RESUMEN
Leukemia is a kind of hematological malignancy originating from bone marrow, which provides essential signals for initiation, progression, and recurrence of leukemia. However, how to specifically deliver drugs to the bone marrow remains elusive. Here, we develop biomimetic vesicles by infusing hematopoietic stem and progenitor cell (HSPC) membrane with liposomes (HSPC liposomes), which migrate to the bone marrow of leukemic mice via hyaluronic acid-CD44 axis. Moreover, the biomimetic vesicles exhibit superior binding affinity to leukemia cells through intercellular cell adhesion molecule-1 (ICAM-1)/integrin ß2 (ITGB2) interaction. Further experiments validate that the vesicles carrying chemotherapy drug cytarabine (Ara-C@HSPC-Lipo) markedly inhibit proliferation, induce apoptosis and differentiation of leukemia cells, and decrease number of leukemia stem cells. Mechanically, RNA-seq reveals that Ara-C@HSPC-Lipo treatment induces apoptosis and differentiation and inhibits the oncogenic pathways. Finally, we verify that HSPC liposomes are safe in mice. This study provides a method for targeting bone marrow and treating leukemia.
Asunto(s)
Apoptosis , Médula Ósea , Citarabina , Sistemas de Liberación de Medicamentos , Células Madre Hematopoyéticas , Leucemia , Liposomas , Animales , Células Madre Hematopoyéticas/efectos de los fármacos , Células Madre Hematopoyéticas/metabolismo , Ratones , Citarabina/farmacología , Médula Ósea/efectos de los fármacos , Médula Ósea/patología , Médula Ósea/metabolismo , Apoptosis/efectos de los fármacos , Leucemia/tratamiento farmacológico , Leucemia/patología , Humanos , Diferenciación Celular/efectos de los fármacos , Membrana Celular/metabolismo , Membrana Celular/efectos de los fármacos , Línea Celular Tumoral , Antígenos CD18/metabolismo , Proliferación Celular/efectos de los fármacos , Receptores de Hialuranos/metabolismo , Ácido Hialurónico/química , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/metabolismoRESUMEN
Knowledge of the molecular pathogenesis of acute myeloid leukemia has advanced in recent years. Despite novel treatment options, acute myeloid leukemia remains a survival challenge for elderly patients. We have recently shown that the triphosphohydrolase SAMHD1 is one of the factors determining resistance to Ara-C treatment. Here, we designed and tested novel and simpler virus-like particles incorporating the lentiviral protein Vpx to efficiently and transiently degrade SAMHD1 and increase the efficacy of Ara-C treatment. The addition of minute amounts of lentiviral Rev protein during production enhanced the generation of virus-like particles. In addition, we found that our 2nd generation of virus-like particles efficiently targeted and degraded SAMHD1 in AML cell lines with high levels of SAMHD1, thereby increasing Ara-CTP levels and response to Ara-C treatment. Primary AML blasts were generally less responsive to VLP treatment. In summary, we have been able to generate novel and simpler virus-like particles that can efficiently deliver Vpx to target cells.
Asunto(s)
Citarabina , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Citarabina/farmacología , Citarabina/uso terapéutico , Proteína 1 que Contiene Dominios SAM y HD/metabolismo , Proteína 1 que Contiene Dominios SAM y HD/genética , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/genética , Línea Celular Tumoral , Lentivirus/genéticaRESUMEN
Parkinson's disease (PD) is a common age-related neurodegenerative disorder, which may be largely due to the mitochondrial dysfunction and impaired mitophagy. Thus, it is of great importance to seek novel therapeutic strategies for PD targeting mitochondrial function and mitophagy. Cytarabine is a marine-derived antimetabolite used in the treatment of acute leukemia, which is also used in the study of the nervous system. In this study, we found that cytarabine pretreatment significantly inhibited the apoptosis and necrosis in the ROT-induced SH-SY5Y cell PD model and reduced the oxidative stress, as evidenced by the reduced MDA levels and the increased levels of SOD, GSH, and total antioxidant capacity. Cytarabine can also enhance mitochondrial vitality, improve mitochondrial respiratory function, and preserve mitochondrial morphology. Cytarabine also enhanced the expression of the mitophagy-related proteins PINK1, Parkin, VDAC1, and DJ-1, and its actions can be reversed by treatment with AMPK inhibitor - Compound C (CC), suggesting that AMPK activation may be involved in cytarabine-enhanced mitophagy. Furthermore, cytarabine can also ameliorate the motor symptoms in the MPTP-induced PD-like mice model, and attenuate the neuropathy in the substantia nigra (SN) of PD mice, while Compound C antagonized cytarabine's beneficial effects. In summary, marine-derived compound cytarabine could resist neurological damage both in vitro and in vivo by activating AMPK to increase PINK1/Parkin-induced mitophagy, serving as a promising disease modulator for treating neurodegenerative disease.