Apoptosis In Acute Promyelocytic Leukemia Research Articles

Retraction Note to: Icariin induces apoptosis in acute promyelocytic leukemia by targeting PIM1.

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1007/s43440-020-00185-0 .

CUDC-101 Overcomes Arsenic Trioxide Resistance Via Caspase-Dependent PML-Rarα Degradation in Acute Promyelocytic Leukemia

While arsenic trioxide (ATO) treatment has transformed acute promyelocytic leukemia (APL) from the most fatal to the most curable hematological cancer, many high-risk APL patients who fail to achieve a complete molecular remission or relapse become resistant to ATO. Studies have suggested that HDAC inhibitors (HDACi) have been targeted as promising agents for the treatment of several cancers. In this study, a novel pan-HDACi 7-(4-(3-Ethynylphenylamino)-7- methoxyquinazolin-6-yloxy)-N-hydroxyheptanamide (CUDC-101) was used to tentatively to treat hematologic disease for the first time. The CCK-8 assay demonstrate that CUDC-101 exerts much stronger anti-proliferative effects than ATO in APL and ATO-resistant APL cell lines. Previous experiments on cell proliferation inhibition prompted investigation of whether CUDC-101 could induce apoptosis in APL and ATO-resistant APL cells. Then the results of apoptosis assay strongly support CUDC-101 as an effective apoptosis inducer that induced apoptotic cell death in both APL and ATO-resistant APL cell lines, more effectively than ATO. Our data also demonstrate that CUDC-101 is a novel drug that has a negligible cytotoxic effect on non-cancerous cell lines including normal CD34+ cells and BMSCs from APL patients. Mechanistically, we conclude that CUDC-101 directly induce hyperacetylation of histone 3, which subsequently lead to the activation of caspase 3, the activated-caspase 3 cleaves the PML-RARα fusion protein subsequently facilitates APL and ATO-resistant APL cells undergoing apoptosis. The precise mechanism of CUDC-101-induced degradation of PML-RARα remains unclear. Yet the apoptosis inducing effect of CUDC-101 offers a potent therapeutic advantage of CUDC-101 and its role in overcoming APL warrants further validation in patient-derived samples in future studies. Finally, using a NB4 xenograft mouse model, we demonstrated that CUDC-101 significantly represses leukemia development in vivo compared with ATO. These findings suggest that the CUDC-101 is efficient in inhibiting tumor growth in vivo and performs better than ATO. To the best of our knowledge, this is the first report that CUDC-101 functions as HDACi and has potential therapeutic value for the treatment of both APL and relapsed APL with ATO resistance in vitro and in vivo. In conclusion, these results suggested that CUDC-101 can serve as a potential candidate drug for APL, particularly for ATO-resistant APL. Disclosures No relevant conflicts of interest to declare.

Metformintreatment Overcomes ATRA-Resistance in Acute Promyelocytic Leukemia and Increases FOXO3A Expression

