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Background/aim: Extracellular signal-regulated kinases (ERK)1/2 are important regulatory proteins that control cell proliferation and survival, playing a significant role in cancer progression, metastasis, and chemoresistance. This study investigated the effects of ERK1/2 inhibitors on the in vitro growth of acute leukemia cell lines., Materials and Methods: Three ERK1/2 inhibitors were used: SCH772984, temuterkib (LY3214996), and ulixertinib (BVD-523). Four acute myeloid leukemia cell lines (OCI/AML3, HL-60, THP-1, and U-937) and two T-lymphoblastic leukemia cell lines (Jurakt and KOPT-K1) were treated with these inhibitors. Cell growth was assessed using a colorimetric assay, and cell-cycle progression and apoptosis were analyzed using flow cytometry. The expression of intracellular signaling proteins was evaluated via immunoblotting. The effects of small interfering RNA (siRNA)-mediated ERK1/2 knockdown were also evaluated., Results: The inhibitors suppressed the growth of three leukemia cell lines (OCI/AML3, HL-60, and THP-1) harboring neuroblastoma rat sarcoma virus (NRAS) mutations. Growth suppression occurred through G0/G1 arrest in all three cell lines and through apoptosis in OCI/AML3 cells. Immunoblotting demonstrated that these inhibitors suppressed the expression of MYC proto-oncogene, bHLH transcription factor (MYC), in the three cell lines. The additional molecular mechanisms of growth suppression varied depending on the specific inhibitor and cell line. The inhibitors had milder suppressive effects on normal lymphocytes compared to the leukemia cell lines., Conclusion: ERK1/2 inhibitors may serve as novel molecular-targeted drugs for treating leukemia with NRAS mutations., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: The renin-angiotensin system (RAS) regulates blood pressure. The RAS is also related to cell growth, and its activation has been reported in various cancer cells. Therefore, we investigated the effects of RAS inhibitors on the in vitro growth of leukemia cell lines., Materials and Methods: THP-1, MV4-11, and TMD7 cells derived from acute myeloid leukemia, K-562 cells from chronic myeloid leukemia, and Jurkat and KOPT-K1 cells from T-lymphoblastic leukemia (T-ALL) with NOTCH1 mutations were used. We used four RAS inhibitors: the renin inhibitor aliskiren, angiotensin-converting enzyme 1 inhibitor captopril, angiotensin II type 1 receptor antagonist azilsartan, and angiotensin II type 2 receptor antagonist PD123319. Cells were cultured with the inhibitors and cell growth was assessed using a colorimetric assay. The expression of signaling proteins was assessed using immunoblotting., Results: Treatment with aliskiren, azilsartan, or PD123319 suppressed the growth of all cell lines. Captopril treatment suppressed the growth of K-562, KOPT-K1, and MV4-11 cells. Flow cytometric analysis revealed that the growth suppression was due to the induction of apoptosis. Their suppressive effects on normal lymphocytes were milder than those on leukemia cells. Treatment with these inhibitors decreased MYC expression, induced caspase3 and PARP cleavage, and suppressed mTOR signaling. The treatment also suppressed NOTCH1 signaling in T-ALL cells., Conclusion: RAS inhibitors can be repurposed as molecular-targeted drugs for leukemia. However, the concentrations of the inhibitors were much higher than those in the plasma of patients with hypertension. Therefore, further investigation is required for their clinical use., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Ferroptosis refers to an iron-dependent mechanism of regulated cell death that is attributable to lipid peroxidation. Ferroptosis has been documented as a therapeutic target for various solid cancers; nonetheless, its implication in leukemia remains ambiguous. Therefore, this study aimed at investigating the impact of ferroptosis inducers and inhibitors on in vitro leukemia cell line proliferation., Materials and Methods: Six leukemia cell lines, including acute myeloid leukemia (AML)-derived MV4-11, THP-1, HL-60, and U-937, and T-lymphoblastic leukemia (T-ALL)-derived Jurkat and KOPT-K1 with activating NOTCH1 mutations, were