓白血病蛋白(PML)表达的影响。方法 Hep2细胞培养于MEM培养基中,用不同浓度As2O3分别处理24或96h。运用原位末端脱氧核苷转换酶(TdT)标记法(TUNEL)和DNA阶梯法检测细胞凋亡。以激光共振聚交显微镜及Western杂交观察PML的表达。结果 TUNEL阳性的凋亡细胞及DNA阶梯片段可见于As203处理组。用2μmol/L As203处理的Hep2细胞核基质蛋白内PML表达减弱。激光共振聚交图像显示,经2μmol/L As203处理的Hep2细胞核中,PML蛋白的表达明显减弱;用5μmol/L As203处理后,PML在Hep2细胞核中的微小点状表达几乎消失。结论 As203在体外可明显抑制Hep2细胞生长,其诱导HePG2细胞凋亡的作用依时间及剂量不同而异。As203可降解HepG2细胞核中PML蛋白,且PML的表达降低与细胞凋亡密切相关。核基质相关蛋白PML可能是As203作用的靶蛋白。     

The effects of arsenic trioxide (As2O3) on human megakaryocytic leukemia cell lines. With a comparison of its effects on other cell lineages

Arsenic trioxide (As2O3) has been demonstrated to be effective for the treatment of acute promyelocytic leukemia (APL) and to inhibit proliferation and produce apoptosis in the APL cell line NB4. The effect of this newly utilized chemotherapeutic agent on other lineages is currently under study to evaluate its efficacy for the treatment of other human malignancies and myeloproliferative syndromes. A recent study described the effects of As2O3 upon viability, proliferation, and induction of apoptosis in four different megakaryocytic leukemia cell lines. At pharmacological concentrations (0.5-2 microM) As2O3 selectively inhibits growth and causes apoptosis in the megakaryocytic leukemia cell lines HEL, Meg-01, UT7 and M07e. Pertinently, these concentrations of As2O3 resulted in identical changes in the characteristics of the APL cell line NB4, suggesting that As2O3 could produce its effects in both cellular lineages via a common mechanism of action. Various mechanisms have been proposed for the As2O3-induced changes in NB4 (including modulation of promyelocytic leukemia proteins (PML) and Bcl-2, modification of the glutathione redox system, caspase activation, and cell cycle arrest) and are currently under investigation in the megakaryocytic leukemia cell lines. Recent preliminary results indicate that As2O3 downregulates Bcl-2 expression and induces cell cycle arrest in megakaryocytic cell lines. The use of As2O3 for the treatment of malignant megakaryocytic disorders also has been considered. The in vitro effects of As2O3 on a chronic megakaryocytic proliferative disorder. i.e., Essential Thrombocythemia (ET), have been analyzed and megakaryocyte progenitors have shown an unexpectedly higher resistance to As2O3, in comparison to normal megakaryocyte colony-forming cells. The effects of As2O3 on ET and other megakaryocytic disorders need to be fully examined, in order to determine the clinical efficacy of As2O3 in the treatment of syndromes affecting the megakaryocytic lineage.     

In acute promyelocytic leukemia NB4 cells, the synthetic retinoid CD437 induces contemporaneously apoptosis, a caspase-3-mediated degradation of PML/RARalpha protein and the PML retargeting on PML-nuclear bodies.

CD437-induced apoptosis has been investigated in NB4, a human t(15;17) acute promyelocytic leukemia (APL) cell line, and in the retinoic acid (RA)-resistant NB4-R1 derivative subclone. Both NB4 and NB4-R1 cells underwent rapid apoptosis in response to low doses of CD437 (10(-7)M). This apoptosis did not require the activation of classical retinoid receptors and like arsenic (As)-induced apoptosis was preceded by the rapid activation of a caspase-3-like enzymatic activity as indicated by the increase of DEVD-pNA hydrolytic activity, by the processing of procaspase-3 protein and by the cleavage of poly(ADP-ribose) polymerase (PARP). Furthermore, it was demonstrated that the caspase-3-like proteolytic activity is responsible for the degradation of both the PML/RARalpha oncogenic protein and the normal RARalpha proteins. In CD437-treated cells, PML proteins were not degraded and PML relocalization on PMLNBs occurred in all the cells before death. CD437-induced apoptosis and receptor degradation were proteasome independent and not influenced either by inhibitors of protein tyrosine kinases (PTK), protein tyrosine phosphatases (PTPases) and serine proteases or by glutathione levels. Moreover, our data suggested that as for As2O3-induced apoptosis Bc12 modulation is not significant for CD437-induced apoptosis of NB4 cells. Since CD437 induces in vitro the rapid apoptosis of both RA-sensitive and -resistant APL cells, it could represent the first retinoid potentially able to eradicate in vivo malignant leukemia blasts.     

