HL-60R Cells Research Articles

Antitumor Effect of Glandora rosmarinifolia (Boraginaceae) Essential Oil through Inhibition of the Activity of the Topo II Enzyme in Acute Myeloid Leukemia.

It was previously shown that the antitumor and cytotoxic activity of the essential oil (EO) extracted from the aerial parts of Glandora rosmarinifolia appears to involve a pro-oxidant mechanism in hepatocellular carcinoma (HCC) and in triple-negative breast cancer (TNBC) cell lines. Its most abundant compound is a hydroxy-methyl-naphthoquinone isomer. Important pharmacological activities, such as antitumor, antibacterial, antifungal, antiviral and antiparasitic activities, are attributed to naphthoquinones, probably due to their pro-oxidant or electrophilic potential; for some naphthoquinones, a mechanism of action of topoisomerase inhibition has been reported, in which they appear to act both as catalytic inhibitors and as topoisomerase II poisons. Our aim was to evaluate the cytotoxic activity of the essential oil on an acute myeloid leukemia cell line HL-60 and on its multidrug-resistant (MDR) variant HL-60R and verify its ability to interfere with topoisomerase II activity. MTS assay showed that G. rosmarinifolia EO induced a decrease in tumor cell viability equivalent in the two cell lines; this antitumor effect could depend on the pro-oxidant activity of EO in both cell lines. Furthermore, G. rosmarinifolia EO reduced the activity of Topo II in the nuclear extracts of HL-60 and HL-60R cells, as inferred from the inability to convert the kinetoplast DNA into the decatenated form and then not inducing linear kDNA. Confirming this result, flow cytometric analysis proved that EO induced a G0-G1 phase arrest, with cell reduction in the S-phase. In addition, the combination of EO with etoposide showed a good potentiation effect in terms of cytotoxicity in both cell lines. Our results highlight the antitumor activity of EO in the HL-60 cell line and its MDR variant with a peculiar mechanism as a Topo II modulator. Unlike etoposide, EO does not cause stabilization of a covalent Topo II-DNA intermediate but acts as a catalytic inhibitor. These data make G. rosmarinifolia EO a potential anticancer drug candidate due to its cytotoxic action, which is not affected by multidrug resistance.

Growth Inhibitory Effects of Ester Derivatives of Menahydroquinone-4, the Reduced Form of Vitamin K2(20), on All-Trans Retinoic Acid-Resistant HL60 Cell Line

The first-choice drug for acute promyelocytic leukemia (APL), all-trans retinoic acid (ATRA), frequently causes drug-resistance and some adverse effects. Thus, an effective and safe agent for ATRA-resistant APL is needed. Menaquinone-4 (MK-4, vitamin K2(20)), used for osteoporosis treatment, does not have serious adverse effects. It has been reported that MK-4 has growth-inhibitory effects on HL60 cells by inducing apoptosis via the activation of Bcl-2 antagonist killer 1 (BAK). However, the effect of MK-4 on ATRA-resistant APL has not been reported. Here, we show that ester derivatives of menahydroquinone-4 (MKH; a reduced form of MK-4), MKH 1,4-bis-N,N-dimethylglycinate (MKH-DMG) and MKH 1,4-bis-hemi-succinate (MKH-SUC), exerted strong growth-inhibitory effects even on ATRA-resistant HL60 (HL-60R) cells compared with ATRA and MK-4. MKH delivery after MKH-SUC treatment was higher than that after MK-4 treatment, and the results indicated apoptosis induced by BAK activation. In contrast, for MKH-DMG, reconversion to MKH was slow and apoptosis was not observed. We suggest that the ester forms, including monoesters of MKH-DMG, exhibit another mechanism independent of apoptosis. In conclusion, the MKH derivatives (MKH-SUC and MKH-DMG) inhibited not only HL60 cells but also HL-60R cells, indicating a potential to overcome ATRA resistance.

In Vitro Modulation of P-Glycoprotein Activity by Euphorbia intisy Essential Oil on Acute Myeloid Leukemia Cell Line HL-60R.

