Proliferation Of K562 Cells Research Articles

Pharmacological assessment of the antineoplastic and immunomodulatory properties of a new spiroindolone derivative (7',8'-Dimethoxy-1',3'-dimethyl-1,2,3',4'-tetrahydrospiro[indole-3,5'-pyrazolo[3,4-c]isoquinolin]-2-one) in chronic myeloid leukemia.

The discovery and development of effective novel compounds is paramount in oncology for improving cancer therapy. In this study, we developed a new derivative of spiroindolone (7',8'-Dimethoxy-1',3'-dimethyl-1,2,3',4'-tetrahydrospiro[indole-3,5'- pyrazolo[3,4-c]isoquinolin]-2-one) and evaluated its anticancer- and immunomodulatory potential in a vitro model of chronic leukemia. We utilized the chronic leukemia cell line K562, as well as non-cancerous peripheral blood mononuclear cells (PBMC) and Vero cells (kidney epithelium of Cercopithecus aethiops). We assessed the cytotoxicity of the compound using the MTT assay, and performed cell cycle assays to determine its impact on different stages of the cell cycle. To evaluate its antineoplastic activity, we conducted a colony formation test to measure the effect of the compound on the clonal growth of cancer cells. Furthermore, we evaluated the immunomodulatory activity of the compound by measuring the levels of pro and anti-inflammatory cytokines. The study findings demonstrate that the spiroindolone-derived compound exerted noteworthy cytotoxic effects against K562 cells, with an IC50 value of 25.27µg/mL. Additionally, it was observed that the compound inhibited the clonal proliferation of K562 cells while displaying minimal toxicity to normal cells. The compound exhibited its antiproliferative activity by inducing G2/M cell cycle arrest, preventing the entry of K562 cells into mitosis. Notably, the compound demonstrated an immunomodulatory effect by upregulating the production of cytokines IL-6 and IL-12/23p40. In conclusion, the spiroindolone-derived compound evaluated in this study has demonstrated significant potential as a therapeutic agent for the treatment of chronic myeloid leukemia. Further investigations are warranted to explore its clinical applications.

Role of Calcium-Activated Potassium Channels in Proliferation, Migration and Invasion of Human Chronic Myeloid Leukemia K562 Cells.

Calcium-activated potassium channels (KCa) are important participants in calcium signaling pathways due to their ability to be activated by an increase in intracellular free calcium concentration. KCa channels are involved in the regulation of cellular processes in both normal and pathophysiological conditions, including oncotransformation. Previously, using patch-clamp, we registered the KCa currents in the plasma membrane of human chronic myeloid leukemia K562 cells, whose activity was controlled by local Ca2+ entry via mechanosensitive calcium-permeable channels. Here, we performed the molecular and functional identification of KCa channels and have uncovered their role in the proliferation, migration and invasion of K562 cells. Using a combined approach, we identified the functional activity of SK2, SK3 and IK channels in the plasma membrane of the cells. Selective SK and IK channel inhibitors, apamin and TRAM-34, respectively, reduced the proliferative, migratory and invasive capabilities of human myeloid leukemia cells. At the same time, the viability of K562 cells was not affected by KCa channel inhibitors. Ca2+ imaging showed that both SK and IK channel inhibitors affect Ca2+ entry and this could underlie the observed suppression of pathophysiological reactions of K562 cells. Our data imply that SK/IK channel inhibitors could be used to slow down the proliferation and spreading of chronic myeloid leukemia K562 cells that express functionally active KCa channels in the plasma membrane.

Frequency and Functional Characterization of RUNX1 Germline Variants in Myeloid Neoplasms

