OBJECTIVE: To investigate the effects of thymoquinone on the proliferation of acute myeloid leukemia (AML) cells and its molecular mechanism, so as to provide theoretical basis for the basic research on the anti-leukemia of traditional Chinese medicine. METHODS: The HL-60 and THP-1 cells were treated with thymoquinone at different concentration gradients, cell proliferation was detected by CCK-8 method, morphological changes were detected by Wright-Giemsa method, apoptosis was detected by Annexin V/PI double staining flow cytometry, and apoptosis and signal pathway protein expression were detected by Western blot. Real-time quantitative fluorescence PCR and Western blot were used to detect the expression changes of high mobility family members of SRY-related proteins (SOX). RESULTS: Thymoquinone inhibited the malignant proliferation of HL-60 and THP-1 cells, up-regulated the expression of pro-apoptotic protein Bax, down-regulated the expression of anti-apoptotic protein Bcl-2 and Survivin, and hydrolyzed Caspase-3 to induce the apoptosis of HL-60 and THP-1 cells. Thymoquinone could also significantly down-regulate the phosphorylation of PI3K, Akt and mTOR, and inhibit the malignant biological characteristics of HL-60 and THP-1 cells by inhibiting the activation of PI3K/Akt/mTOR pathway. After thymoquinone intervention in HL-60 and THP-1 cells, the expression of SOX2 and SOX4 could be down-regulated significantly. At low concentration ( < 10 μmol/L), the expression of SOX12 was weakly affected by thymoquinone. With increasing concentration, the expression of SOX12 could be down-regulated, however, thymoquinone had no effect on SOX11 expression. CONCLUSION Thymoquinone can inhibit the proliferation of AML cells, and its mechanism may be related to inhibiting the activation of PI3K/Akt/mTOR signaling pathway, regulating the expression of apoptotic proteins and core members of SOX family.
Humans ; Benzoquinones/pharmacology* ; Cell Proliferation/drug effects* ; Leukemia, Myeloid, Acute/metabolism* ; Apoptosis/drug effects* ; HL-60 Cells ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Proto-Oncogene Proteins c-bcl-2/metabolism* ; bcl-2-Associated X Protein/metabolism* ; Cell Line, Tumor ; Phosphatidylinositol 3-Kinases/metabolism* ; THP-1 Cells

OBJECTIVE: To investigate the susceptibility of venetoclax-resistant acute myeloid leukemia (AML) cell lines to ferroptosis and to uncover the underlying molecular mechanisms using transcriptomic and metabolomic analysis methods. METHODS: Venetoclax-resistant AML cell lines were constructed using a low-dose concentration escalation method. The sensitivity of cells to chemotherapeutic drugs was detected by CCK-8 assay. The susceptibility of drug-resistant cell lines to ferroptosis was assessed using transcriptomic and metabolomic analysis methods. The expression of cellular GPX4 and SLC7A11 protein was detected by Western blot, and cell death and lipid peroxidation levels were measured by flow cytometry. Depmap database and TCGA cohort were applied to explore the effect of ferroptosis-related genes expression on prognosis. RESULTS: Venetoclax-resistant cell lines exhibited sensitivity to ferroptosis inducers RSL3, APR246, and sorafenib. The ferroptosis inhibitor Fer-1 partially inhibited cell death induced by these inducers. Compared with the parental cells, significant changes in metabolites and gene expression levels related to ferroptosis were observed in the resistant cell lines. In particular, deregulated expression of SLC7A11 and GPX4 may play critical role in ferroptosis susceptibility. Besides, GPX4 was identified as more important for AML cell survival and higher GPX4 expression may predict shortened overall survival, NPM1 mutant and IDH1 R132 mutation positive patients may prone to possess higher GPX4 expression. CONCLUSION Venetoclax-resistant AML cell lines remain susceptible to ferroptosis, higher GPX4 expression maybe a critical marker for poor prognosis. Regulating the expression of ferroptosis-related genes and metabolites may enhance the efficacy of venetoclax and provide new treatment options for AML patients.
Humans ; Ferroptosis ; Leukemia, Myeloid, Acute/metabolism* ; Sulfonamides/pharmacology* ; Bridged Bicyclo Compounds, Heterocyclic/pharmacology* ; Drug Resistance, Neoplasm ; Metabolomics ; Cell Line, Tumor ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Amino Acid Transport System y+/metabolism* ; Transcriptome