Euphorbia species have a large spectrum of traditional medicinal uses. We tested the biological activities of the essential oil (EO) of Euphorbia intisy Drake in an acquired multidrug resistance leukemia model to assess whether the EO obtained by hydrodistillation of stems was able to reverse the resistant phenotype. HL-60R cell lines are characterized by the overexpression of P-glycoprotein (P-gp), inhibitors of apoptosis proteins (IAPs) and constitutive expression of NF-κB. EO chemical composition was determined by GC/MS analysis; cytotoxic activity of EO by MTS assay alone or in combination with doxorubicin; pro-apoptotic effect and doxorubicin accumulation were analyzed by flow cytometry; P-gp ATPase activity was measured by P-gp-Glo™ assay systems kit. The ability to inhibit NF-κB and its target genes was also assessed. E. intisy EO exhibited a comparable cytotoxic effect and ability to block P-gp in both the HL-60 and its MDR variant HL-60R. In addition, EO suppressed P-gp protein expression and significantly downregulated MDR1 mRNA level, as well as some IAPs proteins, probably through the inhibition of NF-κB. Our results suggest that E. intisy EO could reverse P-gp-mediated drug resistance in tumor cells acting as a chemosensitizing agent.

Inhibition of UBE2L6 attenuates ISGylation and impedes ATRA-induced differentiation of leukemic cells.

Ubiquitin/ISG15‐conjugating enzyme E2L6 (UBE2L6) is a critical enzyme in ISGylation, a post‐translational protein modification that conjugates the ubiquitin‐like modifier, interferon‐stimulated gene 15 (ISG15), to target substrates. Previous gene expression studies in acute promyelocytic leukemia (APL) cells showed that all‐trans‐retinoic acid (ATRA) altered the expression of many genes, including UBE2L6 (200‐fold) and other members of the ISGylation pathway. Through gene expression analyses in a cohort of 98 acute myeloid leukemia (AML) patient samples and in primary neutrophils from healthy donors, we found that UBE2L6 gene expression is reduced in primary AML cells compared with normal mature granulocytes. To assess whether UBE2L6 expression is important for leukemic cell differentiation—two cell line models were employed: the human APL cell line NB4 and its ATRA‐resistant NB4R counterpart, as well as the ATRA‐sensitive human AML HL60 cells along with their ATRA‐resistant subclone—HL60R. ATRA strongly induced UBE2L6 in NB4 APL cells and in ATRA‐sensitive HL60 AML cells, but not in the ATRA‐resistant NB4R and HL60R cells. Furthermore, short hairpin (sh)RNA‐mediated UBE2L6 depletion in NB4 cells impeded ATRA‐mediated differentiation, suggesting a functional role for UBE2L6 in leukemic cell differentiation. In addition, ATRA induced ISG15 gene expression in NB4 APL cells, leading to increased levels of both free ISG15 protein and ISG15 conjugates. UBE2L6 depletion attenuated ATRA‐induced ISG15 conjugation. Knockdown of ISG15 in NB4 APL cells inhibited ISGylation and also attenuated ATRA‐induced differentiation. In summary, we demonstrate the functional importance of UBE2L6 in ATRA‐induced neutrophil differentiation of APL cells and propose that this may be mediated by its catalytic role in ISGylation.

Cytoplasmic proliferating cell nuclear antigen connects glycolysis and cell survival in acute myeloid leukemia.

Cytosolic proliferating cell nuclear antigen (PCNA), a scaffolding protein involved in DNA replication, has been described as a key element in survival of mature neutrophil granulocytes, which are non-proliferating cells. Herein, we demonstrated an active export of PCNA involved in cell survival and chemotherapy resistance. Notably, daunorubicin-resistant HL-60 cells (HL-60R) have a prominent cytosolic PCNA localization due to increased nuclear export compared to daunorubicin-sensitive HL-60 cells (HL-60S). By interacting with nicotinamide phosphoribosyltransferase (NAMPT), a protein involved in NAD biosynthesis, PCNA coordinates glycolysis and survival, especially in HL-60R cells. These cells showed a dramatic increase in intracellular NAD+ concentration as well as glycolysis including increased expression and activity of hexokinase 1 and increased lactate production. Furthermore, this functional activity of cytoplasmic PCNA was also demonstrated in patients with acute myeloid leukemia (AML). Our data uncover a novel pathway of nuclear export of PCNA that drives cell survival by increasing metabolism flux.

Anti-leukemic effect of sodium metaarsenite (KML001) in acute myeloid leukemia with breaking-down the resistance of cytosine arabinoside.