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Citarabina , Modelos Animales de Enfermedad , Mitofagia , Proteínas Quinasas , Ubiquitina-Proteína Ligasas , Animales , Mitofagia/efectos de los fármacos , Proteínas Quinasas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Humanos , Ratones , Línea Celular Tumoral , Proteínas Quinasas Activadas por AMP/metabolismo , Masculino , Citarabina/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Ratones Endogámicos C57BL , Estrés Oxidativo/efectos de los fármacos , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Neuronas/efectos de los fármacos , Neuronas/patología , Neuronas/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Apoptosis/efectos de los fármacosRESUMEN
OBJECTIVE: To investigate the effect of TLK2 expression regulated by miR-21 on proliferation and apoptosis of acute myeloid leukemia cells. METHODS: Seventy patients with AML admitted to our hospital from January 2019 to July 2022 were selected, while 30 patients with iron deficiency anemia were selected as the control group. Bone marrow mononuclear cells (BMMNCs) of the patients were obtained using Ficoll density gradient centrifugation. RT-qPCR was used to determine the expression levels of miR-21 and TLK2 mRNA in BMMNCs. Mimics-miR-21, mimics-NC, inhibitor-miR-21, inhibitor-NC and NC were transfected into HL-60 cells using liposome-mediated transfection technology. CCK-8 method was used to determine the activity of transfected HL-60 cells after treatment with cytarabine. The apoptosis rate of HL-60 transfected cells was determined by TUNEL method. The expression of TLK2 mRNA in HL-60 cells transfected with inhibitor-miR-21 was determined by RT-qPCR. RESULTS: The relative expression levels of miR-21 and TLK2 mRNA in BMMNCs of AML patients were significantly higher than those of controls (both P < 0.05). After HL-60 cells were treated with cytarabine, both the cell activity of inhibitor-miR-21 group and mimics-miR-21 group decreased significantly with the increase of cytarabine concentration (both P < 0.05). However, at each concentration point of cytarabine, the cell activity of inhibitor-miR-21 group was lower than that of control group (P < 0.05), while mimics-miR-21 group was higher than control group (P < 0.05). After HL-60 cells were treated with cytarabine, the apoptosis rate of inhibitor-miR-21 group was significantly increased (P < 0.05), while that of mimics-miR-21 group was significantly decreased (P < 0.05). After HL-60 cells were treated with inhibitor-miR-21, the relative expression of TLK2 mRNA decreased significantly (P < 0.05). CONCLUSION: miR-21 is highly expressed in AML patients, which may promote the apoptosis of AML cells by inhibiting the expression of TLK2.
Asunto(s)
Apoptosis , Proliferación Celular , Leucemia Mieloide Aguda , MicroARNs , Humanos , Citarabina/farmacología , Células HL-60 , Leucemia Mieloide Aguda/genética , MicroARNs/genética , TransfecciónRESUMEN
Upregulation of the Wilms' tumour 1 (WT1) gene is common in acute myeloid leukaemia (AML) and is associated with poor prognosis. WT1 generates 12 primary transcripts through different translation initiation sites and alternative splicing. The short WT1 transcripts express abundantly in primary leukaemia samples. We observed that overexpression of short WT1 transcripts lacking exon 5 with and without the KTS motif (sWT1+/- and sWT1-/-) led to reduced cell growth. However, only sWT1+/- overexpression resulted in decreased CD71 expression, G1 arrest, and cytarabine resistance. Primary AML patient cells with low CD71 expression exhibit resistance to cytarabine, suggesting that CD71 may serve as a potential biomarker for chemotherapy. RNAseq differential expressed gene analysis identified two transcription factors, HOXA3 and GATA2, that are specifically upregulated in sWT1+/- cells, whereas CDKN1A is upregulated in sWT1-/- cells. Overexpression of either HOXA3 or GATA2 reproduced the effects of sWT1+/-, including decreased cell growth, G1 arrest, reduced CD71 expression and cytarabine resistance. HOXA3 expression correlates with chemotherapy response and overall survival in NPM1 mutation-negative leukaemia specimens. Overexpression of HOXA3 leads to drug resistance against a broad spectrum of chemotherapeutic agents. Our results suggest that WT1 regulates cell proliferation and drug sensitivity in an isoform-specific manner.