Current estimates suggest that up to 10% of patients with myeloid neoplasms (MN) harbor variants associated with a germline predisposition. A pathogenic variant in the runt-related transcription factor 1 gene (RUNX1) is a frequent cause of germline predisposition to MN. RUNX1 variants detected in tumor tissue at a VAF close to 50% are potentially germline and causative of RUNX1 familial platelet disorder with associated myeloid malignancies. Previous studies have found germline RUNX1 variants in 3% of patients with acute myeloid leukemia; however, the frequency of germline RUNX1 variants in less advanced myeloid neoplasms has not been examined. We screened 590 patients suspected of MN, excluding myeloproliferative neoplasms, for germline variants in RUNX1. We found RUNX1 variants in 83 patients (14%) by targeted sequencing of tumor tissue. In 40 patients (6.8%), the VAF of RUNX1 was above 30%. In 32 of the 40 patients, skin biopsies were available and used for Sanger sequencing to assess the germline status. Two of the tested variants (6.3%) were confirmed as germline, and both variants were curated as variants of unknown significance. To further explore the pathogenicity of these variants, we implemented a novel CRISPR-Select functional genetic assay. The assay demonstrated a profound effect on proliferation in K562 cells for a known pathogenic variant but no effect for the two germline variants detected in the study. We therefore propose that both germline variants are classified as likely benign. In this study, we show that RUNX1 germline variants are rare in Danish patients with MN and use a novel assay for functional classification of germline RUNX1 variants.

Periplaneta americana extract CⅡ-3 induces senescence of leukemia K562 cells via SIRT1/mTOR signaling pathway

This study aims to investigate the role of slient mating-type information regulation 2 homolog 1(SIRT1)/tuberous sclerosis complex 2(TSC2)/mammalian target of rapamycin(mTOR) signaling pathways in the Periplaneta americana extract CⅡ-3-induced senescence of human leukemia K562 cells. K562 cells were cultured in vitro and treated with 0(control), 5, 10, 20, 40, 80, and 160 μg·mL~(-1) of P. americana extract CⅡ-3. Cell counting kit-8(CCK-8) and flow cytometry were employed to examine the proliferation and cell cycle of the K562 cells. Senescence-associated β-galactosidase stain kit(SA-β-gal) was used to detect the positive rate of senescent cells. Mitochondrial membrane potential was detected by flow cytometry. The relative mRNA level of telomerase reverse transcriptase(TERT) was determined by fluorescence quantitative PCR. The mRNA and protein levels of SIRT1, TSC2, and mTOR were determined by fluorescence quantitative PCR and Western blot, respectively. The results showed that CⅡ-3 significantly inhibited the proliferation of K562 cells and the treatment with 80 μg·mL~(-1) CⅡ-3 for 72 h had the highest inhibition rate. Therefore, 80 μg·mL~(-1) CⅡ-3 treatment for 72 h was selected as the standard for subsequent experiments. Compared with the control group, CⅡ-3 increased the proportion of cells arrested in G_0/G_1 phase, decreased the proportion of cells in S phase, increased the positive rate of SA-β-Gal staining, elevated the mitochondrial membrane potential and down-regulated the mRNA expression of TERT. Furthermore, the mRNA expression of SIRT1 and TSC2 was down-regulated, while the mRNA expression of mTOR was up-regulated. The protein expression of SIRT1 and p-TSC2 was down-regulated, while the protein expression of p-mTOR was up-regulated. The results indicated that P. americana extract CⅡ-3 induced the senescence of K562 cells via the SIRT1/mTOR signaling pathway.