Sodium metaarsenite (NaAs2O3: code name KML001) is an orally bioavailable arsenic compound with potential anti-cancer activity. However, the effect of KML001 has not been studied in acute myeloid leukemia (AML). We investigated the anti-leukemic effect of KML001 in AML, and determined the mode of action of KML001. KML001 inhibited the cellular proliferation in all AML cell lines and primary AML blasts as well as HL-60R (cytosine arabinoside-resistant HL-60) cells, while As2O3 was not effective in primary AML blasts and AML cell lines including HL-60R cells. KML001 induced G1 arrest and apoptosis in HL-60 and HL-60R cells. KML001 inhibited the activation of STAT (signal transducer and activator of transcription) 1, 3, 5, NF-κB, AKT and PI3K, while phosphorylated PTEN was upregulated. In addition, activation of ERK, p38 and JNK was observed in KML001-induced growth inhibition of HL-60 and HL-60R cells. Furthermore, KML001 induced telomeric terminal restriction fragment (TRF) length shortening in a time-dependent manner in HL-60 and HL-60R cells. Real‑time PCR with RNA extracted from KML001-treated HL-60 and HL-60R cells showed a significant reduction of catalytic subunit of telomerase, hTERT, in a time-dependent manner. Additionally, γ-H2AX, a sensitive molecular marker of DNA damage, in HL-60 and HL-60R cells was induced by KML001. These results suggest that KML001 inhibits the proliferation of AML cells including cytosine arabinoside-resistant AML cells via various mechanisms such as cell cycle arrest, induction of apoptosis, inhibition of JAK/STAT and PI3K pathways, activation of MAPK pathway and telomere targeting.

Heme oxygenase-1 plays a crucial role in chemoresistance in acute myeloid leukemia

ObjectivesThe heme oxygenase-1 (HO-1) gene may contribute to the development of acquired chemoresistance in solid tumor cells, but its function in acute myeloid leukemia (AML) remains unclear. Therefore, we investigated whether the expressions of HO-1 mRNA and protein were associated with AML chemoresistance.MethodsBone marrow or peripheral blood was obtained from newly diagnosed (n = 26), relapsed (n = 10), and completely remitted (n = 18) patients with AML (M3 exclusion) and healthy donors (n = 10). Small interfering RNA was used to stably silence HO-1 gene expression in AML cell lines. The expressions of HO-1, hypoxia inducible factor-1ɑ (HIF-1ɑ), glucose transporter-1 (GLUT1) mRNA and proteins were measured by quantitative real-time PCR and Western blot. Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis induction was analyzed by flow cytometry.ResultsThe drug-resistant AML cell line HL-60R was significantly less sensitive to cytarabine and daunorubicin than HL-60 cells. HO-1 mRNA and proteins were highly expressed in HL-60R cells. However, down-regulating HO-1 significantly enhanced the sensitivity of HL-60R to chemotherapy, and the expressions of HIF-1ɑ and GLUT1 mRNA and proteins decreased. Meanwhile, the expressions of caspase-3 and caspase-8 proteins increased, while that of bcl-2 decreased. Overexpressions of HO-1, HIF-1ɑ, and GLUT1 were associated with poor response of AML to chemotherapy.ConclusionsOverexpressions of HO-1, HIF-1ɑ, and GLUT1 might be involved in the chemoresistance of AML. HO-1 is a potential target to overcome the drug resistance of AML.

Enhanced chemopreventive activity of hydroxytyrosol on HL60 and HL60R cells by chemical conversion into thio derivatives

Thio derivatives of hydroxytyrosol containing thiol, thioacetate and disulfide functionalities were synthesized from natural hydroxytyrosol (3,4-DHPEA) via 3,4-dihydroxyphenethyl halides. These compounds, containing the combination of catechol moiety and divalent sulfur functions, were tested for the pro-apoptotic and anti-proliferative activities on both parental HL60 and multi-drug resistant HL60R cells. It was found that all synthesized compounds were more effective than 3,4-DHPEA in inducing apoptosis on HL60R cells, and that the hydroxytyrosol disulfide was the most active pro-apoptotic and anti-proliferative compound on both HL60 and HL60R cells. Different from 3,4-DHPEA, all thio derivatives of hydroxytyrosol induced apoptosis by a mechanism not involving the release of H2O2 in the culture medium. The data on HL60R cells suggest that these compounds could be able to reverse the resistance toward the most common drugs in cancer therapy.

Long term cultured HL-60 cells are intrinsically resistant to Ara-C through high CDA activity

Cytarabine (araC) is a highly active antimetabolite against hematological malignancy while the agent shows limited activity for some patients despite maintenance or continued therapy with ara-C-containing regiments. In this study, we focused to elucidate the mechanism of intrinsic resistance to araC. The concentration of intracellular ara-CTP and incorporated ara-CTP were monitored in human leukemia cell line-HL-60 for different passages in parental with its variant HL-60R. The expression of mRNA for deoxycytidine kinase (dCK), cytidine deaminas (CDA), human equilibrative nucleoside transporter 1 (hENT1), and cytosolic 50-nucleotidase II (cN-II) were examined by Real-time PCR in HL-60 and HL-60R for different passages. And activities of two metabolizing enzymes for araC, dCK and CDA were further examined. The results showed that the concentration of intracellular ara-CTP was significantly reduced and the ara-U increased in HL-60 cells for 50 passages compared with the 5 passages, and associated with higher CDA activity. All the factors in HL-60R cells did not change by the incubation of ara-C. In conclusion, the long term cultured cells are intrinsically resistant to ara-C through high CDA activity, but not low DCK activity.