assessed. Erastin, which interrupts cystine uptake and depletes intracellular glutathione, and RAS-selective lethal 3 (RSL3), which suppresses glutathione peroxidase 4 (GPX4), were employed as ferroptosis inducers. Lipid peroxidation-arresting ferrostatin-1 and deferoxamine were used as ferroptosis inhibitors. Cells were cultured with these compounds and cell proliferation was assessed using a colorimetric assay. Additionally, signaling protein expression was monitored using immunoblotting, and the outcome of GPX4 knockdown was evaluated., Results: Ferroptosis inducers suppressed proliferation in all cell lines except THP-1 for Erastin and THP-1 and Jurkat for RSL3. Although the ferroptosis inhibitors did not affect cell proliferation, they rescued inducer-mediated growth suppression. Ferroptosis inducers impeded MYC and cyclin D3 expression in certain cell lines and NOTCH1 signaling in T-ALL cells. GPX4 knockdown and RSL3 treatment interrupted MYC and cyclin D3 expression, respectively, in four cell lines., Conclusion: Ferroptosis inducers may serve as potential candidates for novel molecular therapy against AML and T-ALL., (Copyright © 2024 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: MYC proto-oncogene bHLH transcription factor (MYC) proteins function as transcription factors by binding to MYC-associated factor X (MAX) proteins and are involved in various cancer growth, including leukaemia. This study aimed to examine the effects of synthetic MYC inhibitors, which block the MYC-MAX complex formation, in in vitro human acute leukaemia cell lines., Materials and Methods: Four cell lines, OCI/AML2 derived from acute myeloid leukaemia, NALM-6 from B-lymphoblastic leukaemia, and KOPT-K1 and Jurkat from notch receptor 1 (NOTCH1)-mutated T-lymphoblastic leukaemia (T-ALL), were treated with the small-molecule MYC inhibitors 10058-F4 and MYCi975. The expression of cell proliferation and signalling proteins was studied., Results: These inhibitors suppressed the growth of leukaemia cell lines. Treatment with the two inhibitors down-regulated the protein expression of c-MYC, MAX, and activating enhancer-binding protein 4 (AP4) in all cell lines. Up-regulation of p27 and p21 was observed only in 10058-F4-treated OCI/AML2 cells and MYCi975-treated KOPT-K1 cells. These two inhibitors down-regulated the expression of NOTCH1, cleaved NOTCH1, and hes family bHLH transcription factor 1 (HES1) in both T-ALL cell lines., Conclusion: MYC inhibitors appear to be novel molecular-targeted drugs against acute leukaemia, including NOTCH1-mutated T-ALL. However, it is necessary to elucidate the precise molecular mechanisms of these effects before clinical use., (Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: TYRO3 is a member of the TAM family (TYRO3, AXL, and MERTK) of receptor tyrosine kinases. While the roles of activated AXL and MERTK in the growth of leukaemia cells have been reported, the effect of TYRO3 has not been determined. Therefore, we examined the effects of TYRO3 knockdown on the growth of leukaemia cell lines., Materials and Methods: Three human leukaemia cell lines (AA derived from pure erythroid leukaemia, OCI/AML2, and K562), which express TYRO3 protein were used in this study. To induce TYRO3 knockdown, small interfering RNA (siRNA) against TYRO3 was transfected using an electroporation system. Cell growth was assessed by a colorimetric assay. The expression levels and activation of various signalling proteins were examined by immunoblotting. Changes in comprehensive gene expression after TYRO3 knockdown were examined by microarray analysis., Results: TYRO3 knockdown suppressed cell growth in the leukaemia cell lines tested. Additionally, the knockdown suppressed phosphorylation of signal transducer and activator of transcription-3 in AA cells, and extracellular signal-regulated kinase (ERK) 1/2 in AA and OCI/AML2 cells; both are downstream molecules of TYRO3 signalling. TYRO3 knockdown also suppressed the expression of survivin in all the cell lines. TYRO3 knockdown potently suppressed TYRO3 mRNA expression but not that of AXL and MERTK. Furthermore, TYRO3 knockdown suppressed cyclin D1 mRNA expression, which is a downstream molecule