Abstract 3099: Hnd-01: a potent inhibitor of wild type mutant BTK

Abstract Dasatinib is an oral dual BCR/ABL and Src family tyrosine kinase inhibitor approved for use in patients with chronic myelogenous leukemia and Philadelphia chromosome-positive acute lymphoblastic leukemia. Based on the in vitro and in vivo characterization studies of dasatanib, several metabolites were identified. Dasatinib and its metabolite 20 were the major circulating species identified which represented 25.5% and 12.5% of total radioactivity in human plasma respectively. Here, we report the identification of HND-01, a small molecule inhibitor of Bruton’s tyrosine kinase (BTK) which was achieved by modifying metabolite 20 of dasatinib. Bruton’s tyrosine kinase (BTK) is a non-receptor kinase and a key regulator of the B-cell receptor (BCR) signaling pathway. BCR is critical for proliferation and survival of leukemic cells in many B cell malignancies, including chronic lymphocytic leukemia (CLL). BTK is a key target for the development of small molecule inhibitors in B cell malignancies. There are several covalent BTK inhibitors including ibrutinib that have been approved. These covalent inhibitors bind with high affinity to Cys-481 in the active site of BTK. However, some patients develop acquired resistance to ibrutinib due to Cys-481 mutation in the BTK binding site. The acquired resistance to covalent inhibitors can be overcome with the design of new reversible BTK inhibitors. The reversible BTK inhibitors will bind to different sites other than the cysteine residue of BTK and inactivate BTK. These drugs would potentially be active in the presence or absence of the cysteine-to-serine mutation. HND-01, a novel and potent reversible BTK inhibitor inhibits wild type & C481S mutant BTK with IC50 values of 0.157 nM for WTBTK and 0.032 nM for C481SBTK. HND-01 inhibits K562 cell proliferation (IC50 0.183 nM) and it is more stable than dasatinib in human liver microsomes (in vitro clearance; 142 mL/min/kg for HND-01 vs 211 mL/min/kg for dasatinib). In an established human lymphoma efficacy model (TMD-8 model), HND-01 showed stronger tumor inhibition (80.6%) than Ibrutinib (58%). These data demonstrates that HND-01 is a BTK inhibitor that is more potent with potential desirable ADME properties than ibrutinib. HND-01 was filled for international patents, China patent application # 202080030953.6, US patent application #17/636,476, EU application # EP 20853834.8, and Canada application # CA 3148436. Citation Format: Jing Zhang, Ahmed A. Samatar, Yue Tan, Shiqing Pi, Zhigang Zhou, Yan Xu, Daihong Yang. Hnd-01: a potent inhibitor of wild type mutant BTK [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3099.

Tet2-mediated DNA demethylation regulates the proliferation and apoptosis of human leukemia K562 cells.

TET2 is a member of the TET protein family which is responsible for active DNA demethylation through catalyzing the successive oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), and mutations of Tet2 frequently lead to hematological malignancies. However, the relationship between Tet2-mediated demethylation and hematological malignancies is unclear. The human leukemia K562 cell line is an immortalized leukemia line that serves as an in vitro model of erythroleukemia. In this study, we investigated the effect of Tet2-mediated demethylation on the apoptosis and proliferation of human leukemia K562 cells and found that knockdown of Tet2 promoted and inhibited K562 cell proliferation and apoptosis, respectively, while upregulation of TET2 enzymatic activity via alpha-ketoglutaric acid (α-KG) had the opposite effects. Therefore, the Tet2 gene acts as a potential target for the treatment of leukemia, and small molecules that target the Tet2 gene may be used to screen antitumor drugs for hematological malignancies.

Synthesis, in vitro cytotoxicity evaluation and mechanism of 5′ monosubstituted chalcone derivatives as antitumor agents

A series of 5′ monosubstituted chalcone derivatives were synthesized to explore their antitumor activity and mechanism of action in vitro. The structures of 5′ monosubstituted chalcone derivatives synthesized by reactions such as Suzuki coupling were confirmed by 1H NMR, 13C NMR and MS, and the target compounds were not reported in the literature. The antitumor activity of the aimed compounds was tested by MTT colorimetric method in vitro. Compound 5c has an IC50 value of 1.97 μM for K562 and a value of 2.23 μM for HepG2. Further investigation of the mechanism of action of compound 5c was found to have effects on K562 cell morphology, proliferation, apoptosis, cell cycle, and wound healing of HepG2 cells. The results showed that compound 5c has research value in antitumor activity and mechanism of action in vitro.

Connexin 43-modified bone marrow stromal cells reverse the imatinib resistance of K562 cells via Ca2+-dependent gap junction intercellular communication.