Synthesis and cytotoxic evaluation of novel paraconic acid analogs

A novel class of 2,3-tri- and tetrasubstituted γ-butyrolactones analogous to paraconic acids has been synthesized in one step using a straightforward three-component reaction among aryl bromides, dimethyl itaconate and carbonyl compounds. The in vitro cytotoxic activity of representative compounds has been evaluated against a panel of human cancer cell lines (KB, HCT116, MCF7, HL60). While most molecules exhibit a low to moderate background activity on both KB and HL60 cancer cell lines, one compound shows increased antiproliferative activities against both cell lines with IC(50) values in the 10(-7)-10(-6)mol/L range. An extended evaluation indicated that this compound also inhibits PC3, SK-OV3, MCF7R and HL60R cell growth in the same fashion.

Transcriptional silencing of the ETS1 oncogene contributes to human granulocytic differentiation

Ets-1 is a widely expressed transcription factor implicated in several biological processes including hematopoiesis, where it contributes to the regulation of cellular differentiation. The functions of Ets-1 are regulated by transcription factors as well as by phosphorylation events: phosphorylation of threonine 38 activates Ets-1, whereas phosphorylation of a cluster of serines within exon VII reduces DNA binding activity. This study focuses on the role of Ets-1 during granulocytic differentiation of NB4 promyelocytic and HL60 myeloblastic leukemia cell lines induced by all-trans retinoic acid. Ets-1 expression was measured by real-time reverse transcriptase polymerase chain reaction and western blotting. The role of Ets-1 during all-trans retinoic acid-induced differentiation was analyzed by using a transdominant negative molecule or small interfering RNA. NB4 and HL60 cell lines expressed high levels of p51 Ets-1, while the splice variant isoform that lacks exon VII (p42) was almost undetectable. The addition of all-trans retinoic acid reduced p51 Ets-1 levels and induced inhibitory phosphorylation of the remaining protein. Expression of Ets-1 was also reduced during dimethylsulfoxide-induced differentiation and during granulocytic differentiation of human CD34(+) hematopoietic progenitor cells but not in NB4.R2 and HL60R cells resistant to all-trans retinoic acid. In line with these observations, transduction of a transdominant negative molecule of Ets-1, which inhibited DNA binding and transcriptional activity of the wild-type Ets-1, significantly increased chemical-induced differentiation. Consistently, Ets-1 knockdown by small interfering RNA increased the number of mature neutrophils upon addition of all-trans retinoic acid. Interestingly, p51 Ets-1 over-expression was frequently observed in CD34(+) hematopoietic progenitor cells derived from patients with acute myeloid leukemia, as compared to its expression in normal CD34(+) cells. Our results indicated that a decreased expression of Ets-1 protein generalizes to granulocytic differentiation and may represent a crucial event for granulocytic maturation.

Green Tea Catechin Epigallocatechin-3-Gallate (EGCG) Induces Cell Death in Acute Myeloid Leukemic Cells Via DAPK2 and Potentiates ATRA-Induced Neutrophil Differentiation