of ERK., Conclusion: TYRO3 plays a role in leukaemia cell growth and is a potential therapeutic target for leukaemia., (Copyright © 2022 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Homeobox A9 (HOXA9), a transcription factor regulating haematopoiesis and leukaemia cell proliferation, is suggested as a driver of acute myeloid leukaemia (AML). The aim of this study was to examine the effects of a synthetic HOXA9 inhibitor DB818 on AML cells in vitro., Materials and Methods: AML cell lines OCI/AML3, MV4-11, and THP-1 with gene mutations up-regulating HOXA9 expression were treated with DB818 and analysed for cell proliferation and gene expression. The effects of HOXA9 knockdown were also evaluated., Results: In the three AML cell lines, DB818 suppressed growth, induced apoptosis, and down-regulated the expression of HOXA9 transcriptional target genes: MYB proto-oncogene, transcription factor (MYB), MYC proto-oncogene, bHLH transcription factor (MYC), and BCL2 apoptosis regulator (BCL2), while up-regulating that of Fos proto-oncogene, AP-1 transcription factor subunit (FOS). HOXA9 knockdown showed similar effects, except for MYC expression, which differed between DB818-treated and HOXA9-deficient OCI/AML3 cells, suggesting an off-target effect of DB818., Conclusion: DB818 has potential as a novel molecular targeted drug for treating AML associated with HOXA9 overexpression., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: The deacetylase sirtuin1 (SIRT1) inhibits tumor suppressor p53 and may promote tumorigenesis; however, SIRT1 effects on leukemia cells are controversial. The aim of this study was to clarify the activity of SIRT1 in leukemia cells., Materials and Methods: The effects of SIRT1 inhibition or activation and SIRT1 knockdown or overexpression were examined in two T cell acute lymphoblastic leukemia (T-ALL) cell lines carrying NOTCH1 mutations and three acute myeloid leukemia (AML) cell lines., Results: The growth of T-ALL cells was promoted by SIRT1 inhibition and SIRT1 knockdown but was reduced by SIRT1 activation and overexpression; however, no effects were observed in AML cells. SIRT1 activation decreased NOTCH, NF-κB, and mTOR signaling and inhibited p53, suggesting that the possible mechanisms of T-ALL growth suppression by SIRT1 are independent of p53., Conclusion: SIRT1 activators acting through the down-regulation of NOTCH, NF-κB, and mTOR pathways can be novel targeted drugs for NOTCH1-mutated T-ALLs., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Aim: To examine the influence of hypoxia on the in vitro growth of leukaemia cells and the activity of signalling proteins to better understand the pathophysiology of leukaemia cells in human bone marrow., Materials and Methods: Six human leukaemia cell lines were cultured under normoxic or hypoxic conditions. Cell growth, recovery of clonogenic cells, and the expression and activation of various signalling proteins were examined., Results: Hypoxia suppressed cell growth and the recovery of clonogenic cells. Moreover, hypoxia up-regulated hypoxia-inducible factor (HIF) 1α and HIF2α expression while suppressing the expression and activation of NOTCH1, mechanistic target of rapamycin kinase (mTOR) activation, and nuclear factor-kappa B (NF-κB) phosphorylation., Conclusion: We found that hypoxia up-regulated HIF expression while it suppressed the self-renewal capacity of leukaemia cells, NOTCH activity, and expression of its down-stream signalling molecules, which differs from previous reports mentioning that HIF activates NOTCH signalling. Our findings serve to further elucidate the in vivo pathophysiology of leukaemia cells., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: This study examined whether and how chloroquine inhibits blast progenitor self-renewal (SR) synergistically with phytochemicals or nonsteroidal anti-inflammatory drugs in seven hematological malignant cell lines., Materials and Methods: Vitamin C, resveratrol, cyclo-oxygenase inhibitor NS-398 and indomethacin heptyl ester (Ind) were added to cell culture with or without 3 μM chloroquine., Results: Chloroquine synergistically inhibited blast colony formation in methylcellulose with vitamin