Introduction:FOXO3A is a transcription factor shown to be involved in all-trans retinoic acid (ATRA)-induced granulocytic differentiation and apoptosis in acute promyelocytic leukemia (APL). Its biological activity may be significantly enhanced upon metformin, raising the possibility that ATRA and Metformin may act synergistically; which could be useful to overcome ATRA resistance. Despite progress in APL treatment, approximately 10-15% of patients will relapse after treatment with ATRA and anthracyclines and frequently present with ATRA resistance. Relapsed patients respond well to arsenic trioxide (ATO) treatment, but the cost of ATO is a significant barrier in many countries. Aims: Here, we investigated the effects of in vitro treatment with ATRA plus metformin in APL cell lines and primary blasts, and quantified FOXO3A expression and correlated its levels with treatment outcome in a cohort of patients enrolled in the International Consortium on Acute Leukemia (ICAPL2006) study. Finally, we evaluated the effect of induced overexpression of FOXO3A gene in regards to cell viability and proliferation. Methods: Primary leukemic blasts from hCG-PMLRARα transgenic mice (TM; n=4) and APL patients (age, 25-47y; n=4) were treated with metformin alone (5mM/ 10mM) or in combination with ATRA (1µM) and evaluated for cell viability. In addition, 106 patients (age, 18-82y) with newly diagnosed APL enrolled in the ICAPL2006 study were included. As controls, eight samples of bone marrow mononuclear cells (BMMC) from healthy volunteers (age, 18-60y) were analyzed. To validate our data, FOXO3A transcript levels from an independent cohort was used (31 patients from Amazonia!, Probe n. 204131_s_at, and five normal BMMC samples included in the same databank). NB4/NB4R2 (ATRA-resistant) cell lines were transduced with FOXO3A or empty vector (control). After synchronization using double thymidine block, transduced cells were submitted to proliferation and cell cycle assays. For dose-response curves, cells were treated with graded concentrations of ATRA, ATO and metformin. For the apoptosis analysis, cells were treated with ATRA (1µM), metformin (5mM) and combination for 24, 48 and 72 hours. The granulocytic differentiation in response to ATRA treatment was evaluated based on the CD11b surface levels. Results: In primary cells (from TM/APL patients) a 2-fold reduction in viable cells was observed after metformin and metformin plus ATRA treatment (P Disclosures Tallman:BioSight: Other: Advisory board; Daiichi-Sankyo: Other: Advisory board; ADC Therapeutics: Research Funding; AROG: Research Funding; AbbVie: Research Funding; Cellerant: Research Funding; Orsenix: Other: Advisory board. Ganser:Novartis: Membership on an entity9s Board of Directors or advisory committees. Lowenberg:Editorial Board "European Oncology & Haematology": Membership on an entity9s Board of Directors or advisory committees; Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity9s Board of Directors or advisory committees; Supervisory Board, National Comprehensive Cancer Center (IKNL), Netherlands: Membership on an entity9s Board of Directors or advisory committees; Clear Creek Bio Ltd: Consultancy, Honoraria; international Scientific Advisory Board, Institute Gustave Roussy, Paris: Membership on an entity9s Board of Directors or advisory committees; Chairman Scientific Committee and Member Executive Committee, European School of Hematology (ESH, Paris, France): Membership on an entity9s Board of Directors or advisory committees; Elected member, Royal Academy of Sciences and Arts, The Netherlands: Membership on an entity9s Board of Directors or advisory committees; Editorial Board "The Netherlands Journal of Medicine": Membership on an entity9s Board of Directors or advisory committees; Editorial Board "International Journal of Hematology": Membership on an entity9s Board of Directors or advisory committees; "Up-to-Date", section editor leukemia: Membership on an entity9s Board of Directors or advisory committees; Agios Pharmaceuticals: Membership on an entity9s Board of Directors or advisory committees; Astex: Consultancy; Chairman, Leukemia Cooperative Trial Group HOVON (Netherlands): Membership on an entity9s Board of Directors or advisory committees; Celgene: Membership on an entity9s Board of Directors or advisory committees.

Icariin induces apoptosis in acute promyelocytic leukemia by targeting PIM1.

Acute promyelocytic leukemia (APL) is one type of acute myeloid leukemia (AML) featured by abnormal, heavily granulated promyelocytes. This study aimed to investigate the antitumor activity of icariin in APL cells. APL cell lines (HL-60 and NB4) were used to investigate the effect of icariin in vitro. Cell viability was determined by WST-8 proliferation assay, while cell apoptosis was assessed by flow cytometry. The mRNA and protein expression was determined by quantitative real-time polymerase chain reaction and Western blot, respectively. Moreover, small interfering RNA (siRNA) and overexpressing plasmid were used to manipulate the expression of PIM family kinase 1 (PIM1) to examine the role of PIM1 in icariin-induced apoptosis in APL cells. Icariin could significantly suppress cell growth and induce apoptosis in both model APL cell lines (HL-60 and NB4). It repressed the expression of PIM1 at the molecular level, which was responsible for the antitumor effect of icariin in APL cells. The ectopic overexpression of PIM1 significantly abrogated the inducing effect of icariin on apoptosis. In contrast, the knockdown of PIM1 by siRNA enhanced the antitumor effect of icariin in APL cells. Moreover, the findings indicated that icariin repressed the expression of PIM1 through generating reactive oxygen species and hence modulating the Janus kinase 2(JAK2)/Signal transducer and activator of transcription 3/5 (STAT3/5) signaling pathway. Icariin potently inhibits the cell growth of APL in vitro through inducing caspase-dependent apoptosis. Hence, it can be considered as a promising candidate therapeutic agent for treating APL, although further studies including clinical trials are warranted.

Molecular mechanisms of cisplatin cytotoxicity in acute promyelocytic leukemia cells.