Imatinib mesylate (IM) resistance is an emerging problem for chronic myeloid leukemia (CML). Previous studies found that connexin 43 (Cx43) deficiency in the hematopoietic microenvironment (HM) protects minimal residual disease (MRD), but the mechanism remains unknown. Immunohistochemistry assays were employed to compare the expression of Cx43 and hypoxia-inducible factor 1α (HIF-1α) in bone marrow (BM) biopsies of CML patients and healthy donors. A coculture system of K562 cells and several Cx43-modified bone marrow stromal cells (BMSCs) was established under IM treatment. Proliferation, cell cycle, apoptosis, and other indicators of K562 cells in different groups were detected to investigate the function and possible mechanism of Cx43. We assessed the Ca2+-related pathway by Western blotting. Tumor-bearing models were also established to validate the causal role of Cx43 in reversing IM resistance. Low levels of Cx43 in BMs were observed in CML patients, and Cx43 expression was negatively correlated with HIF-1α. We also observed that K562 cells cocultured with BMSCs transfected with adenovirus-short hairpin RNA of Cx43 (BMSCs-shCx43) had a lower apoptosis rate and that their cell cycle was blocked in G0/G1 phase, while the result was the opposite in the Cx43-overexpression setting. Cx43 mediates gap junction intercellular communication (GJIC) through direct contact, and Ca2+ is the key factor mediating the downstream apoptotic pathway. In animal experiments, mice bearing K562, and BMSCs-Cx43 had the smallest tumor volume and spleen, which was consistent with the in vitro experiments. Cx43 deficiency exists in CML patients, promoting the generation of MRD and inducing drug resistance. Enhancing Cx43 expression and GJIC function in the HM may be a novel strategy to reverse drug resistance and promote IM efficacy.

Potential antiproliferative and apoptotic effects of pilocarpine combined with TNF alpha in chronic myeloid leukemia cells.

Pilocarpine is a selective M1/M3 agonist of muscarinic acetylcholine receptor subtypes. Muscarinic acetylcholine receptors are G protein-coupled receptors. These receptors are different drug targets. The aim of the present work was to investigate the effect of pilocarpine on the expression of M3 muscarinic acetylcholine receptor, the AChE activity, IL-8 release response, and proliferation in K562 cells, via muscarinic receptor activation.Human chronic myeloid leukemic cell cultures were incubated with drugs. Proliferation assays were performed by BrdU assay. Expression of M3 muscarinic acetylcholine receptor and apoptosis proteins such as bcl, bax, cyt C, and caspases was assessed with the semiquantitative Western blotting method.Pilocarpine inhibits chronic myeloid cell proliferation and M3 muscarinic acetylcholine receptor protein expression. Pilocarpine increases caspase-8 and -9 expression levels, upregulating the proapoptotic protein Bax and downregulating the expression levels of the antiapoptotic protein Bcl-2. The apoptotic activity of pilocarpine is associated with an increase in AChE activity. M3 muscarinic acetylcholine receptors can activate multiple signal transduction systems and mediate inhibitory effects on chronic myeloid K562 cell proliferation depending on the presence of 1% FBS conditions. This apoptotic effect of pilocarpine may be due to the concentration of pilocarpine and the increase in AChE level.Our results suggest that inhibition of cell proliferation by inducing apoptosis of pilocarpine in K562 cells may be one of the targets. M3 selective agonist may have therapeutic potential in chronic myeloid leukemia.

Effect of Dichloromethane Extraction Phase of Patrinia Scabiosaefolia Fisch. Stem on Proliferation and Differentiation of K562 Cells

To explore the effect of dichloromethane extraction phase of ethanol extract from stem of Patrinia scabiosaefolia Fisch.(DPSS) on proliferation and differentiation of K562 cells and its related mechanism. MTT assay was used to detect the effects of DPSS at 0, 25, 50, 100 and 200 μg/ml on the proliferation of K562 cells at 24, 48 and 72 hours. Flow cytometry was used to analyze the changes of cell cycle and apoptosis at 24 and 48 hours. Wright-Giemsa staining was used to observe the morphological changes of K562 cells. The cell surface antigens CD33 and CD11b were detected by flow cytometry. The proliferation of K562 cells treated with different concentrations of DPSS was inhibited in a time-dose dependent manner (r=-0.96). Cell cycle analysis showed that with the increase of DPSS concentration, cells in G2/M phase increased (r=0.88), and cells were blocked in G2/M phase. Flow cytometry results showed that with the apoptosis rate of K562 cells was the highest when treated with 200 μg/ml DPSS for 48 h. Morphological observation showed that the K562 cell body increased, the amount of cytoplasm increased, the ratio of nucleus to cytoplasm decreased, and the nuclear chromatin was rough after DPSS treatment. Cell differentiation antigen, CD33 and CD11b, were positively expressed after treated with DPSS. DPSS can induce apoptosis through cell cycle arrest, inhibit the proliferation of K562 cells, and induce K562 cells to differentiate into monocytes, which has a potential anti-leukemia effect.