Arsenic trioxide and the growth of human T-cell leukemia virus type I infected T-cell lines

A novel therapeutic potential for acute promyelocytic leukemia using arsenic trioxide (As(2) O(3) ) has been reported. Recent in vitro studies demonstrated that As(2) O(3) effectively inhibits the growth of some cell lines derived from patients with malignant lymphoma, chronic lymphocytic leukemia and multiple myeloma. Adult T-cell leukemia (ATL) is an aggressive neoplasm of mature T-cell origin caused by human T-cell leukemia virus type-I (HTLV-I) the prognosis of which still remains very poor. A possible role of As(2) O(3) for the treatment of ATL is demonstrated from evidence that As(2) O(3) significantly inhibits the growth of HTLV-I infected T-cell lines and induces apoptosis in fresh ATL cells at clinically achievable concentration of the agent. The growth inhibition of As(2) O(3) treated HTLV-I infected T-cell lines was induced by both apoptosis and G(1) phase accumulation. Cleaved bcl-2 protein and an enhanced expression of bak protein in the cells were coincidentally observed during As(2) O(3) treatment. A broad spectrum caspase inhibitor, z-Val-Ala-DL-Asp-fluoromethylketone inhibited the apoptosis induced by As(2) O(3). Increased expression of p53, Cip1/p21 and Kip1/p27, and dephosphorylation of retinoblastoma protein (pRb) were detected in the As(2) O(3) treated cells. In conclusion, As(2) O(3) might become a new therapeutic tool in the treatment of ATL as As(2) O(3) induces apoptosis by destruction of the bcl-2 protein and enhancement of the bak protein production proceeding to activate caspases, and also induces G(1) phase accumulation by enhancement of p53, Cip1/p21, Kip1/p27 and dephosphorylation of pRb to HTLV-I infected T-cell lines.     

Arsenic enhances the activation of Stat1 by interferon gamma leading to synergistic expression of IRF-1

Arsenic trioxide (As2O3) can induce clinical remission in patients with acute promyelocytic leukemia (APL), including those who have relapsed after treatment with all-trans-retinoic acid (RA). In vitro studies with the APL-derived NB4 cell line showed that As2O3 exerts a dose-dependent dual effect, which induces apoptosis at 1 microM, whereas at a lower concentration of 0.1 microM, a partial differentiation of APL is observed. In non-APL cells, interferon (IFN) alpha and 1 microM As2O3 act synergistically to induce apoptosis. In this report, we show that in NB4 cells and in two RA-resistant NB4-derived cell lines, NB4-R1 and NB4-R2, IFNalpha or IFNgamma combined with 0.1 microM As2O3 lead to an increased maturation effect. Moreover, IFNgamma alone is able to differentiate RA-sensitive and -resistant cells with a higher maturation effect on NB4-R2 cells. In contrast, all these cells underwent apoptosis in the presence of the cytokine and a higher concentration of As2O3. IFNgamma boosted As2O3-induced apoptosis in APL cells as tested by TUNEL, Annexin V staining and activation of caspase 3. As2O3 differently altered IFN-induced gene products; it downregulated PML/RARalpha and PML, did not alter PKR and Stat1, and upregulated interferon regulatory family (IRF)-1. Synergism by IFNgamma and arsenic on IRF-1 expression is mediated by a composite element in the IRF-1 promoter that includes an IFNgamma-activation site (GAS) overlapped by a nonconsensus site for nuclear factor kappa B (NFkappaB). Arsenic has no effect on NFkappaB, whereas it enhances the activation of Stat1 by IFNgamma in NB4 cells leading to an increase in IRF-1 expression.     