Cis-diamminedichloroplatinum (II) (cisplatin) is a widely used anti-tumor drug for the treatment of a broad range of human malignancies with successful therapeutic outcomes for head and neck, ovarian, and testicular cancers. It has been found to inhibit cell cycle progression and to induce oxidative stress and apoptosis in acute promyelocytic leukemia (APL) cells. However, its molecular mechanisms of cytotoxic action are poorly understood. We hypothesized that cisplatin induces cytotoxicity through DNA adduct formation, oxidative stress, transcriptional factors (p53 and AP-1), cell cycle regulation, stress signaling and apoptosis in APL cells. We used the APL cell line as a model, and applied a variety of molecular tools to elucidate the cytotoxic mode of action of cisplatin. We found that cisplatin inhibited cell proliferation by a cytotoxicity, characterized by DNA damage and modulation of oxidative stress. Cisplatin also activated p53 and phosphorylated activator protein (AP-1) component, c-Jun at serine (63, 73) residue simultaneously leading to cell cycle arrest through stimulation of p21 and down regulation of cyclins and cyclin dependent kinases in APL cell lines. It strongly activated the intrinsic pathway of apoptosis through alteration of the mitochondrial membrane potential, release of cytochrome C, and up-regulation of caspase 3 activity. It also down regulated the p38MAPK pathway. Overall, this study highlights the molecular mechanisms that underline cisplatin toxicity to APL cells, and provides insights into selection of novel targets and/or design of therapeutic agents to treat APL.

Downregulation of thymidylate synthase and E2F1 by arsenic trioxide in mesothelioma.

Malignant pleural mesothelioma is a global health issue. Arsenic trioxide (ATO) has been shown to suppress thymidylate synthase (TYMS) in lung adenocarcinoma and colorectal cancer, and induce apoptosis in acute promyelocytic leukemia. With TYMS as a putative therapeutic target, the effect of ATO in mesothelioma was therefore studied. A panel of 5 mesothelioma cell lines was used to study the effect of ATO on cell viability, protein expression, mRNA expression and TYMS activity by MTT assay, western blot, qPCR and tritium-release assay, respectively. The knockdown of TYMS and E2F1 was performed with a specific siRNA. Phosphatidylserine externalization and mitochondrial membrane depolarization were measured by AnnexinV and JC-1 staining respectively. The invivo effect of ATO was studied using a nude mouse xenograft model. Application of ATO demonstrated anticancer effects in the cell line model with clinically achievable concentrations. Downregulation of TYMS protein (except H226 cells and 1.25µM ATO in H2052 cells) and mRNA expression (H28 cells), pRB1 (H28 cells) and E2F1 and TYMS activity (except H226 cells) were also evident. E2F1 knockdown decreased cell viability more significantly than TYMS knockdown. In general, thymidine kinase 1, ribonucleotide reductase M1, c-myc and skp2 were downregulated by ATO. p-c-Jun was downregulated in H28 cells while upregulated in 211H cells. Phosphatidylserine externalization, mitochondrial membrane depolarization, downregulation of Bcl-2 and Bcl-xL, and upregulation of Bak and cleaved caspase-3 were observed. In the H226 xenograft model, the relative tumor growth was aborted, and E2F1 was downregulated while cleaved caspase-3 was elevated and localized to the nucleus in the ATO treatment group. ATO has potent antiproliferative and cytotoxic effects in mesothelioma invitro and invivo, partially mediated through E2F1 targeting (less effect through TYMS targeting). There is sound scientific evidence to support the clinical application of ATO in treatment of mesothelioma.

Mechanisms of resistance to apoptosis in the human acute promyelocytic leukemia cell line NB4.

Current frontline therapies have improved overall survival in acute promyelocytic leukemia (APL) patients to exceptional rates; however, relapse is still a problem among high-risk and old patients. Therefore, the development of better and safer therapies continues to be a goal in the treatment of this disease. In the present work, we examined three different pathways that hinder cell death in the APL cell line NB4, shedding light on the mechanisms that underlie resistance to apoptosis in these cells and that might help provide them with a proliferative advantage. We found that the proteasome inhibitor MG-132 specifically induces in NB4 cells an Nrf2-mediated antioxidant response which counteracts mitochondria-dependent apoptosis induced by the lipophilic cation dequalinium. More importantly, we also demonstrated that high basal autophagy levels and the gain-of-function of mutant p53 are intrinsic mechanisms of resistance to apoptosis in this cell line. According to our results, the pharmacological inhibition of autophagy and p53 mutants are useful tools to explore resistance to apoptosis in APL and other types of cancer and could be the bases of new therapeutic approaches that improve the efficiency and allow dose reduction of the current treatments.