C, resveratrol, NS-398 and Ind in one, two, none and one cell lines, respectively, in a total of four out of 28 conditions. Chloroquine synergistically inhibited blast progenitor SR in suspension with vitamin C, resveratrol, NS-398 and Ind in four, six, one and five cell lines, respectively, in a total of 16 out of 28 conditions. In contrast, chloroquine abolished SR inhibition by another agent in four out of 28 conditions., Conclusion: Chloroquine exerted a marked synergistic inhibition of blast progenitor SR, but not blast colony formation., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Hedgehog (HH), WNT, NOTCH, and mechanistic target of rapamycin (mTOR) signalling pathways are known to regulate the progression of cancer; however, their interaction in leukaemia cells is not fully clarified., Materials and Methods: Myeloid and T-lymphoblastic leukaemia cell lines (NB4, THP-1, Jurkat, and DND-41) were transfected with small interfering RNAs targeting the glioma-associated oncogene homolog 1 (GLI1) and catenin beta-1 (CTNNB1) genes involved in the regulation of HH and WNT pathways, respectively, and we examined cell proliferation and gene expression., Results: The knockdown of GLI1 and CTNNB1 did not significantly affect proliferation of any cell line; however, it up-regulated the expression of NOTCH1, cleaved NOTCH1 fragment, and phosphorylated mTOR in NB4 cells, but not in the other cell lines., Conclusion: Our data suggest that HH and WNT act upstream of NOTCH and mTOR pathways and negatively regulate them in myeloid NB4 cells. Further studies are required to determine the biological significance of this signalling crosstalk in leukaemia., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase that affects cancer cell proliferation. This study evaluated the effects of the synthetic MERTK inhibitors UNC569 and UNC1062 on in vitro growth of acute myeloid leukaemia (AML) cells., Materials and Methods: Four AML cell lines expressing MERTK were treated with UNC569 and UNC1062 and analyzed for cell proliferation, immunoblotting, and gene expression. The effects of MERTK knockdown were also evaluated., Results: Treatment with the inhibitors suppressed cell growth and induced apoptosis in all cell lines. OCI/AML5 and TMD7 cells, in which MERTK was constitutively phosphorylated by autocrine mechanisms, were highly susceptible to these inhibitors. The treatment reduced the phosphorylation of MERTK and its down-stream signalling molecules, v-akt murine thymoma viral oncogene homolog 1 (AKT) and extracellular signal-regulated kinase (ERK). Similar effects were observed after MERTK knockdown. The inhibitors and the knockdown caused similar changes in mRNA expression., Conclusion: These MERTK inhibitors are potential molecular-targeted drugs for treating AML expressing constitutively phosphorylated MERTK., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Myeloid differentiation primary response gene 88 (MYD88), which activates the nuclear factor kappa B (NF-κB) pathway, is important for the growth of lymphoma and leukaemia cells. In this study, we investigated the effects of ST2825, a synthetic peptidomimetic compound which inhibits MYD88 homodimerization, on their growth., Materials and Methods: Seven lymphoma and leukaemia cell lines including TMD8, a B-cell lymphoma line with MYD88-activating mutation, were treated with ST2825 and analysed for cell proliferation and expression of NF-κB signalling-related molecules., Results: ST2825 suppressed the growth of all cell lines by inducing apoptosis and down-regulating phosphorylation of NF-κB pathway components inhibitor of nuclear factor kappa B kinase (IκB) and reticuloendotheliosis oncogene A (RelA), as well as of MYD88 activator Bruton tyrosine kinase (BTK), suggesting that MYD88 may affect BTK activity. ST2825 effects were specific as MYD88-targeting siRNA also suppressed phosphorylation of NF-κB signalling proteins and BTK in TMD8 cells., Conclusion: ST2825 may be a novel drug targeting not only B-lymphoid malignancies with MYD88 mutations, but also lymphoma and leukaemia with wild-type MYD88., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: B cell-specific Moloney murine leukemia virus integration site 