Arsenic trioxide therapy for relapsed acute promyelocytic leukemia: an useful salvage therapy

Arsenic trioxide (As2O3) was recently identified as a very potent agent against acute promyelocytic leukemia (APL). Intravenous infusion of 10 mg As2O3 daily for one to two months can induce significant complete remission (CR) of APL, and there is no cross drug-resistance between As2O3 and other antileukemic agents, including all-trans retinoic acid (ATRA). The CR rate of relapsed and/or refractory APL patients who received As2O3 treatment ranged from 52.3% to 93.3%. The median duration to CR ranged from 38 to 51 days, with accumulative As2O3 dosage of 340-430 mg. Although most adverse reactions of As2O3 treatment were tolerable, certain infrequent but severe toxicities related to As2O3 were observed, including renal failure, hepatic damage, cardiac arrhythmia and chronic neuromuscular degeneration, which should be monitored carefully. As2O3 can induce partial differentiation and subsequent apoptosis of APL cells through degradation of wild type PML and PML/RAR alpha chimeric proteins and possible anti-mitochondrial effects. Like the treatment of ATRA in APL, early relapses from As2O3 treatment within a few months were not infrequently seen, indicating that rapid emerging resistance to As2O3 can occur. Nevertheless, the PML/RAR alpha fusion protein was reported to disappear in some APL patients who received As2O3, and who might earn long-survival. However, the follow-up is still too short to draw the conclusion. Intriguingly, it has been shown that As2O3 can also induce apoptosis of other non-APL tumor cells with clinical achievable concentrations. However, the detailed molecular mechanisms are not yet fully understood. Further studies regarding to the pharmacological characters, clinical efficacies, toxicities, apoptogenic mechanisms, and spectrum of anti-tumor activity of As2O3 are warranted.     

The SUMO E3-ligase PIAS1 regulates the tumor suppressor PML and its oncogenic counterpart PML-RARA

The ubiquitin-like SUMO proteins covalently modify protein substrates and regulate their functional properties. In a broad spectrum of cancers, the tumor suppressor PML undergoes ubiquitin-mediated degradation primed by CK2 phosphorylation. Here, we report that the SUMO E3-ligase inhibitor PIAS1 regulates oncogenic signaling through its ability to sumoylate PML and the PML-RARA oncoprotein of acute promyelocytic leukemia (APL). PIAS1-mediated SUMOylation of PML promoted CK2 interaction and ubiquitin/proteasome-mediated degradation of PML, attenuating its tumor suppressor functions. In addition, PIAS1-mediated SUMOylation of PML-RARA was essential for induction of its degradation by arsenic trioxide, an effective APL treatment. Moreover, PIAS1 suppression abrogated the ability of arsenic trioxide to trigger apoptosis in APL cells. Lastly, PIAS1 was also essential for PML degradation in non-small cell lung carcinoma (NSCLC) cells, and PML and PIAS1 were inversely correlated in NSCLC cell lines and primary specimens. Together, our findings reveal novel roles for PIAS1 and the SUMOylation machinery in regulating oncogenic networks and the response to leukemia therapy.     

Pml and TAp73 interacting at nuclear body mediate imatinib‐induced p53‐independent apoptosis of chronic myeloid leukemia cells

Bcr‐abl signals for leukemogenesis of chronic myeloid leukemia (CML) and activates ras. Since the function of promyelocytic leukemia protein (pml) is provoked by ras to promote apoptosis and senescence in untransformed cells, the function is probably masked in CML. Imatinib specifically inhibits bcr‐abl and induces apoptosis of CML cells. As reported previously, p53^wild CML was more resistant to imatinib than that lacking p53. Here, we searched for an imatinib‐induced p53 independent proapoptotic mechanism. We found imatinib up‐regulated phosphorylation of p38 mitogen‐activated protein kinase (MAPK), checkpoint kinase 2 (chk2) and transactivation‐competent (TA) p73; expression of pml and bax; formation of PML‐nuclear body (NB); and co‐localization of TAp73/PML‐NB in p53‐nonfunctioning K562 and p53^mutant Meg‐01 CML cells, but not in BCR‐ABL^‐ HL60 cells. In K562 cells, with short interfering RNAs (siRNAs), knockdown of pml led to dephosphorylation of TAp73. Knockdown of either pml or TAp73 abolished the imatinib‐induced apoptosis. Inhibition of p38 MAPK with SB203580 led to dephosphorylation of TAp73, abolishment of TAp73/PML‐NB co‐localization, and the subsequent apoptosis. Conversely, interferon α‐2a (IFNα), which increased phosphrylated TAp73 and TAp73/PML‐NB co‐localization, increased additively apoptosis with imatinib. The imatinib‐induced TAp73/PML‐NB co‐localization was accompanied by co‐immpunoprecipitation of TAp73 with pml. The imatinib‐induced co‐localization was also found in primary CML cells from 3 of 6 patients, including 2 with p53^mutant and one with p53^wild. A novel p53‐independent proapoptotic mechanism using p38 MAPK /pml/TAp73 axis with a step processing at PML‐NB and probably with chk2 and bax being involved is hereby evident in some imatinib‐treated CML cells. © 2009 UICC     