Acute promyelocytic leukemia (APL) patients are currently treated with all-trans retinoic acid (ATRA) successfully resolving the differentiation block. However, concurrent chemotherapy is still necessary in combination with ATRA and novel less toxic therapeutic approaches are a major demand. Epigallocatechin-3-gallate (EGCG), the main polyphenolic compound present in green tea, has been reported to have chemopreventive and chemotherapeutic effects in different neoplasms. EGCG inhibits cell growth in vitro and in vivo and induces apoptosis in cancer cells without adversely affecting normal cells. Also, EGCG has been found in earlier studies to effectively kill AML blasts, but it has never been applied in combination with ATRA. We have previously shown that expression of the death-associated protein kinase 2 (DAPK2) enhanced ATRA-induced neutrophil development, and recently it was shown that EGCG specifically killed multiple myeloma cells while inducing DAPK2. Therefore, we first tested whether EGCG treatment induces DAPK2 expression in myeloid leukemic blast cells and whether this will lead to cell death. EGCG treatment of HL60 and NB4 acute myeloid leukemic cells led to a dose-dependent increase of DAPK2 mRNA with a maximum of 5.5- and 3.9-fold, respectively. In parallel, DAPK2 protein was markedly upregulated in both cell lines accompanied by a 72% increase of cell death after 24h, as measured by reduction of tetrazolium salt (XTT assay), and by hallmarks of apoptosis: activation of caspase-3 and phosphatidylserine exposure on the plasma membrane surface. EGCG-induced cytotoxicity was reduced by 48% in HL60 and by 46% in NB4 cells upon stable short hairpin RNA-mediated silencing of DAPK2. Moreover, combined ATRA and EGCG treatment of HL60 cells resulted in cooperative DAPK2 induction and potentiated myeloid differentiation as measured by CD11b and CD15 expression using flow cytometry (Figure A) as well as by C/EBPε and G-CSFR mRNA levels using quantitative RT-PCR. Enhanced differentiation was significantly reversed by knocking down DAPK2. Moreover, EGCG toxicity of NB4 and HL60 cells correlated with 67 kDa laminin receptor (67LR) protein expression which is downregulated during ATRA treatment of HL60 and NB4 cells but not in the ATRA-resistant sublines HL60-R and NB4-R. In line, HL60-R and NB4-R cells are still susceptible to EGCGinduced cell killing, whereas pretreatment of parental HL60 and NB4 cells with ATRA, causing downregulation of 67LR, rendered these cells resistant to EGCG mediated killing. Likewise, susceptibility of primary AML (n=8) samples to EGCG treatment was closely associated with 67LR expression. Moreover, neutrophils and PBMCs from healthy donors did not express 67LR and hence were resistant to EGCG treatment (Figure B). [Display omitted] In summary, we found that DAPK2 is essential for EGCG-induced cell death in myeloid leukemic cells, and that a combination of ATRA and EGCG treatment significantly boosted neutrophil differentiation. This enhanced differentiation is most likely due to preferred EGCG-induced killing of ATRA-resistant leukemic cells expressing the EGCG receptor 67LR. We thus conclude that simultaneous ATRA and EGCG treatment might improve differentiation therapies for APL. In addition, other AML subtypes with high 67LR expression might also benefit from a combined chemotherapy and EGCG regimen.

Retinoid X receptor α is highly phosphorylated in retinoic acid-resistant HL-60R cells and the combination of 9-cis retinoic acid plus MEK inhibitor induces apoptosis in the cells

We examined the effects of 9-cis retinoic acid (RA) on the expression levels of retinoid X receptor α (RXRα) and its phosphorylated form (p-RXRα) in HL-60 and HL-60R cells. 9-cis RA reduced both RXRα and p-RXRα in HL-60 cells, but did neither in HL-60R cells. However, when the HL-60R cells were treated with the combination of 9-cis RA plus PD98059, MEK inhibitor, the p-RXRα and RXRα proteins all markedly decreased. Moreover, the combination of those agents induced apoptosis in HL-60R cells. Phosphorylation of RXRα might be associated with RA-resistance in HL-60R cells.

Novel Tumor Suppressor Protein Programmed Cell Death 4 (PDCD4) Suppresses Activity of PI3K/Akt Pathway and Regulates Expression of p27 (Kip1) and c-myc, DAP5 and Willm's Tumor (WT1) in Acute Myeloid Leukemia.

Programmed cell death 4 (PDCD4) is a recently identied novel tumor suppressor protein that inhibits cap-dependent mRNA translation. PDCD4 inhibits tumor promoter incuced carcinogenesis and transformation by suppressing the helicase activity of eIF4A, leading to translational inhibition. Recently we found that PDCD4 is required for all-trans-retinoic acid (ATRA)-induced granulocytic differentiation of acute promyelocytic leukemia (APL) cells (Ozpolat&Akar et al, Mol Cancer Res, in press), type of acute myeloid leukemia characterized by a t(15;17) and a differentiation block. Here we investigated the downstream mediators or targets of PDCD4 in leukemia cell differentiation. ATRA is currently used as a first line standard treatment in APL. Recently, we reported that ATRA induces translational suppression through multiple posttranscriptional mechanisms that involve suppression of translation initiation, a rate limiting step of protein synthesis (Harris&Ozpolat et al, Blood , 104 (5) 2004). We found that ATRA treatment induced PDCD4 expression in NB4 APL, HL60 AML, primary APL patient leukemia cells and normal human CD34+ bone marrow progenitors cells during granulocytic differentiation. However, ATRA/maturation resistant NB4.R1 and HL60R cells failed to express and translocate PDCD4 into nucleus after ATRA treatment. To identify downstream targets of PDCD4 we knock downed PDCD4 expression by siRNA and examined changes in the expression of target proteins that are known to be regulated by ATRA by Western blot analysis in NB4 cells. We found that PDCD4 represses c-myc, and WT1 expression however, it is required for the expression of DAP5 (death associated protein 5), and cyclin dependent kinase inhibitor p27KIP1, and but not c-jun, p21Cip1, and tissue transglutaminase (TG2). Inhibition of PDCD4 by siRNA resulted in upregulation of phospho-P70S6K, suggesting that PDCD4 inhibits activity of PI3K/Akt pathway. RT-PCR analysis revealed that mRNA of these proteins did not change suggesting that PDCD4 tumor suppressor protein regulates expression of these important cellular proteins at translational level and suppresses PI3K/Akt pathway. Furthermore we rapamycin, a specific mTOR inhibitor currently in clinical trials in AML, induced a marked expression of PDCD4, which regulates c-myc and p27 Kip1, revealing a novel mechanism of action of rapamycin, providing new rationale for targeting translational pathways as a therapeutic intervention in the treatment of AML. Overall, data suggest that PDCD4 regulates expression of critical cellular proteins involved in differentiation of leukemia cells and PDCD4 mediated translational control may be an important regulatory mechanism for regulation of gene expression.