Effects of miR-144-3p on Proliferation, Cell Cycle and Apoptosis of K562 Cells

To investigate the effects of miR-144-3p on cell proliferation, cell cycle and apoptosis of blast phase chronic myelogenous leukemia (CML) K562 cells. K562 cells were cultured in vitro and mimics negative control, hsa-miR-144-3p mimics, inhibitor negative control and miR-144-3p inhibitor were respectively transfected into K562 cells with transfection reagents. The cells were divided into five groups including blank control, mimics negative control, miR-144-3p mimics, inhibitor negative control and miR-144-3p inhibitor. After transfection, the cell proliferation activity was detected by CCK-8 assay. The cell cycle distribution and apoptosis were detected by flow cytometry. Compared with the blank control and mimics negative control groups, the proliferation rate of miR-144-3p mimics group was significantly decreased (P<0.05), the proportion of S phase cells was markedly increased (P<0.05), while the proportion of G1 phase cells was obviously decreased (P<0.05), and the apoptosis rate was significantly increased (P<0.05). Compared with the blank control and inhibitor negative control groups, the proliferation rate of miR-144-3p inhibitor group was obviously increased (P<0.05), the proportion of S phase cells was markedly decreased (P<0.05), while the proportion of G1 phase cells was obviously increased (P<0.05), and the apoptosis rate was significantly decreased (P<0.05). miR-144-3p can inhibit the proliferation and promote apoptosis of K562 cells, affect the cell cycle, and block K562 cells in S phase, which indicates that miR-144-3p is involved in the cell cycle activity of CML during blastic phase.

Chemical Components of Volatile Oil Extracted from the Fermentation Broth of Ganoderma lingzhi (Agaricomycetes) Coupled with Its Antitumor and Antioxidant Activities In Vitro

Volatile oil extracted from fermentation broth of Ganoderma lingzhi by hydrodistillation was analyzed based on gas chromatography-mass spectrometry (GC-MS). Its antitumor activity was tested on K562, SW620, A549, HepG2 cells in vitro. In addition, the antioxidant activity of the oil was determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay. In total, 16 constituents were identified accounting for about 99.99% of the total volatile oil in the fermentation broth of G. lingzhi. Among these components, 1-propanol (33.33%), phenylacetaldehyde (24.24%), 2-hexyl-1-decanol (12.12%) were found to be the major constituents. The antitumor results showed that the IC50 of the inhibition to the proliferation of K562, SW620, A549, HepG2 cells were 32.2, 78.9, 96.4, 99.0 μg/mL, respectively. And the oil could inhibit the proliferation of K562 cells by apoptosis induction and cell cycle arrest at S phase. Moreover, the oil exhibited radical scavenging activity (IC50 = 0.1469 mg/mL) in DPPH assay.

Construction of Lentiviral Vector for miR-217 Overexpression and Knockdown and Its Effect on CML.

We attempted to construct a myeloid leukemia cell strain for stable overexpression and knock-down of miR-217 and explored the possible mechanism underlying miR-217 in chronic myeloid leukemia (CML). MiR-217 overexpression and the knock-down lentiviral vector with puromycin resistance were constructed and packaged within recombinant lentivirus. Stably transfected K562 cells were obtained through puromycin screening, and the qPCR assay detected the relative expression of the target gene. The proliferation, apoptosis, and methylation level of PER2 within cultured cells were detected using the CCK-8 assay, flow cytometry, and TaqMan real‑time fluorescence quantitative methylation-specific PCR. qPCR and Western blot detected the expression of miR-217-related genes within the constructed K562 cell model. Colony PCR and sequencing proved that recombinant lentivirus expression vectors pSE16 and pSE17 were correctly constructed. The lentivirus titer was 2.95 × 109 and 2.61 × 109IU/mL. The miR-217 expression level was high in pSE5316-K562 cells, and that of the miR-217 sponge was high in pSE5317-K562 cells. Overexpressed miR-217 could inhibit the K562 cell proliferation and induce apoptosis. Inhibition of miR-217 enhanced the expression of DNMT3A, decreased the PER2 expression, and elevated the degree of PER2 methylation. The miR-217 overexpression and knock-down of the K562 cell line were successfully constructed, providing a tool for further exploring the miR-217 mechanism in CML. DNMT3A could be the molecular target of miR-217 by regulating PER2 gene methylation and getting involved with the occurrence and development of CML.

Effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells

To investigate the effects of matrine combined with LY294002 on proliferation, apoptosis and cell cycle of human myeloid leukemia K562 cells and explore the underlying mechanism. The effects of different concentrations of matrine alone and in combination with LY294002 on the proliferation of K562 cells were examined with CCK-8 assay. The changes in morphology of K562 cells were observed following treatment for 48 h with 0.4 g/L matrine and 10 μmol/L Y294002, either alone or in combination, and cell apoptosis was detected using flow cytometry with annexin V-FITC/PI double labeling; the changes in cell cycle was detected with PI labeling. Western blotting was performed to examine the effect of matrine combined with LY294002 on expressions of p-mTOR, p-PI3K, Akt, p-Akt, cyclinD1, Bcl-2 and caspase-9 in the cells. Treatment with different concentrations of matrine, both alone and in combination with LY294002, inhibited the proliferation of K562 cells in a time- and concentration-dependent manner. Compared with matrine treatment alone, the combined treatment caused more obvious morphological changes of the cells, significantly increased cell apoptosis (P < 0.01), and induced cell cycle arrest in G0/G1 (P < 0.01). Western blotting showed that the protein expression levels of p-mTOR, cyclinD1, p-PI3K, p-Akt and Bcl-2 in K562 cells increased while the expression level of caspase-9 decreased significantly after the combined treatment (P < 0.01). Matrine combined with LY294002 produces a synergistic inhibitory effect on K562 cells possibly by down-regulating the p-Akt expression in PI3K/Akt signaling pathway, reducing the expressions of p-mTOR, cyclinD1 and Bcl-2, and increasing the expression of caspase-9.

Convallatoxin Inhibits Cell Proliferation and Induces Cell Apoptosis by Attenuating the Akt-E2F1 Signaling Pathway in K562 Cells

Objective: To determine the effect of convallatoxin on K562 cell proliferation and apoptosis. Methods: CCK-8 assay was used to detect cell proliferation; PI staining, JC-1 staining, and Annexin V-FITC/PI double staining were used to analyze the cell cycle, cell mitochondrial membrane potential, and cell apoptosis; and Western blotting was used to detect cleaved caspase-9, cleaved caspase-3, Bcl-2, Bax, and E2F1 expression and Akt phosphorylation. Subsequently, AutoDock software was used to determine the interaction between convallatoxin and Akt1. Results: Upon treatment with convallatoxin, the proliferation of K562 cells was inhibited, the cells were arrested at the S and G2/M phases, and cell apoptosis was significantly induced. In addition, Akt phosphorylation and E2F1 expression were significantly decreased, whereas E2F1 overexpression rescued convallatoxin-induced cell proliferation and apoptosis. In addition, a molecular docking assay indicated that convallatoxin could bind to Akt1. Conclusion: Convallatoxin inhibited cell proliferation and induced mitochondrial-related apoptosis in K562 cells by reducing the Akt-E2F1 signaling pathway, indicating that it is a potential agent for treating leukemia.