MicroRNAs Contribute to Promyelocyte Apoptosis in As2O3-Treated APL Cells

Background: Arsenic trioxide (As₂O₃), an ancient drug used in traditional Chinese medicine, has substantial anticancer activities, especially in the treatment of patients suffering from acute promyelocytic leukemia (APL); however the underlying mechanisms are not well understood. Methods: MTT assay was used to detect the cell viability. Flow Cytometry analysis and caspase-3 activity assay were used to measure apoptosis of APL cells. Caspase-3 and Bax levels were analyzed by western blot and let-7d and miR-766 levels were determined by real-time RT-PCR. Results: As₂O₃ significantly inhibited cell viability and induced apoptosis in APL cells. Several microRNAs, including let-7d and miR-766, were dysregulated in APL cells treated with As₂O₃. The expression of caspase-3 and Bax, which are targets of let-7d and miR-766, respectively, were up-regulated in As₂O₃ treated cells. Transfection of let-7d and miR-766 into NB4 cells decreased the expression of caspase-3 and Bax, respectively. Correspondingly, transfection of these microRNAs increased NB4 cell viability. As₂O₃ induced degradation of promyelocytic leukemia (PML), and then induced the down-regulation of both let-7d and miR-766 in NB4 cells. Conclusions: We construct a dysregulated microRNA network involved in As₂O₃-induced apoptosis in APL. Targeting this network may be a new strategy for the prevention of side effects associated with APL treatment with As₂O₃.

Mechanism of Regulating Fas Signaling by Promyelocytic Leukemia Protein (PML) and Its Dominant-Negative Mutant PMLRARα

Abstract 1327 Objective:Chemotherapies and irradiation depend on an intact Fas signaling system to eradicate cancer cells. Defective Fas signaling is an important cause of acquired resistance to cancer therapy. If we were able to restore Fas apoptosis or sensitize cancer cells to Fas-mediated apoptosis, we could improve the efficacy of many current cancer therapies. To elucidate defects of Fas signaling in cancer cells, we sought to identify potential modulators of Fas selectively expressed in cancers cells and target them to sensitize cancer cells to Fas-mediated apoptosis as a component of chemotherapy. Methods:Liquid chromatography tandem mass spectroscopy was used to identify Fas-associated proteins. Co-immunoprecipitation (co-IP) and Western Blot (WB) were used to detect/confirm interactions of PML and PMLRARα with Fas, and components of death-inducing signaling complex (DISC) FADD, c-FLIP, and caspase-8 cleavage in tissues from PML wild-type (WT) and knock-out (KO) mice and acute promyelocytic leukemia (APL) cells. Deletional mutagenesis was used to map protein interacting domains. PML shRNA lentivirus and As2O3 were used to downregulate PML and PMLRARα. Flow cytometry analysis of propidium iodide- and Annexin-V-stained cells was used to detect apoptosis in response to Fas stimulation. Mice transfected with PMLRARα were monitored for survival after a lethal challenge with agonistic Fas antibody Jo2 and tissues were analyzed for apoptosis by staining for cleaved caspase-3 and TUNEL. Results:The promyelocytic leukemia protein (PML) was identified as a Fas-binding protein by mass spectroscopy analysis. Using co-IP/WB analysis of tissues from PML WT and KO mice, we found PML interaction with Fas and FADD in PML WT MEF cells and liver cells but absent in KO MEF and liver cells; PML-Fas complexes were exclusively present in the membrane/cytoplasmic extracts but not in the nuclear extracts. The B-box domain of PML was found to be required for Fas binding. Knockdown of PML was associated with suppressed rates of Fas-mediated apoptosis compared to non-targeted knockdown cells; PML KO cells reconstituted with cytoplasmic PML were sensitized to Fas apoptosis. Furthermore, we found that liver cells from PML KO mouse showed impaired assembly of the Fas death-inducing signaling complex (DISC) in response to Fas activation when compared to PML WT. PML functions are known to be blocked by its dominant-negative form PMLRARα. We found PMLRARα interaction with Fas in primary human and transgenic mouse APL cells blocked Fas-mediated apoptosis. Blockage of apoptosis was mediated through PMLRARα -dependent recruitment of c-FLIPL/Sto and exclusion of procaspase-8 from the DISC. PMLRARα effects were also observed in vivo, as expression of PMLRARα protected mice against a lethal effect of agonistic anti-Fas antibody (P<.001). Livers from PMLRARα -transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with control vector-transfected mice. Conclusions:PML binds to Fas and promotes Fas-mediated apoptosis through enhancing Fas DISC formation while binding of PMLRARα to Fas blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex enriched in c-FLIP. Our data suggest that PML plays a critical role in initiation of Fas signaling; in contrast, the dominant-negative mutant PMLRARα is a confirmed cancer specific inhibitor of Fas-mediated apoptosis. Thus, deficiency of PML or expression of PMLRARα can contribute to cancer development and resistance to chemotherapy. The newly discovered PML-Fas and PMLRARα -Fas complexes can be sites for modulation of apoptosis. Thus, by neutralizing the inhibitory effect of Fas-binding proteins such as PMLRARα and/or promoting positive Fas modulators such as PML, we can improve responses to chemotherapy that depend on activation of death receptors for effective elimination of cancer cells. Disclosures:No relevant conflicts of interest to declare.