Programmed Cell Death-4 Tumor Suppressor Protein Contributes to Retinoic Acid–Induced Terminal Granulocytic Differentiation of Human Myeloid Leukemia Cells

Programmed cell death-4 (PDCD4) is a recently discovered tumor suppressor protein that inhibits protein synthesis by suppression of translation initiation. We investigated the role and the regulation of PDCD4 in the terminal differentiation of acute myeloid leukemia (AML) cells. Expression of PDCD4 was markedly up-regulated during all-trans retinoic acid (ATRA)-induced granulocytic differentiation in NB4 and HL60 AML cell lines and in primary human promyelocytic leukemia (AML-M3) and CD34(+) hematopoietic progenitor cells but not in differentiation-resistant NB4.R1 and HL60R cells. Induction of PDCD4 expression was associated with nuclear translocation of PDCD4 in NB4 cells undergoing granulocytic differentiation but not in NB4.R1 cells. Other granulocytic differentiation inducers such as DMSO and arsenic trioxide also induced PDCD4 expression in NB4 cells. In contrast, PDCD4 was not up-regulated during monocytic/macrophagic differentiation induced by 1,25-dihydroxyvitamin D3 or 12-O-tetradecanoyl-phorbol-13-acetate in NB4 cells or by ATRA in THP1 myelomonoblastic cells. Knockdown of PDCD4 by RNA interference (siRNA) inhibited ATRA-induced granulocytic differentiation and reduced expression of key proteins known to be regulated by ATRA, including p27(Kip1) and DAP5/p97, and induced c-myc and Wilms' tumor 1, but did not alter expression of c-jun, p21(Waf1/Cip1), and tissue transglutaminase (TG2). Phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian target of rapamycin (mTOR) signaling pathway was found to regulate PDCD4 expression because inhibition of PI3K by LY294002 and wortmannin or of mTOR by rapamycin induced PDCD4 protein and mRNA expression. In conclusion, our data suggest that PDCD4 expression contributes to ATRA-induced granulocytic but not monocytic/macrophagic differentiation. The PI3K/Akt/mTOR pathway constitutively represses PDCD4 expression in AML, and ATRA induces PDCD4 through inhibition of this pathway.

The Combination of 9-cis Retinoic Acid Plus MEK Inhibitor Induces Apoptosis Via Dephosphorylation of Retinoid X Receptor α in HL-60R Cells.