1 (BMI1) is up-regulated in several cancers; therefore, we investigated the effects of BMI1 inhibitors on leukemia cells., Materials and Methods: Four acute myeloid leukemia and two T-lymphoblastic leukemia cell lines were treated with BMI1 inhibitors artemisinin, PRT4165, and PTC-209 and analyzed for cell proliferation and gene expression by microarray and immunoblotting., Results: PTC-209 and PRT4165 suppressed the growth of all cell lines through apoptosis.Artemisinin acted only on Jurkat cells. BMI1 inhibitors and BMI1-specific siRNA down-regulated the expression of NOTCH signaling proteins NOTCH1, HES1, and MYC. All but one cell lines did not have the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene targeted by BMI1, thus the inhibitors acted through CDKN2A-independent pathways., Conclusion: BMI1 inhibition suppressed proliferation of leukemia cells through NOTCH signaling which functions downstream of BMI1, suggesting that BMI1 inhibitors can be candidate targeted drugs against leukemia., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Hedgehog (HH) and NOTCH pathways are involved in the regulation of cancer stem cells and haematopoietic malignancies. However, the effects of HH stimulation on cell growth and NOTCH signalling in acute T-lymphoblastic leukaemia (T-ALL) cells have not been elucidated., Materials and Methods: Two T-ALL cell lines, Jurkat and KOPT-K1 harbouring activating NOTCH1 mutations, were cultured with recombinant Sonic (S) HH and analysed for proliferation, colony formation, and expression of NOTCH-regulated genes and proteins., Results: SHH stimulation did not affect cell growth but suppressed colony formation, increased the levels of cleaved NOTCH1 fragment characteristic for NOTCH1 activation, and upregulated mRNA expression of HES1, while decreasing that of MYC in Jurkat cells. However, no such effects were observed in KOPT-K1 cells., Conclusion: Our results indicate that SHH stimulation activates NOTCH signalling in Jurkat cells, thus disclosing a novel relationship between HH and NOTCH pathways., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

NOTCH activation plays oncogenic roles in acute T-lymphoblastic leukaemia (T-ALL). However, whether NOTCH is oncogenic or tumor-suppressive in acute myeloid leukaemia (AML) is still controversial. Herein, the roles of NOTCH in AML are reviewed. AML cells express NOTCH and NOTCH ligands; however, cell-autonomous activation is not observed. Activating NOTCH1 mutations are rare in AML, unlike in T-ALL. NOTCH ligand stimulation generally suppresses the in vitro growth of AML cells but promotes transient growth of some samples. Conversely, knockdown of NOTCH1 and NOTCH2 does not affect the growth of AML cells, whereas it suppresses the growth of T-ALL cells. These findings suggest that NOTCH is dispensable or suppressive for AML cell growth. However, the effects of NOTCH differ depending on cell conditions, and various stemness-related signals modify these effects; hence, forced NOTCH activation in vitro may not exhibit effects in bone marrow. Thus, further understanding is required for the development of AML therapies targeting NOTCH signalling., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Signaling induced by binding of erythropoietin-producing hepatoma-amplified sequence (EPH) receptors to their cell-surface ephrin ligands is implicated in hematopoiesis and growth of various cancer cells. However, the roles of EPH-ephrin signaling in leukemia have not been elucidated. We investigated the effects of EPHB4 and ephrin B2 on the growth of leukemia cells., Materials and Methods: Seven human leukemia cell lines were used to examine the effects of recombinant ephrin B2 and EPHB4 on cell proliferation by colorimetric WST-1 assay and colony assays; on protein tyrosine phosphorylation; and on mRNA expression by reverse transcription-polymerase chain reaction and microarray analysis., Results: In an erythroid leukemia-derived cell line AA, exogenous ephrin B2 induced proliferation and colony formation; in addition, it up-regulated protein tyrosine phosphorylation and the expression of growth-related genes such as FBJ murine osteosarcoma viral oncogene homolog B and v-src