scholarly journals Role of Folate Receptor 3 in Tyrosine Kinase Inhibitor Discontinuation in Chronic Myeloid Leukemia

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2998-2998
Author(s):  
Na Shen ◽  
Xiaojian Zhu ◽  
Shu Zhou ◽  
Yong You ◽  
Zhaodong Zhong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Cell Differentiation ◽  
Chronic Myeloid Leukemia ◽  
Tumor Cell ◽  
Transcriptome Analysis ◽  
Myeloid Leukemia ◽  
Folate Receptor ◽  
K562 Cells ◽  
Tumor Cell Differentiation

Abstract Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by Philadelphia chromosome and its molecular counterpart, the BCR-ABL1 fusion gene. Discontinuation of tyrosine kinase inhibitors (TKIs) therapy after achieving a persistent deep molecular response (DMR) is an urgently-needed treatment goal for patients and it was written in NCCN guideline Version 2.2017 for CML. Indeed, various studies have confirmed the feasibility of stopping TKIs therapy in many regions. However, increasing evidence demonstrated that TKIs are unable to eliminate quiescent leukemic stem cells (LSCs), which lead to treatment resistance or relapse after discontinuation of TKIs treatment. Multiple works have confirmed that approximately 50-60% of DMR patients stopping TKIs will lose their response and require retreatment. The patients who will lose the treatment-free remission (TFR) remains unknown currently. We selected bone marrow of 14 patients (matched 7 relapse and 7 non-relapse patients) who discontinued TKIs therapy in our center for transcriptome analysis to discover the differences between relapse and non-relapse patients. Bioinformatics indicated that folate receptor 3 (FOLR3) was highly expressed in non-relapse patients and only in the non-relapsers (Figure 1A). Further work found these samples all carried a common SNP mutation. Then we respectively transferred newly diagnosed chronic phase (CP) CML patients derived bone marrow CD34+ cells and CML K562 cells with lentiviral vectors containing FOLR3 SNP, FOLR3 SNP shRNA, and wild type FOLR3. The result indicated that FOLR3 SNP significantly promoted the clonogenicity of CD34+/K562 cells, while loss of FOLR3 SNP hindered cell differentiation (Figure 1B and D are data of CD34, Figure 1C and E are data of K562). K562 subcutaneous tumor formation in balb/c node mice confirmed that tumors weight and volume of FOLR3 SNP shRNA group were higher than control. While, small animal PET scanning showed that maximum standardized uptake value (SUVmax) of 18F-FDG of FOLR3 SNP+ group was higher than the rest. Experiments in vivo and vitro synergistically proved FOLR3 SNP promoted tumor cell differentiation, delayed tumor growth. To better understand how FOLR3 SNP promote tumor cell differentiation and delay tumor growth, we performed second transcriptome analysis. Consistently, both in FOLR3 SNP+ CD34+ and K562 cells, enrichment analysis revealed that differentially expressed genes were enriched in mitochondria associated Gene Ontology (GO) biological process (Figure F), in which mitochondria complex V matched genes were most significant, such as ATP5 family. In vitro, we demonstrated that ATP syntheses, maximal respiration and spare respiratory capacity of FOLR3 SNP+ CD34+/K562 cells were significantly higher than control and shFOLR3 counterpart through seahorse XF cell mito stress test (Figure G and H refer to CD34 and K562, separately). Electron microscope also exhibited an increase of mitochondrial in FOLR3 SNP+ cells. Mitochondria play an essential role in energy generation, cell signalling, differentiation, death and senescence in eukaryotic cells. We detected a series of genes related to aging, cell cycle and mitochondria unfolded protein response. The results showed that cell cycle kinase such as CDK4 decreased in FOLR3 SNP+ CD34+/K562 cells. On the other hand, senescence associated genes seemed increased. In conclusion, we highlighted the connection of FOLR3 and post-cessation relapse. FOLR3 SNP could be an indicator of TFR. Its internal mechanism might be the mitochondrion activation induced aging of residual leukemia cells. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

Role of eIF3a in 4-amino-2-trifluoromethyl-phenyl retinate-induced cell differentiation in human chronic myeloid leukemia K562 cells

Gene ◽  
2019 ◽  
Vol 683 ◽  
pp. 195-209 ◽  
Author(s):  
Ge Li ◽  
Ke Wang ◽  
Yue Li ◽  
Jinging Ruan ◽  
Cong Wang ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
K562 Cells

scholarly journals A Novel Methoxybenzyl 5-Nitroacridone Derivative Effectively Triggers G1 Cell Cycle Arrest in Chronic Myelogenous Leukemia K562 Cells by Inhibiting CDK4/6-Mediated Phosphorylation of Rb