OBJECTIVE: To investigate the impacts of sulforaphane (SPN) on cell proliferation and apoptosis in acute promyelogenous leukemia by regulating the PI3K/Akt/mTOR signaling pathway. METHODS: NB4 cells were divided into 5 μmol/L SPN group, 10 μmol/L SPN group, 20 μmol/L SPN group, 740 Y-P (10 μmol/L) group and 20 μmol/L SPN+740 Y-P group, and the untreated NB4 cells were used as the control group. CCK-8, Hoechst 33342 staining, flow cytometry and monodansulfonylpentanediamine (MDC) were used to detect cell proliferation, apoptosis and autophagy, respectively. The expression levels of Bcl-2, Bax, cyclin D1 and LC3B mRNA were detected by qRT-PCR. Western blot was used to detect the expression levels of PI3K/Akt/mTOR pathway-related proteins in NB4 cells. RESULTS: Compared with the control group, the proliferation rate, Bcl-2, cyclin D1 mRNA expressions, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR ratio were greatly increased in the 740 Y-P group (P < 0.05), the apoptosis rate, percentage of MDC positive, Bax and LC3B mRNA expression levels were greatly decreased (P < 0.05). The proliferation rate, Bcl-2, cyclin D1 mRNA expression levels, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR ratio were greatly decreased in the 5 μmol/L SPN group, 10 μmol/L SPN group, and 20 μmol/L SPN group (P < 0.05), the apoptosis rate, percentage of MDC positive,Bax and LC3B mRNA expression levels were greatly increased, there were differences among different SPN treatment groups (P < 0.05). Compared with the 20 μmol/L SPN group, the proliferation rate, Bcl-2, cyclin D1 mRNA expression levels, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR ratio were greatly increased in the 20 μmol/L SPN+740 Y-P group(P < 0.05), the apoptosis rate, percentage of MDC positive, Bax and LC3B mRNA expression levels were greatly decreased (P < 0.05). Compared with the 740 Y-P group, the proliferation rate, Bcl-2, cyclin D1 mRNA expression levels, p-PI3K/PI3K, p-Akt/Akt, and p-mTOR/mTOR ratio in the 20 μmol/L SPN+740 Y-P group were greatly reduced (P < 0.05), the apoptosis rate, percentage of MDC positive, Bax and LC3B mRNA expression levels were greatly increased (P < 0.05). CONCLUSION SPN reduces the proliferation of acute promyelocytic leukemia cells and promotes cells apoptosis by inhibiting the PI3K/Akt/mTOR signaling pathway.
Cell Proliferation/drug effects* ; Apoptosis/drug effects* ; Humans ; TOR Serine-Threonine Kinases/metabolism* ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Isothiocyanates/pharmacology* ; Phosphatidylinositol 3-Kinases/metabolism* ; Sulfoxides ; Cell Line, Tumor ; Cyclin D1/metabolism*

OBJECTIVE: To investigate the effect of Arsenic trioxide (ATO) combined with Norcantharidin (NCTD) on the proliferation and apoptosis of Jurkat cells, and to evaluate its effect on the proliferation and apoptosis of acute T-lymphoblastic leukemia (T-ALL) based on the Wnt/β-catenin signaling pathway. METHODS: Jurkat cell lines were used as the study subjects and treated with different concentrations of ATO (0, 2, 4, 8, 16 μmol/L) and NCTD (0, 10, 25, 50, 100 μmol/L) for 72 hours, and the cell proliferation was detected by CCK-8. Meanwhile, flow cytometry was used to detect the apoptosis rate, EdU staining to detect cell proliferation viability, cell clone formation assay to assess cell cloning ability, Transwell assay to assess cell invasion ability, and Western blot to detect apoptosis and the expression of Wnt/β-catenin signaling pathway-related proteins. RESULTS: Compared with the control group, both ATO and NCTD effectively inhibited Jurkat cell proliferation when used alone, and the inhibition effect was more significant when used in combination ( P < 0.05). The combination significantly increased the apoptosis rate of Jurkat cells ( P < 0.05). Meanwhile, the combination significantly decreased the proliferation vitality and clone formation ability of the cells ( P < 0.05), and inhibited the invasion ability of Jurkat cells ( P < 0.05). Western blot analysis showed that the combination of ATO and NCTD significantly up-regulated the expression of pro-apoptotic proteins Bax and E-cadherin, and down-regulated the expression of anti-apoptotic proteins Bcl-2, c-myc and Cyclin D1 ( P < 0.05). CONCLUSION The combination of ATO and NCTD had a synergistic effect in inhibiting proliferation and promoting apoptosis in Jurkat cells, which may be related to the inhibition of Wnt/β-catenin signaling pathway.
Humans ; Apoptosis/drug effects* ; Jurkat Cells ; Cell Proliferation/drug effects* ; Arsenic Trioxide ; Wnt Signaling Pathway/drug effects* ; Bridged Bicyclo Compounds, Heterocyclic/pharmacology* ; beta Catenin/metabolism* ; Arsenicals/pharmacology* ; Oxides/pharmacology*