avian sarcoma viral oncogene homolog., Conclusion: Growth-promoting effects of ephrin B2 were observed in an erythroid leukemia cell line, suggesting that the EPH-ephrin signaling may be involved in the pathology of leukemia., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Aim: The effects of small interfering RNA (siRNA)-mediated knockdown of NOTCH1 and NOTCH2 on cell proliferation and downstream signaling pathways in leukemia cells were examined., Materials and Methods: Two T-lymphoblastic leukemia (T-ALL) cell lines and two acute myeloblastic leukemia (AML) cell lines were transfected with siRNAs targeting NOTCH1 and NOTCH2. The effects of knockdown on cell proliferation and protein expression were examined by colorimetric WST-8 assay and immunoblotting, respectively., Results: In T-ALL cell lines, NOTCH1 knockdown as well as NOTCH2 knockdown suppressed cell proliferation and induced apoptosis. v-Myc avian myelocytomatosis viral oncogene homolog (MYC) protein expression was down-regulated in NOTCH1-knockdown cells but not affected in NOTCH2-knockdown cells. In AML cell lines, cell proliferation was not significantly affected by NOTCH siRNAs. NOTCH2 knockdown increased the level of cleaved NOTCH1 fragment without increasing NOTCH1 expression. NOTCH knockdown reduced the level of mechanistic target of rapamycin (mTOR) protein in the monoblastic leukemia cell line THP-1. Contrastingly, NOTCH activation by NOTCH ligand stimulation increased the expression of mTOR in THP-1 cells., Conclusion: These novel findings on NOTCH signaling may contribute to the development of effective NOTCH-targeted therapies against leukemia.

Aim: To examine the effects of echinomycin, a compound that inhibits DNA-binding activity of hypoxia-inducible factor-1 (HIF1), on leukaemia cell growth., Materials and Methods: Three acute myeloid leukaemia cell lines and three T-lymphoblastic leukaemia cell lines were cultured with echinomycin. Cell growth, mRNA and protein expression levels were examined by WST-1 assay, reverse-transcription polymerase chain reaction and immunoblotting, respectively., Results: HIF1α protein was expressed in all cell lines under normoxia. Treatment with echinomycin suppressed cell growth and induced apoptosis in association with decreased mRNA expression of HIF1 targets, glucose transporter-1 (GLUT1) and B-cell CLL/lymphoma-2 (BCL2). Echinomycin also suppressed the protein expression of NOTCH1, cleaved NOTCH1, v-myc myelocytomatosis viral oncogene homolog (MYC), v-akt murine thymoma viral oncogene homolog-1 (AKT), phosphorylated AKT, mechanistic target of rapamycin (mTOR), and phosphorylated mTOR and increased that of cleaved caspase-3 in some cell lines., Conclusion: Echinomycin suppresses leukaemia cell growth in association with reduced NOTCH1 expression. This is the first report to show that HIF inhibitor treatment suppresses NOTCH1 signalling. HIF inhibitors could be novel candidates for a molecular-targeted therapy against leukaemia.

Aim: PP242 is a compound which inhibits both mammalian target of rapamycin complex-1 (mTORC1) and mTORC2. We examined the effects of PP242 and rapamycin on mTOR signalling and evaluated potential crosstalk with the NOTCH signalling in eight leukemia cell lines., Materials and Methods: We examined the effects of treatment with these inhibitors on cell growth and protein expression., Results: PP242 suppressed growth more potently than did rapamycin. In two cell lines poorly sensitive to PP242, PP242 failed to inhibit v-akt murine thymoma viral oncogene homolog (AKT) phosphorylation. Suppression of mTOR phosphorylation was weaker in myeloid cell lines. Rapamycin induced eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) hyperphosphorylation in three cell lines. Phosphorylation of both isoforms (p70 and p85) of S6 kinase (S6K) was suppressed in three cell lines; only p70 was suppressed in the others. NOTCH1 expression and activation were up-regulated by PP242 in one cell line but down-regulated in another., Conclusion: PP242 is a candidate for molecular-targeted leukemia therapy, although its effects must be evaluated on a case-by-case basis. Crosstalk was found between the mTOR and NOTCH signalling pathways.