Resistance against retinoic acid (RA) is a serious problem of differentiation-induction therapy for acute promyelocytic leukemia (APL) in clinical practice. RA exerts its biological activities primarily through the nuclear receptor dimmer, consisting of retinoic acid receptor (RAR) and retinoid X receptor (RXR). Both receptors consist of three subtypes (α, β and γ) and form heterodimeric RAR/RXR and homodimeric RXR/RXR complexes. 9-cis RA, which is a high-affinity ligand for RXR but also binds to RAR, induces cell differentiation in wild type HL-60 human myeloid leukemia cells. However, in HL-60R cells, the RA-resistant subclone of HL-60, 9-cis RA can not induce cell differentiation and apoptosis. We recently reported that malfunction of RXRα due to posttranslational modification by phosphorylation to be associated with carcinogenesis of hepatocellular carcinoma (HCC). Phosphorylated form of RXRα (p-RXRα) at serine 260 by Ras/MAPK is resistant to ubiquitin/proteasome-mediated degradation and the accumulation of p-RXRα interferes with the function of remaining normal RXRα in a dominant negative manner, thereby promoting growth of HCC cells. We also found that in the presence of MEK inhibitor PD98059, 9-cis RA can induce the degradation of p-RXRα and thus restoring the function of this receptor in RXRα-phosphorylated human HCC cells. Based on the results as described above, we initiated this study to examine whether 9-cis RA can exert growth inhibitory effects on RA-resistant HL-60R cells when combined with MEK inhibitor, with focusing on the inhibition of expression of p-RXRα protein. We found that RXRα protein was originally expressed in both HL-60 and HL-60R cells, and that the expression level of RXRα protein was inhibited by about 60% and 20%, respectively, when those cells were treated with 0.5 μM 9-cis RA. Not only total RXRα protein, but also the level of p-RXRα protein was constitutively expressed in both HL-60 and HL-60R cells, and was significantly decreased in HL-60 cells by treatment with 9-cis RA alone. On the other hand, in HL-60R cells, 9-cis RA alone nor 20 μM PD98059 alone did not cause a down regulation of these proteins. However, when HL-60R cells were treated with the combination of 9-cis RA plus PD98059, the expression level of p-RXRα protein was markedly decreased. Moreover, the combination of these agents induced apoptosis in HL-60R cells, whereas similar effect was not obtained when the cells were treated with either agent alone. The combined treatment of these agents also cooperatively inhibited the growth of HL-60 R cells. The present findings suggest that the accumulation of p-RXRα might impair the function of normal RXRα as a master regulator of nuclear receptors and, thus, contributing to the resistance to RA in HL-60R cells. Combination of 9-cis RA plus MEK inhibitor might be an effective regimen for patients with RA resistant APL.

Antioxidant and anticancer activities of novel p-alkylaminophenols and p-acylaminophenols (aminophenol analogues)

Novel compounds were designed based on fenretinide, N-(4-hydroxyphenyl)retinamide ( 2), which is a synthetic amide of all- trans-retinoic acid ( 1) that is a potent antioxidant and anticancer agent. Our recent findings indicated that antioxidant and anticancer activities were due to p-methylaminophenol moiety ( 8) in 2, and that p-octylaminophenol ( 7), which has an elongated alkyl chain, was more potent than 8. This finding lets us to investigate whether compounds containing alkyl or acyl chains linked to an aminophenol residue as long as 2 and 1, would show activities greater than 2. For this purpose, we prepared p-dodecanoylaminophenol ( 3), p-decanoylaminophenol ( 4), p-dodecylaminophenol ( 5), and p-decylaminophenol ( 6). The p-alkylaminophenols, 5 and 6, exhibited superoxide scavenging activities, but not p-acylaminophenols, 3 and 4. Elongation of the alkyl chain length reduced superoxide trapping capability ( 8 > 7 > 6 > 5). In contrast, lipid peroxidation in rat liver microsomes was reduced by 5 and 6 in dose-dependent manner. Compounds 3 and 4 were poor lipid peroxidation inhibitors, being approximately 400- to 1300-fold lower than 5 and 6. In addition, all compounds inhibited cell growth of human leukemia cell lines, HL60 and HL60R, in dose-dependent manners ( 5 > 6 > 3 = 4). The HL60R cell line is resistant against 1. Growth of both cell lines was suppressed by 5 and 6 in a fashion dependent on the length of the aminophenol alkyl chain, but not by 3 and 4. These results indicate that 5, a potent anticancer agent greater than 2, may potentially have clinical utility, and that its anticancer activity is correlated with inhibitory potency against lipid peroxidation, but not with superoxide scavenging activity.

Novel Stilbene-Based Antileukemic Agents Active in P-Glycoprotein Expressing and Apoptosis-Resistant Acute Leukaemia Cell Lines.