Abstract 4965: Oncoprotein PMLRARα directly suppresses Fas-mediated apoptosis through forming an apoptotic inhibitory complex with c-FLIP in vivo

Abstract Objective: Many genotoxic therapies, including radiation, depend on intact Fas signaling to eradicate cancer cells. Defective Fas signaling is an important cause of cancer resistance to therapy. Restoring Fas apoptosis or sensitizing cancer cells to Fas-mediated apoptosis would improve the efficacy of many cancer therapies. To elucidate a role for specific regulators of Fas signaling in cancer cells, we sought to identify potential modulators of Fas expressed in cancers and target them to selectively sensitize cancer cells to Fas-mediated apoptosis as a component of chemotherapy. Methods: Liquid chromatography tandem mass spectrometry was used to identify Fas-associated proteins; co-immunoprecipitation and Western blot were used to detect interactions of PMLRARα, PML, c-FLIP and Fas, and to examine the components of death-inducing signaling complex (DISC) and caspase-8 cleavage. Deletional mutagenesis was used to map the interaction domains. PML shRNA lentivirus and As2O3 were used to knock down PML and PMLRARα. Flow cytometry analysis of propidium iodide- and Annexin-V-stained cells was used to detect apoptosis. Mice were transfected with PMLRARα, monitored for survival, and tissues were analyzed for apoptosis by staining for cleaved caspase-3 and TUNEL. Results: We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PMLRARα. We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)-derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIPL/S and excluded procaspase-8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P&amp;lt;.001) and the protected tissues contained Fas-PMLRARα-cFLIP complexes. Livers from PMLRARα-transfected mice contained fewer cleaved caspase-3 positive/apoptotic cells when compared with control vector-transfected mice. Conclusions: PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex with c-FLIP. These data suggest that PMLRARα is a cancer specific Fas-binding inhibitor of Fas-mediated apoptosis and thus, can contribute to cancer development and resistance to therapy. The newly discovered PMLRARα-Fas and PML-Fas complexes can be sites for modulation of apoptosis. By neutralizing the effect of death receptor inhibitors, such as PMLRARα, we can improve responses to many chemotherapeutic treatments that depend on activation of death receptors for effective elimination of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4965. doi:1538-7445.AM2012-4965

PMLRARα binds to Fas and suppresses Fas-mediated apoptosis through recruiting c-FLIP in vivo

AbstractDefective Fas signaling leads to resistance to various anticancer therapies. Presence of potential inhibitors of Fas which could block Fas signaling can explain cancer cells resistance to apoptosis. We identified promyelocytic leukemia protein (PML) as a Fas-interacting protein using mass spectrometry analysis. The function of PML is blocked by its dominant-negative form PML–retinoic acid receptor α (PMLRARα). We found PMLRARα interaction with Fas in acute promyelocytic leukemia (APL)–derived cells and APL primary cells, and PML-Fas complexes in normal tissues. Binding of PMLRARα to Fas was mapped to the B-box domain of PML moiety and death domain of Fas. PMLRARα blockage of Fas apoptosis was demonstrated in U937/PR9 cells, human APL cells and transgenic mouse APL cells, in which PMLRARα recruited c-FLIPL/S and excluded procaspase 8 from Fas death signaling complex. PMLRARα expression in mice protected the mice against a lethal dose of agonistic anti-Fas antibody (P < .001) and the protected tissues contained Fas-PMLRARα-cFLIP complexes. Taken together, PMLRARα binds to Fas and blocks Fas-mediated apoptosis in APL by forming an apoptotic inhibitory complex with c-FLIP. The presence of PML-Fas complexes across different tissues implicates that PML functions in apoptosis regulation and tumor suppression are mediated by direct interaction with Fas.