Asunto(s)
Resistencia a Antineoplásicos , Proteínas de Homeodominio , Leucemia Mieloide Aguda , Regulación hacia Arriba , Proteínas WT1 , Humanos , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos CD/biosíntesis , Línea Celular Tumoral , Citarabina/farmacología , Citarabina/uso terapéutico , Resistencia a Antineoplásicos/genética , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Nucleofosmina , Isoformas de Proteínas , Receptores de Transferrina , Proteínas WT1/genética , Proteínas WT1/metabolismo , Proteínas WT1/biosíntesisRESUMEN
Acute Myeloid Leukemia (AML) is a life-threatening disease whose induction treatment consists of combination chemotherapy with Idarubicin and Cytarabine for fit patients. Treatment failures are frequent, urging the need for novel treatments for this disease. The DNA Damage Response Mechanism (DDR) comprises numerous molecules and pathways intended to arrest the cell cycle until DNA damage is repaired or else drive the cell to apoptosis. AML-derived cell lines after treatment with Idarubicin and Cytarabine were used for studying the expression profile of 84 DDR genes, through PCR arrays. Utilizing de novo AML patient and control samples we studied the expression of PPP1R15A, CDKN1A, GADD45A, GADD45G, and EXO1. Next, we performed PPP1R15A silencing in AML cell lines in two separate experiments using siRNA and CRISPR-cas9, respectively. Our findings highlight that DDR regulators demonstrate increased expression in patients with high cytogenetic risk possibly reflecting increased genotoxic stress. Especially, PPP1R15A is mainly involved in the recovery of the cells from stress and it was the only DDR gene upregulated in AML patients. The PPP1R15A silencing resulted in decreased viability of Idarubicin and Cytarabine-treated cell lines, in contrast to untreated cells. These findings shed light on new strategies to enhance chemotherapy efficacy and demonstrate that PPP1R15A is an important DDR regulator in AML and its downregulation might be a safe and effective way to increase sensitivity to chemotherapy in this disease.
Asunto(s)
Citarabina , Daño del ADN , Silenciador del Gen , Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Daño del ADN/efectos de los fármacos , Citarabina/farmacología , Línea Celular Tumoral , Proteína Fosfatasa 1/genética , Proteína Fosfatasa 1/metabolismo , Idarrubicina/farmacología , Idarrubicina/administración & dosificación , Masculino , Femenino , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Reparación del ADN/efectos de los fármacos , Persona de Mediana Edad , Adulto , Anciano , Regulación Leucémica de la Expresión Génica/efectos de los fármacosRESUMEN
Nucleoside analogue drugs are pervasively used as antiviral and chemotherapy agents. Cytarabine and gemcitabine are anti-cancer nucleoside analogue drugs that contain C2' modifications on the sugar ring. Despite carrying all the required functional groups for DNA synthesis, these two compounds inhibit DNA extension once incorporated into DNA. It remains unclear how the C2' modifications on cytarabine and gemcitabine affect the polymerase active site during substrate binding and DNA extension. Using steady-state kinetics, static and time-resolved X-ray crystallography with DNA polymerase η (Pol η) as a model system, we showed that the sugar ring C2' chemical groups on cytarabine and gemcitabine snugly fit within the Pol η active site without occluding the steric gate. During DNA extension, Pol η can extend past gemcitabine but with much lower efficiency past cytarabine. The Pol η crystal structures show that the -OH modification in the ß direction on cytarabine locks the sugar ring in an unfavorable C2'-endo geometry for product formation. On the other hand, the addition of fluorine atoms on gemcitabine alters the proper conformational transition of the sugar ring for DNA synthesis. Our study illustrates mechanistic insights into chemotherapeutic drug inhibition and resistance and guides future optimization of nucleoside analogue drugs.