The stilbene scaffold is a basic element for a number of biologically active natural and synthetic compounds and in accordance with Evans' definition it can be considered as a privileged structure. One of the most relevant and studied stilbenes is Resveratrol, a phytoalexin present in grapes, endowed with chemopreventive and chemotherapeutic properties and able to induce apoptosis in different cancer cell lines. Since reduced apoptosis has been implicated in the development and progression of malignant tumors and in the occurrence of chemoresistant phenotypes, resveratrol-induced apoptosis might therefore contribute to its antitumor activity. However, resveratrol is a not potent cytotoxic compound if compared with others chemotherapeutic drugs and it is scarcely active in P-glycoprotein expressing (MDR) and Bcr-Abl expressing leukaemia cells. With the aim to find new stilbene compounds active in resistant leukaemia cells we synthesized a small library of resveratrol analogs, bearing the 3,5-dimethoxy motif at the A phenyl ring and amino, methoxy and hydroxy moieties at the 3′-and/or 4′-positions. Moreover, we synthesized analogues which incorporate a phenyl ring as bioisosteric substitution of the alkenyl bridge. Among these new stilbenes we identified two compounds endowed with interesting antileukemic properties: a) a methoxylated cis derivative active at nanomolar concentrations in P-glycoprotein expressing HL60-R and CEM VBL100 acute leukaemia cell lines and in P-glycoprotein and Bcr-Abl expressing K562-ADR cell line which is resistant to apoptosis induced by most common anticancer agents, and b) a terphenyl derivative active in MDR and Bcr-Abl expressing cell lines. Both compounds induced apoptosis prevalently through the mitochondrial pathway. Differently from resveratrol and other stilbenes, the therphenyl derivative induced a block of cells in G0-G1 phase of cell cycle which was associated to the shift of the phosphorylation state of pRb from hyperphosphorylated to hypophosphorylated. Morover, low concentrations of this compound were able to induced a potent granulocytic and monocytic differentiation of HL60 cells.

Anticancer and superoxide scavenging activities of p-alkylaminophenols having various length alkyl chains

A series of p-alkylaminophenols including 3, p-butylaminophenol; 4, p-hexylaminophenol; 5, p-octylaminophenol; and 6, N-( p-methoxybenzyl)aminophenol were synthesized based on the structure of fenretinide, N-(4-hydroxyphenyl)retinamide ( 1). This latter agent is a synthetic amide of all- trans-retinoic acid (RA), which is a cancer chemopreventive and antiproliferative agent. It was found that elongation of the alkyl chain length in these compounds increased antioxidative activity and inhibition of lipid peroxidation. These findings led us to investigate whether antiproliferative activity against cancer cells was effected by the length of alkyl chains linked to the aminophenol residue. All p-alkylaminophenols inhibited growth of HL60 and HL60R cells in a dose-dependent manners. The HL60R line is a resistant clone against RA. Growth of various cancer cell lines (HL60, HL60R, MCF-7, MCF-7/Adr R, HepG2, and DU-145) was suppressed by p-alkylaminophenols in a fashion dependent on the aminophenol alkyl chain length ( 5 > 4 > 3 > p-methylaminophenol ( 2)), with 5 being the most potent inhibitor of cell growth against HL60R, MCF-7/Adr R, and DU-145 cells among p-alkylaminophenols tested, including 1. In particular, with the exception of compound 2, antiproliferative activity against DU-145 cells by these p-alkylaminophenols was greater than by 1. In HL60 cells, growth inhibition was associated with apoptosis. On the other hand, elongation of the alkyl chain length reduced superoxide trapping capability ( 2 > 3 > 4 > 5) in contrast to the effects on inhibition of lipid peroxidation. These results indicate that anticancer activity of p-alkylaminophenols correlated with the inhibitory activity of lipid peroxidation, but not with the superoxide scavenging activity.

Induction of Apoptosis by the Adenosine Derivative IB-MECA in Parental or Multidrug-Resistant HL-60 Leukemia Cells: Possible Relationship to the Effects on Inhibitor of Apoptosis Protein Levels

Background: The effects of the A₃ adenosine receptor (A₃AR) agonist IB-MECA were examined in HL-60 leukemia and in its multidrug-resistant variant HL-60R cells. Methods: Cytotoxicity was evaluated by MTS assays and apoptosis by flow cytometry analyses of DNA fragmentation and phosphatidylserine exposure. The mRNAs of A₃AR and inhibitor of apoptosis proteins (IAPs) were determined by RT-PCR. Results: A₃AR expression was similar in HL-60 and HL-60R cells. At ≧100 µM, IB-MECA exhibited strong cytotoxic and apoptotic effects in HL-60, but not in HL-60R cells. This activity was not modified by the A₃AR antagonist VUF5574, the P-glycoprotein inhibitor verapamil, the adenosine uptake inhibitor NBTI or the anti-Fas antibody ZB4. HL-60R cells showed higher levels of different IAPs than HL-60 cells. IB-MECA 100 µM downregulated HIAP1, NAIP and survivin mRNAs in HL-60, but not in HL-60R cells. Conclusions: The antitumor effects of IB-MECA are not mediated by A₃AR in HL-60 cells, where the proapoptotic mechanism of the compound may involve downregulation of IAPs. The resistance of HL-60R cells to IB-MECA may depend on their elevated levels of IAPs.