Design and Stereoselective Synthesis of Retinoids with Ferrocene or N‐Butylcarbazole Pharmacophores that Induce Post‐Differentiation Apoptosis in Acute Promyelocytic Leukemia Cells

New ferrocene and N-alkylcarbazole retinoids were designed and synthesized stereoselectively in good yields. A number of these synthesized ligands, in particular 2, 3, and 11, were found to exhibit a high RARα activation potential and to effectively induce post-differentiation apoptosis in NB4 acute promyelocytic leukemia (APL) cells. Increasing the length of the side chain attached to the heterocycle of the carbazole arotinoids creates new opportunities for altered compound catabolism and for fine-tuning of the apoptosis-inducing potential of the ligand. In the carbazole series of new retinoids, maximal activity was established for N-butylcarbazole analogue 11 in all assays (i.e., RARα activation, differentiation induction, and apoptosis induction). Study of the mechanism of apoptosis revealed an activation of initiator caspases-8 and -9, followed by efficient cleavage of effector caspase-3 on day 6 of treatment. Subsequent induction of a caspase cascade in NB4 cells triggered ultimate leukemic cell death. The selected ligands 2, 3, and 11 may provide alternate options for the treatment of APL in cases of life-threatening ATRA syndrome, resistance, and high toxicity to conventionally used retinoids.

Glutathione depletion enhances arsenic trioxide‐induced apoptosis in lymphoma cells through mitochondrial‐independent mechanisms

Arsenic trioxide (ATO) is an effective therapeutic agent for acute promyelocytic leukemia (APL) (Evens et al, 2004). In APL, ATO induces differentiation at low concentrations, while inducing apoptosis at higher concentrations (Miller, et al 2002). In addition, ATO-induced apoptosis in APL is mediated through the mitochondrial apoptotic pathway, resulting in part from the production of reactive oxygen species (ROS) such as hydrogen peroxide (Dai, et al 1999, Yi, et al 2002). High intracellular levels of glutathione (GSH) confer resistance to ATO in part through the detoxification of ROS. Compounds that promote ROS and/or deplete protective metabolites such as GSH are able to sensitize tumor cells to oxidative cytolysis. Buthionine sulfoximine (BSO), a selective inhibitor of gamma glutamylcysteine synthetase, is known to effectively deplete cellular GSH (Davison, et al 2003, Gartenhaus, et al 2002). We evaluated herein the cytotoxic activity and cell death pathways induced by ATO alone and combined with BSO in non-Hodgkin’s lymphoma (NHL) cell lines and primary lymphoproliferative cells.

FOXO3A as a key molecule for all-trans retinoic acid–induced granulocytic differentiation and apoptosis in acute promyelocytic leukemia

AbstractAll-trans retinoic acid (ATRA) induces granulocytic differentiation and apoptosis in acute promyelocytic leukemia (APL) cells, although the detailed mechanisms are not fully understood. We investigated ATRA-induced cellular responses mediated by the transcription factor FOXO3A in APL cells. FOXO3A was constitutively phosphorylated and localized in the cytoplasm in both APL-derived NB4 cells and primary APL cells. Upon treating the cells with ATRA, FOXO3A phosphorylation was reduced and FOXO3A translocated into the nucleus. In addition, the expression of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), a target molecule for FOXO3A, was increased at the transcriptional and protein levels. As expected, transfection of a short hairpin RNA (shRNA) oligonucleotide specific for FOXO3A significantly inhibited ATRA-induced granulocytic differentiation and apoptosis in NB4 cells. In NB4-derived ATRA-resistant NB4/RA cells, neither FOXO3A nuclear localization nor subsequent TRAIL induction was observed after ATRA treatment. Furthermore, forced expression of active FOXO3A in the nucleus induced TRAIL production and apoptosis in NB4/RA cells. We conclude that activation of FOXO3A is an essential event for ATRA-induced cellular responses in NB4 cells. FOXO3A is a promising target for therapeutic approaches to overcome ATRA resistance in APL.