Asunto(s)
Citarabina , ADN Polimerasa Dirigida por ADN , Desoxicitidina , Gemcitabina , Desoxicitidina/análogos & derivados , Desoxicitidina/química , Desoxicitidina/farmacología , Citarabina/química , Citarabina/farmacología , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , Humanos , Cristalografía por Rayos X , ADN/química , ADN/metabolismo , ADN/biosíntesis , Dominio Catalítico , Replicación del ADN/efectos de los fármacos , CinéticaRESUMEN
Phosphoinositide-3-kinase-γ (PI3Kγ) is implicated as a target to repolarize tumour-associated macrophages and promote antitumour immune responses in solid cancers1-4. However, cancer cell-intrinsic roles of PI3Kγ are unclear. Here, by integrating unbiased genome-wide CRISPR interference screening with functional analyses across acute leukaemias, we define a selective dependency on the PI3Kγ complex in a high-risk subset that includes myeloid, lymphoid and dendritic lineages. This dependency is characterized by innate inflammatory signalling and activation of phosphoinositide 3-kinase regulatory subunit 5 (PIK3R5), which encodes a regulatory subunit of PI3Kγ5 and stabilizes the active enzymatic complex. We identify p21 (RAC1)-activated kinase 1 (PAK1) as a noncanonical substrate of PI3Kγ that mediates this cell-intrinsic dependency and find that dephosphorylation of PAK1 by PI3Kγ inhibition impairs mitochondrial oxidative phosphorylation. Treatment with the selective PI3Kγ inhibitor eganelisib is effective in leukaemias with activated PIK3R5. In addition, the combination of eganelisib and cytarabine prolongs survival over either agent alone, even in patient-derived leukaemia xenografts with low baseline PIK3R5 expression, as residual leukaemia cells after cytarabine treatment have elevated G protein-coupled purinergic receptor activity and PAK1 phosphorylation. Together, our study reveals a targetable dependency on PI3Kγ-PAK1 signalling that is amenable to near-term evaluation in patients with acute leukaemia.
Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib , Leucemia , Transducción de Señal , Quinasas p21 Activadas , Animales , Humanos , Ratones , Línea Celular , Fosfatidilinositol 3-Quinasa Clase Ib/genética , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Citarabina/farmacología , Citarabina/uso terapéutico , Leucemia/tratamiento farmacológico , Leucemia/enzimología , Leucemia/genética , Leucemia/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fosforilación Oxidativa/efectos de los fármacos , Quinasas p21 Activadas/antagonistas & inhibidores , Quinasas p21 Activadas/metabolismo , Fosforilación , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Acute myeloid leukemia is the second most frequent type of leukemia in adults. Due to a high risk of development of chemoresistance to first-line chemotherapy, the survival rate of patients in a 5-year period is below 30%. One of the reasons is that the AML population is heterogeneous, with cell populations partly composed of very primitive CD34+CD38- hematopoietic stem/progenitor cells, which are often resistant to chemotherapy. First-line treatment with cytarabine and idarubicin fails to inhibit the proliferation of CD34+CD38- cells. In this study, we investigated Metformin's effect with or without first-line conventional chemotherapy, or with other drugs like venetoclax and S63845, on primitive and undifferentiated CD34+ AML cells in order to explore the potential of Metformin or S63845 to serve as adjuvant therapy for AML. We found that first-line conventional chemotherapy treatment inhibited the growth of cells and arrested the cells in the S phase of the cell cycle; however, metformin affected the accumulation of cells in the G2/M phase. We observed that CD34+ KG1a cells respond better to lower doses of cytarabine or idarubicin in combination with metformin. Also, we determined that treatment with cytarabine, venetoclax, and S63845 downregulated the strong tendency of CD34+ KG1a cells to form cell aggregates in culture due to the downregulation of leukemic stem cell markers like CD34 and CD44, as well as adhesion markers. Also, we found that idarubicin slightly upregulated myeloid differentiation markers, CD11b and CD14. Treatment with cytarabine, idarubicin, venetoclax, metformin, and S63845 upregulated some cell surface markers like HLA-DR expression, and metformin upregulated CD9, CD31, and CD105 cell surface marker expression. In conclusion, we believe that metformin has the potential to be used as an adjuvant in the treatment of resistant-to-first-line-chemotherapy AML cells. Also, we believe that the results of our study will stimulate further research and the potential use of changes in the expression of cell surface markers in the development of new therapeutic strategies.
Asunto(s)
Antígenos CD34 , Citarabina , Resistencia a Antineoplásicos , Leucemia Mieloide Aguda , Metformina , Humanos , Metformina/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Resistencia a Antineoplásicos/efectos de los fármacos , Antígenos CD34/metabolismo , Línea Celular Tumoral , Citarabina/farmacología , Proliferación Celular/efectos de los fármacos , Sulfonamidas/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Idarrubicina/farmacologíaRESUMEN
Research on the relationships between oligoelements (OE) and the development of cancer or its prevention is a field that is gaining increasing relevance. The aim was to evaluate OE and their interactions with oncology treatments (cytarabine or etoposide) to determine the effects of this combination on biogenic amines and oxidative stress biomarkers in the brain regions of young Wistar rats. Dopamine (DA), 5-Hydroxyindoleacetic acid (5-Hiaa), Glutathione (Gsh), Tiobarbituric acid reactive substances (TBARS) and Ca+2, Mg+2 ATPase enzyme activity were measured in brain regions tissues using spectrophometric and fluorometric methods previously validated. The combination of oligoelements and cytarabine increased dopamine in the striatum but decreased it in cerebellum/medulla-oblongata, whereas the combination of oligoelements and etoposide reduced lipid peroxidation. These results suggest that supplementation with oligoelements modifies the effects of cytarabine and etoposide by redox pathways, and may become promising therapeutic targets in patients with cancer.