OBJECTIVE: To investigate the role of DYRK1A in the cytarabine (Ara-C) resistance mechanism of acute myeloid leukemia (AML) cells. METHODS: Overexpression and silencing of DYRK1A gene in THP-1 cells were used to observe whether the sensitivity of THP-1 cells to Ara-C was altered. RT-PCR was used to detect the changes in mRNA expression of related genes during Ara-C transport or metabolism. Western blot and RT-PCR were used to detect SAMHD1 expression after regulating DYRK1A expression in Ara-C treated cells. Co-IP technology was used to detect the interaction between Cyclin L2, DYRK1A, and SAMHD1. RESULTS: Overexpression of DYRK1A decreased Ara-C sensitivity in THP-1 cells while silencing DYRK1A increased it. Overexpression and silencing of DYRK1A did not affect Ara-C transport or metabolic gene expression. Overexpression of DYRK1A could increase the expression of SAMHD1 protein in cells, while silencing DYRK1A reduced SAMHD1 expression. Cyclin L2 interacted with DYRK1A and SAMHD1 in THP-1 cells. CONCLUSION DYRK1A is involved in Ara-C resistance in AML cells, and its mechanism may be related to increased expression of SAMHD1 by interacting with Cyclin L2.
Humans ; Cytarabine/pharmacology* ; Protein-Tyrosine Kinases/metabolism* ; Leukemia, Myeloid, Acute ; Dyrk Kinases ; Drug Resistance, Neoplasm ; Protein Serine-Threonine Kinases/metabolism* ; SAM Domain and HD Domain-Containing Protein 1 ; Cell Line, Tumor

OBJECTIVE: To investigate the effects of histone deacetylase (HDAC) levels on the proliferation and apoptosis of Burkitt lymphoma cells, and the changes in related signaling molecules in the PI3K/AKT/mTOR signaling pathway, so as to explore the pathogenesis of Burkitt lymphoma. METHODS: HDAC levels in Burkitt lymphoma were detected by RT-PCR and Western blot. CA46 and RAJI cells were treated with the HDAC selective inhibitor VPA. CCK8 assay was used to detect the proliferation ability of cells. Western Blot was used to measure the expression of apoptosis-related proteins, PI3K/AKT/mTOR signaling pathway proteins and their phosphorylation levels. RESULTS: The expression levels of classⅠ HDAC in Burkitt lymphoma were higher than those in normal cells, and the HDAC1 inhibitor VPA could inhibit the proliferation of CA46 and RAJI cells. VPA decreased HDAC expression in CA46 and RAJI cells, inhibited the phosphorylation of PI3K/AKT/mTOR pathway molecules AKT and p70S6K, increased the expression of apoptotic proteins Cleaved Caspase-3, Cleaved Caspase-8, Cleaved Caspase-9 and Bax, and decreased the expression of anti-apoptotic proteins Bcl-2 and PARP. CONCLUSION Inhibition of HDAC activity can Attenuate the proliferation of Burkitt lymphoma cells and induce apoptosis by inhibiting the PI3K/AKT/mTOR signaling pathway activity.
Humans ; Burkitt Lymphoma/pathology* ; Apoptosis ; Cell Proliferation ; Signal Transduction ; Proto-Oncogene Proteins c-akt/metabolism* ; Phosphatidylinositol 3-Kinases/metabolism* ; Cell Line, Tumor ; Histone Deacetylases/metabolism* ; TOR Serine-Threonine Kinases/metabolism* ; Histone Deacetylase Inhibitors/pharmacology* ; Phosphorylation