Arsenic trioxide induces apoptosis and G2/M phase arrest by inducing Cbl to inhibit PI3K/Akt signaling and thereby regulate p53 activation

Arsenic trioxide (ATO) strongly induces apoptosis in acute promyelocytic leukemia (APL), but it induces cell cycle arrest in most solid tumors. In this study, we investigated the mechanism of ATO action on APL-derived NB4 cells and gastric cancer cell lines. ATO decreased the viability of both cell lines, but gastric cancer cells were much less susceptible. ATO-induced G2/M phase arrest and p53 degradation in gastric cancer MGC803 cells. In contrast, ATO-induced apoptosis in NB4 cells without degradation of p53. Both processes were accompanied by transient activation of Akt. The PI3K/Akt inhibitor LY294002 significantly increased the amount of p53 protein and ATO-induced apoptosis in both cell lines and decreased G2/M phase arrest of MGC803 cells. In addition, ATO up-regulated the expression of Cbl proteins in both cell lines. Inhibition of Cbl with the proteasome inhibitor Ps341 decreased apoptosis in NB4 cells and increased the G2/M phase arrest of MGC803 cells, and it also prolonged the activation of PI3K/Akt by ATO. Consistent results with those in MGC803 cells were showed in gastric cancer cell BGC823 and SGC7901 after ATO treatment. These results demonstrate that inhibition of PI3K/Akt signaling by Cbl is involved in both ATO-induced apoptosis of NB4 cells and ATO-induced G2/M phase arrest of gastric cancer cells. Cbl achieved these effects probably via its regulating PI3K/Akt pathway, and thereby modulated p53 activation.

Downregulation of the c-MYC target gene, peroxiredoxin III, contributes to arsenic trioxide-induced apoptosis in acute promyelocytic leukemia.

Arsenic trioxide (ATO) induces differentiation and apoptosis in acute promyelocytic leukemia (APL). Several reports indicate that in APL cells apoptosis occurs mainly by a mechanism that involves the inhibition of glutathione peroxidase, one of the enzymes that regulates mitochondrial levels of H(2)O(2). Peroxiredoxin (Prx) III, a c-MYC target gene, is also a mitochondria-specific H(2)O(2)-scavenger enzyme. We studied here the role of Prx III during ATO-induced apoptosis in APL-derived NB4 cells, since these cells express high levels of Prx III. The protein and mRNA levels of Prx III decreased during ATO-induced apoptosis of NB4 cells. The downregulation of Prx III occurred before reactive oxygen species accumulation, reduction in the mitochondrial membrane potential and apoptosis. Depletion of Prx III enhanced mitochondrial-dependent apoptosis events. In contrast, overexpression of Prx III led to reduced levels of ATO-induced apoptosis. c-MYC was also downregulated in ATO-treated NB4 cells. Furthermore, depletion of c-MYC also reduced the Prx-III expression levels. Finally chromatin immunoprecipitation and luciferase reporter assays confirmed that downregulation of Prx-III was caused by the reduction of c-MYC levels during ATO-induced apoptosis of NB4 cells. These findings demonstrate a novel apoptotic-response pathway whereby downregulation of Prx-III potentiates ATO-induced apoptosis in APL cells.

Inhibition of Class I Phosphoinositide 3-Kinase Activity Impairs Proliferation and Triggers Apoptosis in Acute Promyelocytic Leukemia without Affecting Atra-Induced Differentiation

We have investigated the role of phosphoinositide 3-kinases (PI3Ks) in the in vitro pathophysiology of acute promyelocytic leukemia (APL) and in the response to treatment with all-trans-retinoic-acid (ATRA), utilizing a range of novel inhibitors that target individual or all catalytic class I isoforms of PI3K (p110alpha, p110beta, p110delta, and p110gamma). ATRA-induced phosphorylation of the Akt kinase and ribosomal S6 protein in APL cells was sensitive to class I PI3K, and p110beta or p110delta inhibitors, and to the mammalian target of rapamycin (mTOR) inhibitor rapamycin. In primary APL, inhibition of p110beta or p110delta triggered apoptosis in the absence or presence of ATRA. Class I PI3K inhibition could also reverse ATRA-induced protection of these cells against doxorubicin and arsenic trioxide, correlating with impaired induction of the antiapoptotic MCL-1 protein. The differentiation-inducing effects of ATRA were not dependent on class I PI3K/mTOR. In summary, class I PI3K signaling, mediated by p110beta and p110delta, plays an important role in basal and ATRA-induced cell survival mechanisms in APL. Addition of PI3K inhibitors to induction treatment regimens may provide therapeutic benefit.

Death-associated protein 5 (DAP5/p97/NAT1) contributes to retinoic acid-induced granulocytic differentiation and arsenic trioxide-induced apoptosis in acute promyelocytic leukemia.