Asunto(s)
Encéfalo , Citarabina , Dopamina , Etopósido , Estrés Oxidativo , Ratas Wistar , Animales , Etopósido/farmacología , Estrés Oxidativo/efectos de los fármacos , Citarabina/farmacología , Dopamina/metabolismo , Ratas , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Masculino , Peroxidación de Lípido/efectos de los fármacos , Suplementos Dietéticos , Glutatión/metabolismoRESUMEN
Acute myeloid leukemia (AML) is one of the most prevalent blood cancers, characterized by a dismal survival rate. This poor outcome is largely attributed to AML cells that persist despite treatment and eventually result in relapse. Relapse-initiating cells exhibit diverse resistance mechanisms, encompassing genetic factors and, more recently discovered, nongenetic factors such as metabolic adaptations. Leukemic stem cells (LSC) rely on mitochondrial metabolism for their survival, whereas hematopoietic stem cells primarily depend on glycolysis. Furthermore, following treatments such as cytarabine, a standard in AML treatment for over four decades, drug-persisting leukemic cells exhibit an enhanced reliance on mitochondrial metabolism. In this issue of Cancer Research, two studies investigated dependencies of AML cells on two respiratory substrates, α-ketoglutarate and lactate-derived pyruvate, that support mitochondrial oxidative phosphorylation (OXPHOS) following treatment with the imipridone ONC-213 and the BET inhibitor INCB054329, respectively. Targeting lactate utilization by interfering with monocarboxylate transporter 1 (MCT1 or SLC16A1) or lactate dehydrogenase effectively sensitized cells to BET inhibition in vitro and in vivo. In addition, ONC-213 affected αKGDH, a pivotal NADH-producing enzyme of the TCA cycle, to induce a mitochondrial stress response through ATF4 activation that diminished the expression of the antiapoptotic protein MCL1, consequently promoting apoptosis of AML cells. In summary, targeting these mitochondrial dependencies might be a promising strategy to kill therapy-naïve and treatment-resistant OXPHOS-reliant LSCs and to delay or prevent relapse. See related articles by Monteith et al., p. 1101 and Su et al., p. 1084.
Asunto(s)
Leucemia Mieloide Aguda , Humanos , Leucemia Mieloide Aguda/genética , Citarabina/farmacología , Ciclo del Ácido Cítrico , Lactatos , RecurrenciaRESUMEN
Acute myeloid leukaemia (AML) is a fatal haematopoietic malignancy and is treated with the conventional combination of cytarabine (Ara-C) and daunorubicin (Dau). The survival rate of AML patients is lower due to the cardiotoxicity of daunorubicin. Clinically, homoharringtonine (HHT) plus Ara-C has been reported to be equally effective as Dau plus Ara-C in some types of AML patients with less toxic effects. We utilized the clinical use of homoharringtonine in combination with Ara-C to test its combination mechanism. We found that the insensitivity of AML cells to cytarabine-induced apoptosis is associated with increased Mcl-1 stability and p38 inactivation. HHT downregulates Mcl-1, phosphorylates H2AX and induces apoptosis by activating p38 MAPK. Inactivation of p38 through inhibitors and siRNA blocks apoptosis, H2AX phosphorylation and Mcl-1 reduction. HHT enhances Ara-C activation of the p38 MAPK signalling pathway, overcoming Ara-C tolerance to cell apoptosis by regulating the p38/H2AX/Mcl-1 axis. The optimal ratio of HHT to Ara-C for synergistic lethality in AML cells is 1:4 (M/M). HHT synergistically induces apoptosis in combination with Ara-C in vitro and prolongs the survival of xenografts. We provide a new mechanism for AML treatment by regulating the p38 MAPK/H2AX/Mcl-1 axis to improve cytarabine therapy.