Treatment of acute promyelocytic leukemia with arsenic trioxide: clinical and basic studies

Arsenic trioxide (As2O3) has recently been identified as an effective drug in the treatment of newly diagnosed and relapsed acute promyelocytic leukemia (APL) without cross-resistance to all-trans retinoic acid and achieved complete remission rates of 80-90% according to most reports. With intravenous infusion at a dose of 0.08-0.16 mg/kg daily, a course of 28-42 days is required to induce remission. As2O3 in combination with chemotherapy as postremission therapy results in longer survival than arsenic alone. In vitro, As2O3 exerts dose-dependent dual effect; triggering apoptosis at relatively high concentration (0.5-2.0 micromol/l), which is associated with the disruption of mitochondrial transmembrane potentials, while inducing partial differentiation at low concentration (0.1-0.5 micromol/l), which might be related to retinoic acid signaling pathway. Importantly, at both concentrations, As2O3 can degrade PML (promyelocytic leukemia) -RAR alpha (retinoic acid receptor), an oncoprotein that has a central role in leukemogenesis.     

Arsenic trioxide induces apoptosis in HTLV-I infected T-cell lines and fresh adult T-cell leukemia cells through CD95 or tumor necrosis factor alpha receptor independent caspase activation

Arsenic trioxide (As2O3) has been reported to induce apoptosis in human T-cell leukemia virus type-I (HTLV-I) infected T-cell lines and fresh adult T-cell leukemia (ATL) cells and to induce G1 phase accumulation in HTLV-I infected T-cell lines. The present study aimed to clarify the pathway of As2O3-induced apoptosis in HTLV-I infected T-cell lines, MT-1 and MT-2, and fresh ATL cells separated from peripheral blood of patients with acute or chronic type ATL. Cells were treated up to 72 h at clinically tolerable concentrations of As2O3 (1-2 micromol/l) shown to be safe in patients with acute promyelocytic leukemia (APL). Activation of caspases 3, 8, and 9, loss of mitochondrial transmembrane potential and cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP) were observed during As2O3 treatment. Furthermore, prior exposure to a broad-spectrum caspase inhibitor blocked As2O3-induced apoptosis but not G1 phase accumulation. While pre-treatment with a CD95 receptor-blocking antibody (Ab) or a TNF-alpha neutralizing Ab did not show such inhibitions in these cells. In conclusion, As2O3 induces apoptosis in HTLV-I infected T-cell lines and fresh ATL cells through CD95 or TNF-alpha receptor independent caspase activation.     

Sustained activation of c-jun-terminal kinase (JNK) is closely related to arsenic trioxide-induced apoptosis in an acute myeloid leukemia (M2)-derived cell line, NKM-1.

High concentrations (greater than 5 microM) of arsenic trioxide (As(2)O(3)) have been reported to be able to induce apoptosis in several malignant cells. We explored cell lines in which apoptosis was induced with a therapeutic concentration (1-2 microM) of As(2)O(3), and found that 1 microM of As(2)O(3) induced apoptosis in the NKM-1 cell line, which was established from a patient with acute myeloid leukemia (M2). Apoptosis induced by 1 microM of As(2)O(3) in NKM-1 cells was accompanied by an increased cellular content of H(2)O(2), a decreased mitochondrial membrane potential (Deltapsim), and activation of caspase-3. C-Jun-terminal kinase (JNK) was activated only in NKM-1 cells and arsenic-sensitive NB4 cells, but not in arsenic-insensitive HL-60 cells. Activation of JNK in NKM-1 was sustained from 6 to 24 h after As(2)O(3) treatment, and preceded changes in cellular H(2)O(2), Deltapsim, and caspase-3 activation. Moreover, addition of a JNK inhibitor reduced the percentage of apoptotic cells after the As(2)O(3) treatment. Taken together, in the M2 cell line NKM-1, 1 microM of As(2)O(3) induced sustained activation of JNK and apoptosis. This finding may provide a basis to select a subgroup other than acute promyelocytic leukemia, which can benefit from As(2)O(3) treatment.     

Glutathione depletion overcomes resistance to arsenic trioxide in arsenic-resistant cell lines.