OBJECTIVE: To investigate the effects of bortezomib (BTZ) combined with polyphyllin Ⅶ (PP7) on proliferation, apoptosis and oxidative stress of myeloma cell line ARH-77. METHODS: MTT assay was used to detect the inhibitory effects of different concentrations of BTZ, PP7 monotherapy, and their combination on the proliferation of ARH-77 cells. In subsequent experiments, the cells were divided into 4 groups: control group (no drug added), BTZ (15 nmol/L) group, PP7 (1.5 μmol/L) group and BTZ(15 nmol/L)+PP7 (1.5 μmol/L) group. The effects of the two drugs on the morphology of ARH-77 cells were observed. Flow cytometry was used to detect the apoptosis rate of the cells in each group. Calcein-AM/PI double staining kit was used to observe the status of the cells and the cell viability were evaluated. The expression of apoptosis-related proteins were detected by Western blot. DCFH-DA fluorescent probe was used to detect the levels of reactive oxygen species (ROS). RESULTS: Both BTZ and PP7 monotherapy, as well as their combination, could inhibit the growth of ARH-77 cells in a dose-dependent manner (r_BTZ=-0.9717, r_PP7=-0.9941, r_BTZ+PP7=-0.9951), and the combination of BTZ and PP7 exhibited a synergistic effect within a certain concentration range. Compared with the BTZ group and PP7 group, the apoptosis rate of the BTZ+PP7 group was significantly increased (P < 0.01), the expressions of pro-apoptotic proteins Bax, Smac and P53 were significantly upregulated (P < 0.05), the expression of anti-apoptotic protein Bcl-2 was significantly downregulated (P < 0.01), and the ratio of Bax/Bcl-2 was significantly increased (P < 0.01). Compared with the control group, the level of ROS in the BTZ, PP7 monotherapy group and BTZ+PP7 group were significantly increased (P < 0.05). CONCLUSION BTZ combined with PP7 can inhibit the proliferation and induce apoptosis of ARH-77 cells, and increase the level of intracellular ROS.
Apoptosis/drug effects* ; Bortezomib ; Humans ; Cell Proliferation/drug effects* ; Oxidative Stress/drug effects* ; Multiple Myeloma/metabolism* ; Cell Line, Tumor ; Saponins/pharmacology*

OBJECTIVE: To screen anti-tumor drugs that improve antigen processing and presentation in acute myeloid leukemia (AML) cells. METHODS: A TCR-like or TCR mimic antibody that can specifically recognize HLA-A*0201:WT1_126-134 ( RMFPNAPYL) complex (hereafter referred to as HLA-A2:WT1) was synthesized to evaluate the function of antigen processing and presentation machinery (APM) in AML cells. AML cell line THP1 was incubated with increasing concentrations of IFN-γ, hypomethylating agents (HMA), immunomodulatory drugs (IMiD), proteasome inhibitors (PI) and γ-secretase inhibitors (GSI), followed by measuring of HLA-ABC, HLA-A2 and HLA-A2:WT1 levels by flow cytometry at consecutive time points. RESULTS: The TCR-like antibody we generated only binds to HLA-A*0201⁺WT1⁺ cells, indicating the specificity of the antibody. HLA-A2:WT1 level of THP-1 cells detected with the TCR-like antibody was increased significantly after co-incubation with IFN-γ, showing that the HLA-A2:WT1 TCR like antibody could evaluate the function of APM. Among the anti-tumor agents screened in this study, GSI (LY-411575) and HMA (decitabine and azacitidine) could significantly increase the HLA-A2:WT1 level. The IMiD lenalidomide and pomalidomide could aslo upregulate the expression of HLA-A2:WT1 complex under certain concentrations of the drugs and incubation time. As proteasome inhibitors, carfilzomib could significantly decreased the expression of HLA-A2:WT1, while bortezomib had no significant effect on HLA-A2:WT1 expression. CONCLUSION HLA-A2:WT1 TCR-like antibody can effectively reflect the APM function. Some of the anti-tumor drugs can affect the APM function and immunogenicity of tumor cells.
Humans ; Leukemia, Myeloid, Acute/immunology* ; Antineoplastic Agents/pharmacology* ; Antigen Presentation/drug effects* ; HLA-A2 Antigen/immunology* ; Receptors, Antigen, T-Cell/immunology* ; Cell Line, Tumor ; Interferon-gamma