Asunto(s)
Apoptosis , Citarabina , Histonas , Homoharringtonina , Leucemia Mieloide Aguda , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Quinasas p38 Activadas por Mitógenos , Humanos , Homoharringtonina/farmacología , Citarabina/farmacología , Proteína 1 de la Secuencia de Leucemia de Células Mieloides/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Leucemia Mieloide Aguda/genética , Apoptosis/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Ratones , Histonas/metabolismo , Línea Celular Tumoral , Sinergismo Farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Fosforilación/efectos de los fármacos , FemeninoRESUMEN
Adjuvant treatment for Glioblastoma Grade 4 with Temozolomide (TMZ) inevitably fails due to therapeutic resistance, necessitating new approaches. Apoptosis induction in GB cells is inefficient, due to an excess of anti-apoptotic XPO1/Bcl-2-family proteins. We assessed TMZ, Methotrexate (MTX), and Cytarabine (Ara-C) (apoptosis inducers) combined with XPO1/Bcl-2/Mcl-1-inhibitors (apoptosis rescue) in GB cell lines and primary GB stem-like cells (GSCs). Using CellTiter-Glo® and Caspase-3 activity assays, we generated dose-response curves and analyzed the gene and protein regulation of anti-apoptotic proteins via PCR and Western blots. Optimal drug combinations were examined for their impact on the cell cycle and apoptosis induction via FACS analysis, paralleled by the assessment of potential toxicity in healthy mouse brain slices. Ara-C and MTX proved to be 150- to 10,000-fold more potent in inducing apoptosis than TMZ. In response to inhibitors Eltanexor (XPO1; E), Venetoclax (Bcl-2; V), and A1210477 (Mcl-1; A), genes encoding for the corresponding proteins were upregulated in a compensatory manner. TMZ, MTX, and Ara-C combined with E, V, and A evidenced highly lethal effects when combined. As no significant cell death induction in mouse brain slices was observed, we conclude that this drug combination is effective in vitro and expected to have low side effects in vivo.
Asunto(s)
Amidas , Antineoplásicos , Compuestos Bicíclicos Heterocíclicos con Puentes , Glioblastoma , Pirimidinas , Sulfonamidas , Animales , Ratones , Temozolomida/farmacología , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Metotrexato/farmacología , Metotrexato/uso terapéutico , Citarabina/farmacología , Citarabina/uso terapéutico , Antineoplásicos Alquilantes/farmacología , Línea Celular Tumoral , Antineoplásicos/farmacología , ApoptosisRESUMEN
Over the years, the overall survival of older patients diagnosed with acute myeloid leukemia (AML) has not significantly increased. Although standard cytotoxic therapies that rapidly eliminate dividing myeloblasts are used to induce remission, relapse can occur due to surviving therapy-resistant leukemic stem cells (LSCs). Hence, anti-LSC strategies have become a key target to cure AML. We have recently shown that previously approved cardiac glycosides and glucocorticoids target LSC-enriched CD34+ cells in the primary human AML 8227 model with more efficacy than normal hematopoietic stem cells (HSCs). To translate these in vitro findings into humans, we developed a mathematical model of stem cell dynamics that describes the stochastic evolution of LSCs in AML post-standard-of-care. To this, we integrated population pharmacokinetic-pharmacodynamic (PKPD) models to investigate the clonal reduction potential of several promising candidate drugs in comparison to cytarabine, which is commonly used in high doses for consolidation therapy in AML patients. Our results suggest that cardiac glycosides (proscillaridin A, digoxin and ouabain) and glucocorticoids (budesonide and mometasone) reduce the expansion of LSCs through a decrease in their viability. While our model predicts that effective doses of cardiac glycosides are potentially too toxic to use in patients, simulations show the possibility of mometasone to prevent relapse through the glucocorticoid's ability to drastically reduce LSC population size. This work therefore highlights the prospect of these treatments for anti-LSC strategies and underlines the use of quantitative approaches to preclinical drug translation in AML.
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Leucemia Mieloide Aguda , Células Madre Neoplásicas , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/patología , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Modelos Teóricos , Citarabina/uso terapéutico , Citarabina/farmacologíaRESUMEN
This study investigated the effect of rapamycin alone and in combination with chemotherapy (doxorubicin and cytarabine) on AML. Human acute monocytic leukemia cell line SHI-1 and NPG AML model mice created by intravenous injection of SHI-1 cell were treated with rapamycin, chemotherapy, or rapamycin plus chemotherapy. Analysis by cell counting kit-8, western blot, flow cytometry, and immunohistochemistry was performed, and results suggested that both rapamycin and chemotherapy inhibited proliferation of SHI-1 cells both in vitro and in vivo, suppressed neoplasm growth in vivo, and promoted survival of NPG AML mice. The antitumor effect of rapamycin plus chemotherapy was better than that of rapamycin alone and chemotherapy alone. In addition, western blot results demonstrated that rapamycin inhibited the phosphorylation of mTOR downstream targets 4EBP1 and S6K1 in SHI-1 cells, and increased the pro-apoptosis-related protein Bax and autophagy-associated proteins Beclin-1, LC3B-II, and ATG5 while reducing the anti-apoptosis-related protein Bcl-2. In conclusion, the results of this study indicate that rapamycin acts synergistically with doxorubicin and cytarabine in AML treatment, and its underlying mechanism might be associated with mTORC1 pathway-mediated apoptosis and autophagy.