All-trans-retinoic acid (ATRA) and arsenic trioxide (ATO) induce differentiation and apoptosis in acute promyelocytic leukemia (APL) cells. Here we investigated the role and regulation of death-associated protein-5 (DAP5/p97/NAT1), a novel inhibitor of translational initiation, in APL cell differentiation and apoptosis. We found that ATRA markedly induced DAP5/p97 protein and gene expression and nuclear translocation during terminal differentiation of APL (NB4) and HL60 cells but not differentiation-resistant cells (NB4.R1 and HL60R), which express very low levels of DAP5/p97. At the differentiation inducing concentrations, ATO (<0.5 microM), dimethyl sulfoxide, 1,25-dihydroxy-vitamin-D3, and phorbol-12-myristate 13-acetate also significantly induced DAP5/p97 expression in NB4 cells. However, ATO administered at apoptotic doses (1-2 microM) induced expression of DAP5/p86, a proapoptotic derivative of DAP5/p97. ATRA and ATO-induced expression of DAP5/p97 was associated with inhibition of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Furthermore, DAP5/p97 expression was upregulated by inhibition of the PI3K/Akt/mammalian target of rapamycin (mTOR) pathway via LY294002 and via rapamycin. Finally, knockdown of DAP5/p97 expression by small interfering RNA inhibited ATRA-induced granulocytic differentiation and ATO-induced apoptosis. Together, our data reveal new roles for DAP5/p97 in ATRA-induced differentiation and ATO-induced apoptosis in APL and suggest a novel regulatory mechanism by which PI3K/Akt/mTOR pathway inhibition mediates ATRA- and ATO-induced expression of DAP5/p97.

Downregulation of Peroxiredoxin III Contributes to Arsenic Trioxide-Induced Apoptosis in APL.

Arsenic trioxide (ATO) induces differentiation and apoptosis in acute promyelocytic leukemia (APL). Compared with other leukemia cells, the APL-derived NB4 cells are particularly sensitive to ATO-induced apoptosis. Several reports indicate that in NB4 cells, apoptosis occurs in part by a mechanism that involves the inhibition of glutathione peroxidase, one of the enzymes that regulate the levels of H2O2 in the mitochondria, and that is present only at low levels in NB4 cells. Peroxirredoxin III (Prx III) is a mitochondria-specific H2O2-scavenger member of the thioredoxin-dependent peroxidases. NB4 cells express high levels of Prx III; however the role of Prx III in ATO-induced apoptosis in NB4 cells has not been investigated. We studied here whether Prx III is regulated during ATO-induced apoptosis in NB4 cells and whether the depletion of Prx III further sensitized these cells to ATO-induced apoptosis. The protein and mRNA levels of Prx III were decreased during ATO-induced apoptosis of NB4 cells. The down-regulation of Prx III occurred prior to the accumulation of reactive oxygen species, reduction in the mitochondrial membrane potential and apoptosis. Depletion of Prx III enhanced the ATO-induced mitochondrial damage, and also promoted cytochrome-c release, and caspase-3 and caspase-9 activation. Prx III is downregulated early during ATO-induced apoptosis, facilitating in this way the mitochondria-depending apoptotic events triggered by the accumulation of H2O2.

Trolox selectively enhances arsenic-mediated oxidative stress and apoptosis in APL and other malignant cell lines

AbstractAlthough arsenic trioxide (As2O3) is an effective therapy in acute promyelocytic leukemia (APL), its use in other malignancies is limited by the toxicity of concentrations required to induce apoptosis in non-APL tumor cells. We looked for agents that would synergize with As2O3 to induce apoptosis in malignant cells, but not in normal cells. We found that trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid), a widely known antioxidant, enhances As2O3-mediated apoptosis in APL, myeloma, and breast cancer cells. Treatment with As2O3 and trolox increased intracellular oxidative stress, as evidenced by heme oxygenase-1 (HO-1) protein levels, c-Jun terminal kinase (JNK) activation, and protein and lipid oxidation. The synergistic effects of trolox may be specific to As2O3, as trolox does not add to toxicity induced by other chemotherapeutic drugs. We explored the mechanism of this synergy using electron paramagnetic resonance and observed the formation of trolox radicals when trolox was combined with As2O3, but not with doxorubicin. Importantly, trolox protected nonmalignant cells from As2O3-mediated cytotoxicity. Our data provide the first evidence that trolox may extend the therapeutic spectrum of As2O3. Furthermore, the combination of As2O3 and trolox shows potential specificity for tumor cells, suggesting it may not increase the toxicity associated with As2O3 monotherapy in vivo.