Targeted silencing of CDX2 gene with siRNA enhanced vincristine-induced inhibition of proliferation and induction of apoptosis of leukemia K562 cells

Purpose: To determine the effect of targeted silencing of CDX2 with siRNA on vincristine-induced inhibition of proliferation and apoptosis induction in leukemia K562 cells.&#x0D; Methods: K562 cells were divided into untreated group, vincristine group, blank group, and CDX2-siRNA group. The expression of CDX2 gene after CDX2-siRNA transfection was determined by reverse-transcription-polymerase chain reaction (RT-PCR) and immunoblotting, while MTT and flow cytometric procedures were used to evaluate the effect of vincristine on proliferation and apoptotic changes in K562 cells.&#x0D; Results: Protein expression of CDX2 did not change significantly in non-treated cells, vincristine group and blank group, while the expression of CDX2 protein in cells of CDX2-siRNA group was decreased significantly (p &lt; 0.05) while MTT assay results showed that the absorbance of CDX2-siRNA cells was significantly lower than those of the other three groups at 12, 24 and 48 h after CDX2 gene silencing. Flow cytometry showed markedly higher percentage apoptosis of CDX2-siRNA group than in other groups at 12, 24 and 48 h after CDX2 gene silencing. There was no difference in apoptosis level between vincristine group and blank group, but apoptosis was higher in these groups than in untreated group (p &lt; 0.05).&#x0D; Conclusion: Silencing CDX2 gene via targeting with siRNA enhances vincristine-induced suppression of growth and apoptotic changes in leukemia K562 cells, thereby enhancing the anti-tumor effect of vincristine.

Effect of Curcumin on the Proliferation, Apoptosis, and Cell Cycle of Human Acute Myeloid Leukemia Cell Line K562

To investigate the effects of curcumin on the proliferation, apoptosis, and cell cycle of human acute myeloid leukemia cell line K562. MTT method was used to detect the proliferation inhibition of logarithmic growth phase human acute myeloid leukemia K562 cells, flow cytometry was used to detect the cell cycle, Annexin V-FITC was used to detect the apoptosis rate, and real-time fluorescent quantitative PCR and Western blot were used to detect the expression of Bax, BCL-2 and caspase-3 mRNA and protein, respectively. The inhibition rate of cell proliferation in curcumin 10, 20, and 40 μmol/L group for 24 h and 48 h were higher than that in the control group (curcumin 0 μmol/L), and the cell proliferation inhibition rate was concentration-time dependent (r=0.879, r=0.914). The proportion of G0/G1 cells and apoptosis rate of K562 cells in the curcumin 10, 20, and 40 μmol/L group were higher than those in the control group, and showed drug concentration dependent (r=0.856, r=0.782). The expression of Bax and Caspase-3 mRNA in the curcumin 10, 20, and 40 μmol/L group was higher, while BCL-2 mRNA was lower than those in the control group, and showed drug concentration dependent (r=0.861, r=0.748, r=-0.817). The gray value of Bax protein expression in the curcumin 10, 20, and 40 μmol/L group was higher than that in the control group, while the gray value of BCL-2 and Caspase-3 protein expression was lower than that in the control group, and showed drug concentration dependent (r=0.764, r=-0.723, r=-0.831). Curcumin can inhibit the proliferation of human acute myeloid leukemia cell line K562 cells, block the cell cycle at G0/G1 phase, promote cell apoptosis, and induce apoptosis by regulating Bax, BCL-2, and Caspase-3.

Effects of Vinblastine and Vincristine on the function of chronic myeloid leukemic cells through expression of A20 and CYLD.

Chronic myelogenous leukemia (CML) is characterised by the translocation of regions of the BCR and ABL genes, leading to the fusion gene BCR-ABL forming the Philadelphia (Ph) chromosome. Vinblastine (Vinb) and Vincristine (Vinc) are Vinca alkaloids and frequently used in combination chemotherapy in leukemias and lymphomas. Deubiquitinating enzyme (DUB) genes such as A20, Otubain 1 and CYLD are known as inhibitors of functional activation of immune cells mediated through the NF-κB/STAT pathway. Little is known about the regulatory role of Vinb/Vinc on the function of CML cells and the contribution of the DUBs to those effects. In the end, the gene expression profile was determined by quantitative RT-PCR, physiological properties of CML cells by flow cytometry and cytokine production by ELISA. As a result, inactivated expression of the DUBs A20, CYLD, Otubain 1 and Cezanne and enhanced activation of CD11b+ and CD4T cells were observed in CML patients. Importantly, Vinc enhanced the expression of A20 and CYLD and inhibited the proliferation and survival of CML (K562) cells. The effects were abolished in the presence of A20 siRNA, while cell proliferation only depended on the presence of CYLD. In conclusion, the up-regulation of A20 by Vinc could involve inhibitory effects on the proliferation and survival of K562 cells. The events might contribute to the anticancer effect of Vinc on A20-sensitive CML cells.