Asunto(s)
Apoptosis , Autofagia , Doxorrubicina , Diana Mecanicista del Complejo 1 de la Rapamicina , Transducción de Señal , Sirolimus , Animales , Autofagia/efectos de los fármacos , Apoptosis/efectos de los fármacos , Humanos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Sirolimus/farmacología , Línea Celular Tumoral , Doxorrubicina/farmacología , Transducción de Señal/efectos de los fármacos , Citarabina/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Sinergismo Farmacológico , Ensayos Antitumor por Modelo de Xenoinjerto , Proliferación Celular/efectos de los fármacos , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéuticoRESUMEN
BACKGROUND/AIM: Methionine metabolism contributes to supplying sulfur-containing amino acids, controlling the methyl group transfer reaction, and producing polyamines in cancer cells. Polyamines play important roles in various cellular functions. Methylthioadenosine phosphorylase (MTAP), the key enzyme of the methionine salvage pathway, is reported to be deficient in 15-62% of cases of hematological malignancies. MTAP-deficient cancer cells accumulate polyamines, resulting in enhanced cell proliferation. The aim of this study was to investigate the combined effects of the polyamine synthesis inhibitor SAM486A and the anticancer antimetabolite cytarabine in MTAP-deficient leukemic cells in vitro. MATERIALS AND METHODS: The leukemia cell line U937 and the subline, U937/MTAP(-), in which MTAP was knocked down by shRNA, were used. The experiments were performed in media supplemented with 20% methionine (low methionine), which was the minimum concentration for maintaining cellular viability. RESULTS: The knockdown efficiency test confirmed a 70% suppression of the expression of the MTAP gene in U937/MTAP(-) cells. Even in the media with low methionine, the intracellular methionine concentration was not reduced in U937/MTAP(-) cells, suggesting that the minimum supply of methionine was sufficient to maintain intracellular levels of methionine. Both U937/MTAP(+) and U937/MTAP(-) cells were comparably sensitive to anticancer drugs (cytarabine, methotrexate, clofarabine and 6-thioguanine). The combination of SAM486A and cytarabine was demonstrated to have synergistic cytotoxicity in U937/MTAP(-) cells with regard to cell growth inhibition and apoptosis induction, but not in U937/MTAP(+) cells. Mechanistically, SAM486A altered the intracellular polyamine concentrations and reduced the antiapoptotic proteins. CONCLUSION: Methionine metabolism and polyamine synthesis can be attractive therapeutic targets in leukemia.
Asunto(s)
Amidinas , Antineoplásicos , Indanos , Leucemia , Humanos , Citarabina/farmacología , Purina-Nucleósido Fosforilasa/genética , Purina-Nucleósido Fosforilasa/metabolismo , Poliaminas , Metionina/farmacología , Metionina/metabolismo , Leucemia/tratamiento farmacológicoRESUMEN
African swine fever (ASF) caused by African swine fever virus (ASFV) is a highly infectious and lethal swine disease. Currently, there is only one novel approved vaccine and no antiviral drugs for ASFV. In the study, a high-throughput screening of an FDA-approved drug library was performed to identify several drugs against ASFV infection in primary porcine alveolar macrophages. Triapine and cytarabine hydrochloride were identified as ASFV infection inhibitors in a dose-dependent manner. The two drugs executed their antiviral activity during the replication stage of ASFV. Furthermore, molecular docking studies showed that triapine might interact with the active center Fe2+ in the small subunit of ASFV ribonucleotide reductase while cytarabine hydrochloride metabolite might interact with three residues (Arg589, Lys593, and Lys631) of ASFV DNA polymerase to block new DNA chain extension. Taken together, our results suggest that triapine and cytarabine hydrochloride displayed significant antiviral activity against ASFV in vitro.