OBJECTIVE: To investigate the mechanism of circular RNA RAD18 (CircRAD18 ) in regulating daunorubicin (DNR) resistance in acute myeloid leukemia (AML) cells through the miR-185-5p/hepatoma-derived growth factor ( HDGF) axis. METHODS: Real-time fluorescence quantitative PCR and immunoblotting were applied to detect the expression of CircRAD18 , miR-185-5p, and HDGF in human AML cell lines HL-60, U937, and human AML drug-resistant cell line KG1a. KG1a cells were cultured in vitro and randomly divided into control group, DNR group, DNR+negative control group, DNR+CircRAD18 knockdown group, and DNR+CircRAD18 knockdown+miR-185-5p inhibitor group. After transfection, real-time fluorescence quantitative PCR and immunoblotting were applied to detect the expression of CircRAD18 , miR-185-5p, and HDGF of cells, CCK-8 method and Ki-67 immunofluorescence staining were applied to detect cell proliferation, flow cytometry was applied to detect cell apoptosis, and immunoblotting was applied to detect the expression of cell proliferation, apoptosis and drug resistance related proteins in each group. The double luciferase reporter gene experiment was applied to detect the targeting regulation of CircRAD18 on miR-185-5p, and miR-185-5p on HDGF in KG1a cells. RESULTS: Compared with HL-60 and U937 cells, the expression of CircRAD18 , and HDGF mRNA and protein in KG1a cells increased (all P <0.05), while miR-185-5p decreased ( P <0.05). Compared with the control group, the CircRAD18 expression, HDGF mRNA and protein expression, cell viability, proliferation rate, and PCNA, Bcl-2, BCRP, and P-gp protein expression in the DNR+CircRAD18 knockdown group decreased (all P <0.05), while miR-185-5p expression, apoptosis rate, and Bax protein expression increased (all P <0.05). There were no obvious changes in all indicators of cells in the DNR group compared with control group ( P >0.05). Compared with the DNR group, the CircRAD18 expression, HDGF mRNA and protein expression, cell viability, proliferation rate, PCNA, Bcl-2, BCRP, and P-gp protein expression in the DNR+CircRAD18 knockdown group decreased (all P < 0.05), while miR-185-5p expression, apoptosis rate, and Bax protein expression increased (all P < 0.05). There were no obvious changes in all indicators of cells in the DNR+negative control group compared with DNR group (P >0.05). Compared with the DNR+CircRAD18 knockdown group, the HDGF mRNA and protein expression, cell viability, proliferation rate, PCNA, Bcl-2, BCRP, and P-gp protein expression in the DNR+CircRAD18 knockdown+miR-185-5p inhibitor group increased (all P < 0.05), while miR-185-5p expression, apoptosis rate, and Bax protein expression decreased (all P < 0.05). CircRAD18 was able to target and down-regulate the expression of miR-185-5p in KG1a cells, and miR-185-5p was able to target and down-regulate the HDGF expression. CONCLUSION Knocking down CircRAD18 can reduce HDGF expression by up-regulating miR-185-5p, thereby weakening DNR resistance in AML cells, inhibiting KG1a cell proliferation under DNR treatment, and promoting apoptosis.
Humans ; MicroRNAs/metabolism* ; Leukemia, Myeloid, Acute ; Daunorubicin/pharmacology* ; Drug Resistance, Neoplasm ; Apoptosis ; RNA, Circular ; Intercellular Signaling Peptides and Proteins/metabolism* ; Cell Proliferation ; HL-60 Cells ; Cell Line, Tumor

OBJECTIVE: Photodynamic therapy has become an important method in clinical tumor treatment. This study aimed to investigate the effects of hematoporphyrin on multiple myeloma (MM) and its potential applications. METHODS: The MM cell line RPMI 8226 was treated with hematoporphyrin derivative (HPD), and CCK-8 assay was used to determine cell viability, apoptosis was detected by flow cytometry, intracellular reactive oxygen species (ROS) levels were measured using a detection kit combined with flow cytometry, and Western blot assay was used to detect apoptosis-related proteins and key signaling pathway protein levels. RESULTS: The optimal incubation time for the maximum absorption of HPD in RPMI 8226 cells was 4 hours. HPD significantly inhibited the proliferation of RPMI 8226 cells in a dose- and illumination time-dependent manner ( r =0.981; r =0.961). Additionally, HPD induced apoptosis in RPMI 8226 cells, but had no significant inhibitory effect on peripheral blood mononuclear cells derived from healthy individuals. HPD combined with illumination treatment significantly increased the intracellular ROS level, upregulated the expression of apoptosis-related proteins such as cleaved PARP, cleaved caspase-3 and Bax, and down-regulated the expression of proteins that maintain cell survival, such as NF-κB and Akt. CONCLUSION The HPD can inhibit the proliferation and induce apoptosis of multiple myeloma cells.
Humans ; Multiple Myeloma/pathology* ; Hematoporphyrins/pharmacology* ; Apoptosis/drug effects* ; Cell Line, Tumor ; Reactive Oxygen Species/metabolism* ; Cell Proliferation/drug effects* ; Photochemotherapy ; Cell Survival/drug effects* ; Signal Transduction