Background: Bone morphogenetic protein 4 (BMP4) signaling is involved in the maintenance of hematopoietic stem cells. However, the effects of BMP4 on leukemia and lymphoma cells are unknown., Materials and Methods: The effects of recombinant BMP4 on the in vitro growth of 12 leukemia and lymphoma cell lines were examined., Results: BMP4 treatment promoted the short-term growth of three cell lines and suppressed the growth of one. Induction of differentiation was not observed. BMP4 treatment suppressed the clonogenicity of four out of the six examined cell lines. BMP4 treatment promoted the growth of Jurkat cells but suppressed their ability to form colonies. BMP4 treatment up-regulated the phosphorylation of SMAD1/5/8 complex, indicating that BMP4 mediated signal transduction in the cells., Conclusion: BMP4 suppressed the clonogenicity of selected leukemia and lymphoma cell lines. The regulation of BMP4 signaling may be a useful therapeutic approach for leukemia and lymphoma, if appropriate cases are selected.

Background: Hedgehog (Hh) signaling is involved in cancer cell growth. However, the effects of Hh stimulation on leukemia cells are unknown., Materials and Methods: The effects of recombinant sonic Hedgehog (Shh) protein on the in vitro growth of one B-lymphoma and four myeloid leukemia cell lines were examined., Results: Shh stimulation had no significant effect on the short-term growth of whole cell populations in any of the five cell lines. However, Shh promoted clonogenic cell recovery after suspension culture, suggesting promotion of leukemia stem or progenitor cell amplification in three cell lines. The lack of Hh receptors in one cell line and endogenous Shh expression in another were possible reasons for the lack of effects of Shh in these cases., Conclusion: These results suggest that Shh stimulation promotes the self-renewal capacity of leukemia stem cells in some cell lines. Inhibition of Hh signaling could represent a novel therapeutic approach in leukemia.

Background: The Notch inhibitors, γ-secretase inhibitors (GSIs), are promising candidates for molecular targeted therapy against leukemia. However, they show only limited effectiveness. We thought that the efficacy of GSIs might be improved by their combination with Hedgehog inhibitors and Wnt inhibitors because these signaling pathways are also important for the growth of leukemia cells., Materials and Methods: The effects of the combination of GSI-XXI plus the Hedgehog inhibitor, cyclopamine (Cy), or the Wnt inhibitor, quercetin (Qu), on the in vitro cell growth, colony formation and Notch1 protein expression of three T-cell acute lymphoblastic leukemia (T-ALL) cell lines with NOTCH1 mutations and three acute myeloid leukemia cell lines were examined., Results: The addition of Cy or Qu to GSI suppressed the growth of DND-41 T-ALL cells additively or synergistically, respectively. Interestingly, Cy treatment and Qu treatment reduced Notch1 protein and its active fragment in DND-41 cells, which suggests a relationship between Notch signaling and Hedgehog or Wnt signaling. The addition of Cy or Qu to GSI promoted the decrease of Notch1 activation and expression., Conclusion: The anti-leukemic effects of a GSI could be promoted by its combination with Cy or Qu, in appropriate cases selected by in vitro drug sensitivity test.

Background: Notch signaling regulates the fate of hematopoietic stem cells and leukemia cells. However, the role of Notch in erythroid differentiation remains unclear., Materials and Methods: We examined the effects of three γ-secretase inhibitors (GSI-IX, GSI-XII and GSI-XXI) that inhibit Notch signaling on the in vitro growth and differentiation of HEL and AA erythroid leukemia cell lines., Results: GSI treatment induced morphologic erythroid differentiation and promoted hemoglobin production. GSI treatment suppressed short-term growth and colony formation, while treatment with GSI-XXI promoted the growth of AA cells. The degree of differentiation induced by each GSI roughly correlated with the reduction in HES1 mRNA expression., Conclusion: GSIs have potential uses in differentiation induction therapy for erythroid leukemia in the future. Before clinical use, in vitro sensitivity tests should be performed because the effects of GSIs are diverse depending upon the combination of leukemia cells and GSIs.