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Virus de la Fiebre Porcina Africana , Fiebre Porcina Africana , Piridinas , Tiosemicarbazonas , Porcinos , Animales , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/metabolismo , Fiebre Porcina Africana/prevención & control , Simulación del Acoplamiento Molecular , Antivirales/farmacología , Antivirales/metabolismo , Citarabina/metabolismo , Citarabina/farmacología , Replicación ViralRESUMEN
The aryl hydrocarbon receptor (AHR) has been identified as a significant driver of tumorigenesis. However, its clinical significance in acute myeloid leukemia (AML) remains largely unclear. In this study, RNA-Seq data from AML patients (bone marrow samples from 173 newly diagnosed AML patients) obtained from the TCGA database, and normal human RNA-Seq data (bone marrow samples from 70 healthy individuals) obtained from the GTEX database are downloaded for external validation and complementarity. The data analysis reveals that the AHR signaling pathway is activated in AML patients. Furthermore, there is a correlation between the expressions of AHR and mitochondrial oxidative phosphorylation genes. In vitro experiments show that enhancing AHR expression in AML cells increases mitochondrial oxidative phosphorylation and induces resistance to cytarabine. Conversely, reducing AHR expression in AML cells decreases cytarabine resistance. These findings deepen our understanding of the AHR signaling pathway's involvement in AML.
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Citarabina , Leucemia Mieloide Aguda , Humanos , Citarabina/farmacología , Fosforilación Oxidativa , Receptores de Hidrocarburo de Aril/genética , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismoRESUMEN
We recently demonstrated that a small subset of cells in FLT3-mutated acute myeloid leukemia (AML) cell lines exhibit SORE6 reporter activity and cancer stem-like features including chemoresistance. To study why SORE6+ cells are more chemoresistant than SORE6- cells, we hypothesized that these cells carry higher autophagy, a mechanism linked to chemoresistance. We found that cytarabine (Ara-C) induced a substantially higher protein level of LC3B-II in SORE6+ compared to SORE6- cells. Similar observations were made using a fluorescence signal-based autophagy assay. Furthermore, chloroquine (an autophagy inhibitor) sensitized SORE6+ but not SORE6- cells to Ara-C. To decipher the molecular mechanisms underlying the high autophagic flux in SORE6+ cells, we employed an autophagy oligonucleotide array comparing gene expression between SORE6+ and SORE6- cells before and after Ara-C treatment. ULK2 was the most differentially expressed gene between the two cell subsets. To demonstrate the role of ULK2 in conferring higher chemoresistance in SORE6+ cells, we treated the two cell subsets with a ULK1/2 inhibitor, MRT68921. MRT68921 significantly sensitized SORE6+ but not SORE6- cells to Ara-C. Using our in vitro model for AML relapse, we found that regenerated AML cells contained higher ULK2 expression compared to pretreated cells. Importantly, inhibition of ULK2 using MRT68921 prevented in vitro AML relapse. Lastly, using pretreatment and relapsed AML patient bone marrow samples, we found that ULK2 expression was higher in relapsed AML. To conclude, our results supported the importance of autophagy in the relapse of FLT3-mutated AML and highlighted ULK2 in this context.
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Resistencia a Antineoplásicos , Leucemia Mieloide Aguda , Proteínas Serina-Treonina Quinasas , Humanos , Autofagia/genética , Bioensayo , Enfermedad Crónica , Citarabina/farmacología , Resistencia a Antineoplásicos/genética , Tirosina Quinasa 3 Similar a fms/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
ABSTRACT: Sterile alpha motif and histidine-aspartate (HD) domain-containing protein 1 (SAMHD1) is a deoxynucleoside triphosphate triphosphohydrolase with ara-CTPase activity that confers cytarabine (ara-C) resistance in several hematological malignancies. Targeting SAMHD1's ara-CTPase activity has recently been demonstrated to enhance ara-C efficacy in acute myeloid leukemia. Here, we identify the transcription factor SRY-related HMG-box containing protein 11 (SOX11) as a novel direct binding partner and first known endogenous inhibitor of SAMHD1. SOX11 is aberrantly expressed not only in mantle cell lymphoma (MCL), but also in some Burkitt lymphomas. Coimmunoprecipitation of SOX11 followed by mass spectrometry in MCL cell lines identified SAMHD1 as the top SOX11 interaction partner, which was validated by proximity ligation assay. In vitro, SAMHD1 bound to the HMG box of SOX11 with low-micromolar affinity. In situ crosslinking studies further indicated that SOX11-SAMHD1 binding resulted in a reduced tetramerization of SAMHD1. Functionally, expression of SOX11 inhibited SAMHD1 ara-CTPase activity in a dose-dependent manner resulting in ara-C sensitization in cell lines and in a SOX11-inducible mouse model of MCL. In SOX11-negative MCL, SOX11-mediated ara-CTPase inhibition could be mimicked by adding the recently identified SAMHD1 inhibitor hydroxyurea. Taken together, our results identify SOX11 as a novel SAMHD1 interaction partner and its first known endogenous inhibitor with potentially important implications for clinical therapy stratification.