Arsenic trioxide (As(2)O(3)) is an effective treatment for acute promyelocytic leukemia (APL), but is less effective against other leukemias. Although the response of APL cells to As(2)O(3) has been linked to degradation of the PML/RARalpha fusion oncoprotein, there is evidence that PML/RARalpha expression is not the only mediator of arsenic sensitivity. Indeed, we found that exogenous expression of PML/RARalpha did not sensitize a non-APL leukemic line to As(2)O(3). To evaluate possible other determinants of sensitivity of leukemic cells to As(2)O(3), we derived two arsenic-resistant NB4 subclones. Despite being approximately 10-fold more resistant to arsenic than their parental cell line, PML/RARalpha protein was still degraded by As(2)O(3) in these cells, providing further evidence that loss of expression of the oncoprotein does not confer arsenic sensitivity. Both arsenic-resistant clones contained high glutathione (GSH) levels, however, and we found that GSH depletion coupled with As(2)O(3) treatment dramatically inhibited their growth. Annexin V-staining and TUNEL analysis confirmed a synergistic induction of apoptosis. In addition, these cells failed to accumulate ROS in response to arsenic treatment, in contrast to their arsenic-sensitive parental cells, unless cotreated with buthionine sulfoximine. While other malignant cells did not show a good correlation between arsenic sensitivity and GSH content, GSH depletion nevertheless sensitized all cell lines examined, regardless of their initial response to arsenic alone. These findings suggest that PML/RARalpha expression is not a determinant of arsenic sensitivity, and further support the coupling of GSH depletion and arsenic treatment as a novel treatment for human malignancies that are unresponsive to arsenic alone.     

N-(beta-Elemene-13-yl)tryptophan methyl ester induces apoptosis in human leukemia cells and synergizes with arsenic trioxide through a hydrogen peroxide dependent pathway

beta-Elemene is an active component of herb medicine Curcuma Wenyujin and N-(beta-elemene-13-yl)tryptophan methyl ester (ETME) was synthesized for increasing its antitumor activity. ETME induced apoptosis in human leukemia HL-60 and NB4 cells at concentrations less than 40muM. The apoptosis induction ability of ETME was associated with the production of hydrogen peroxide (H(2)O(2)), the decrease of mitochondrial membrane potential, and the activation of caspase-3 that was blocked by catalase. ETME in combination with arsenic trioxide (As(2)O(3)), an agent used to treat acute promyelocytic leukemia, synergistically induced apoptosis in both cell lines by enhanced production of H(2)O(2). These data suggest that ETME induces apoptosis and synergizes with As(2)O(3) in leukemia cells through a H(2)O(2)-dependent pathway.     

Role of the p38 mitogen-activated protein kinase pathway in the generation of arsenic trioxide-dependent cellular responses

Arsenic trioxide (As(2)O(3)) induces differentiation and apoptosis of leukemic cells in vitro and in vivo, but the precise mechanisms that mediate such effects are not known. In the present study, we provide evidence that the kinases MAPK kinase 3 (Mkk3) and Mkk6 are activated during treatment of leukemic cell lines with As(2)O(3) to regulate downstream engagement of the p38 mitogen-activated protein kinase. Using cells with targeted disruption of both the Mkk3 and Mkk6 genes, we show that As(2)O(3)-dependent activation of p38 is defective in the absence of Mkk3 and Mkk6, establishing that these kinases are essential for As(2)O(3)-dependent engagement of the p38 pathway. Pharmacologic inhibition of p38 enhances As(2)O(3)-dependent activation of the c-jun NH(2)-terminal kinase (JNK) and subsequent induction of apoptosis of chronic myelogenous leukemia (CML)- or acute promyelocytic leukemia (APL)-derived cell lines. In addition, in APL blasts, inhibition of p38 enhances myeloid cell differentiation in response to As(2)O(3), as well as suppression of Bcl-2 expression and loss of mitochondrial membrane potential. Similarly, induction of As(2)O(3)-dependent apoptosis is enhanced in mouse embryonic fibroblasts (MEF) with targeted disruption of both the Mkk3 and Mkk6 genes, establishing a key role for this pathway in the regulation of As(2)O(3)-induced apoptosis. In other studies, we show that the small-molecule p38 inhibitors SD-282 and SCIO-469 potentiate As(2)O(3)-mediated suppression of myeloid leukemic progenitor growth from CML patients, indicating a critical regulatory role for p38 in the induction of antileukemic responses. Altogether, our data indicate that the Mkk3/6-p38 signaling cascade is activated in a negative regulatory feedback manner to control induction of As(2)O(3)-mediated antileukemic effects.