Background: Notch activation is involved in the growth of leukemia cells. gamma-Secretase inhibitors (GSIs), which block Notch activation, may be candidates for molecular target therapy against leukemia., Materials and Methods: The effects of three kinds of GSIs: GSI-IX, GSI-XII and GSI-XXI, on the in vitro growth of various leukemia cell lines were examined., Results: The effects of GSI were diverse depending upon the combination of cells and GSI. GSI treatment suppressed the growth of most of the cell lines examined. Conversely, the growth of some cell lines were promoted by GSI-XXI. GSI-XXI treatment reduced the amount of cleaved Notch1 protein and HES1 mRNA in the cells, which means that it suppressed Notch activity. The treatment up-regulated mRNA of nuclear factor kappa-B1 (NFKB1) and v-rel reticuloendotheliosis viral oncogene homolog A (RELA), which can be a cause of growth promotion., Conclusion: The diverse effects of GSIs must be elucidated before clinical use because they can stimulate the growth of leukemia cells.

Background: Hedgehog (Hh) and Wnt signaling pathways are involved in the stimulation of growth of leukemia and lymphoma cells. In the present study, whether or not the Hh inhibitor, cyclopamine, and the Wnt inhibitor, quercetin, suppress cell growth was investigated., Materials and Methods: The effects of cyclopamine and quercetin on the in vitro growth and protein expression of ten acute leukemia and B-cell lymphoma cell lines were examined., Results: Cyclopamine and quercetin suppressed cell growth and induced apoptosis in seven and eight cell lines respectively. Cyclopamine decreased the level of Gli1 protein, a target gene product of Hh signaling. Quercetin decreased the level of Notch1 protein and its active fragment in the DND-41 T-lymphoblastic leukemia cell line with constitutive Notch activation., Conclusion: Cyclopamine and quercetin suppress the growth of a number of leukemia and lymphoma cells. This finding suggests the potential use of these compounds in molecularly-targeted therapy for leukemia and lymphoma.

Background: Notch activation by ligand stimulation regulates the growth of acute myeloid leukemia (AML) cells. However, the molecular mechanisms underlying this have not been fully elucidated., Materials and Methods: Two AML cell lines, THP-1 and TMD7, and three Notch ligands, Jagged1, Dll1 and Dll4, were used. The effects of the ligands on cell growth, cleavage of Notch1, and the expression of various genes were examined by cell culture, immunoblotting, and quantitative RT-PCR, respectively., Results: Ligand stimulation suppressed the growth of the THP-1 cells but promoted that of the TMD7 cells. Immunoblots showed two cleaved Notch1 bands, which became intense with different peak time from the start of stimulation. Ligand stimulation changed the expression levels of various genes related to cell proliferation. Some genes changed in opposite directions in the two cell lines. The three ligands had similar effects on gene expression., Conclusion: The time course for Notch1 cleavage following ligand stimulation and the effects of stimulation on gene expression are shown.

Background: Wnt/beta-catenin signaling is involved in the growth of various types of cancer cells. Wnt3A has been reported to promote the self-renewal of hematopoietic stem cells., Materials and Methods: The effects of recombinant Wnt3A protein on the in vitro growth of four acute myeloid leukemia (AML) and four acute T-lymphoblastic leukemia (T-ALL) cell lines was examined., Results: Wnt3A stimulation either had no effect on, or slightly suppressed, the short-term growth of these cell lines. In three cell lines, Wnt3A promoted clonogenic cell recovery after suspension culture, suggesting the promotion of the self-renewal capacity of leukemic stem or progenitor cells. Immunoblot analysis showed that Wnt3A stimulation reduced phosphorylated beta-catenin and increased beta-catenin in these cells, indicating that Wnt3A stimulation activated Wnt/beta-catenin signaling., Conclusion: Wnt3A stimulation did not promote the growth of whole cell populations, but did promote the self-renewal of leukemic stem/progenitor cells in some AML and T-ALL cell lines.