Upregulated mitosis-associated genes CENPE, CENPF, and DLGAP5 predict poor prognosis and chemotherapy resistance of Acute Myeloid Leukemia.

Mitosis-associated genes are dysregulated in many types of cancers and play important roles in disease progression and chemotherapy resistance. However, their expression and functions in chemotherapy-resistant Acute Myeloid Leukemia (AML) are still largely undetermined. This study aims to explore the roles of spindle assembly checkpoint (SAC) genes CENPE, CENPF, and DLGAP5 in chemotherapy-resistant AML. RNA-sequencing (RNA-seq) was performed in patients with chemotherapy-resistant AML and chemotherapy-sensitive AML. AML mRNA data from 151 patients with recurrence were downloaded from TCGA. Integrated analysis of the differentially expressed genes (DEGs), GO and KEGG pathways. CENPE, CENPF, or DLGAP5 knockdown cell lines were used to analyse proliferation, apoptosis and cell cycle alterations. A total of 87 DEGs (48 upregulated and 39 downregulated) were obtained through gene analysis of R/R-AML and a total of 329 DEGs (202 upregulated and 127 downregulated) were obtained in refractory S-AML. Upregulated DEGs were mainly enriched in cell cycle (GO: 0007049, hsa04110) and mitotic cell cycle (GO: 0000278) processes and pathway. Venn diagram analysis identified the most upregulated DEGs (including CENPE, CENPF, and DLGAP5) in chemoresistant AML. The expression of CENPE, CENPF and DLGAP5 in R-AML (TCGA) was significantly higher than that of primary AML (GEO). The proliferation of K562 cells after CENPE and DLGAP5 knockdown was significantly decreased (P= 0.0001 and P= 0.0006). In THP-1 cells, the CCK-8 values after CENPE, CENPF and DLGAP5 knockdown were significantly decreased (P= 0.01, P= 0.0395 and P= 0.0362). Knockdown of CENPE, CENPF and DLGAP5 significantly increased cell apoptosis by regulating Caspase-9, BAX, TP-53 and bcl-2, and induced cell cycle arrested by regulating CDK1, CDK2, CDKN1A, and CyclinD1. In conclusion, the mitotic cell cycle-associated genes CENPE, CENPF, and DLGAP5 were upregulated in chemotherapy-resistant AML patients and might be useful for predicting poor prognosis.

A novel coumarin derivative DBH2 inhibits proliferation and induces apoptosis of chronic myeloid leukemia cells

With the development of tyrosine kinase inhibitor (TKI) resistance, finding the novel effective chemotherapeutic agent is of seminal importance for chronic myelogenous leukemia (CML) treatment. This study aims to find the effective anti-leukemic candidates and investigate the possible underlying mechanism. We synthesized the novel coumarin derivatives and evaluated their anti-leukemic activity. Cell viability assay revealed that compound DBH2 exhibited the potent inhibitory activity on the proliferation of CML K562 cells and TKI resistant K562 cells. Morphological observation and flow cytometry confirmed that DBH2 could selectively induce cell apoptosis and cell cycle arrest at G2/M phase of the K562 cells, which was further confirmed on the bone marrow cells from CML transgenic model mice and CD34+ bone marrow leukemic cells from CML patients. Treatments of DBH2 in combination with imatinib could prolong the survival rate of SCL-tTA-BCR/ABL transgenic model mice significantly. Quantitative RT-PCR revealed that DBH2 inhibited the expression of STAT3 and STAT5 in K562 cells, and caspase-3 knockout alleviated the DBH2 induced apoptosis. Furthermore, DBH2 could induce the expression of PARP1 and ROCK1 in K562 cells, which may play the important role in caspase-dependent apoptosis. Our results concluded that coumarin derivative DBH2 serves as a promising candidate for the CML treatment, especially in the combination with imatinib for the TKI resistant CML, and STAT/caspase-3 pathway was involved in the molecular mechanism of anti-leukemic activity of DBH2.