2020 ◽  
Vol 21 (14) ◽  
pp. 5077
Author(s):  
Bin Zhang ◽  
Ting Zhang ◽  
Tian-Yi Zhang ◽  
Ning Wang ◽  
Shan He ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chronic Myeloid Leukemia ◽  
Cell Cycle Arrest ◽  
Chronic Myelogenous Leukemia ◽  
Myeloid Leukemia ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
G1 Phase ◽  
Cycle Arrest ◽  
G1 Cell Cycle Arrest

Chronic myeloid leukemia (CML) is a malignant tumor caused by the abnormal proliferation of hematopoietic stem cells. Among a new series of acridone derivatives previously synthesized, it was found that the methoxybenzyl 5-nitroacridone derivative 8q has nanomolar cytotoxicity in vitro against human chronic myelogenous leukemia K562 cells. In order to further explore the possible anti-leukemia mechanism of action of 8q on K562 cells, a metabolomics and molecular biology study was introduced. It was thus found that most of the metabolic pathways of the G1 phase of K562 cells were affected after 8q treatment. In addition, a concentration-dependent accumulation of cells in the G1 phase was observed by cell cycle analysis. Western blot analysis showed that 8q significantly down-regulated the phosphorylation level of retinoblastoma-associated protein (Rb) in a concentration-dependent manner, upon 48 h treatment. In addition, 8q induced K562 cells apoptosis, through both mitochondria-mediated and exogenous apoptotic pathways. Taken together, these results indicate that 8q effectively triggers G1 cell cycle arrest and induces cell apoptosis in K562 cells, by inhibiting the CDK4/6-mediated phosphorylation of Rb. Furthermore, the possible binding interactions between 8q and CDK4/6 protein were clarified by homology modeling and molecular docking. In order to verify the inhibitory activity of 8q against other chronic myeloid leukemia cells, KCL-22 cells and K562 adriamycin-resistant cells (K562/ADR) were selected for the MTT assay. It is worth noting that 8q showed significant anti-proliferative activity against these cell lines after 48 h/72 h treatment. Therefore, this study provides new mechanistic information and guidance for the development of new acridones for application in the treatment of CML.

Bcr-Abl-Mediated Suppression of Normal Hematopoiesis in Chronic Myeloid Leukemia: Involvement of a Modified Form of NGAL.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2869-2869
Author(s):  
Hui Lin ◽  
Xiaohong Leng ◽  
Tong Sun ◽  
Giuseppe Monaco ◽  
Clifton Stephens ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Hematopoietic Cells ◽  
K562 Cells ◽  
Soft Agar ◽  
Leukemia Cells ◽  
Active Process ◽  
Culture Studies ◽  
Induced Apoptosis

Abstract The BCR-ABL oncogene plays an essential role in chronic myeloid leukemia (CML). In NOD/scid mice injected with soft agar clones of a human CML cell line (K562), we observed a leukemia syndrome involving not only leukemia but also a severe reduction of normal mouse hematopoiesis (Lin et al., Oncogene, 2001). Some of these mice died of a wasting syndrome that involved suppression of hematopoiesis without extensive tumor cell invasion of the spleen and marrow. In CML patients, since normal hematopoietic cells in marrow and spleen are replaced with proliferating leukemic blasts, we postulate that this is an active process mediated by the leukemia cells. The lipocalin 24p3 is secreted by mouse hematopoietic cells deprived of IL-3, resulting in apoptosis induction in a variety of hematopoietic cells including bone marrow cells (Devireddy et al., Science, 2001). We found that BCR-ABL+ mouse hematopoietic cells induce a persistent secretion of a modified form of 24p3 (21 kDa). Co-culture studies show that BCR-ABL+ cells induced apoptosis in BCR-ABL negative cells. Importantly, BCR-ABL+ hematopoietic cells are resistant to apoptosis under the same conditions. Conditioned medium (CM) from BCR-ABL+ cells expressing anti-sense/siRNA 24p3 or CM mixed with 24p3 antibody have reduced apoptotic activity for target cells. We also found that the expression of the Bcr-Abl oncoprotein and its tyrosine kinase are required for induction of 24p3 expression. Leukemic mice induced by BCR-ABL+ cells expressing anti-sense/siRNA 24p3 have increased levels of normal hematopoiesis (marrow and spleen erythropoiesis and blood platelet levels) and reduced invasion of leukemia cells in marrow and spleen tissues, but the leukemia cells readily invade liver and the abdomen as ascites (Lin et al, Oncogene, 2005). These findings indicate that suppression of normal hematopoiesis in BCR-ABL induced leukemia is an active process involving the apoptotic factor 24p3, raising the possibility that similar factors are involved in BCR-ABL+ CML patients. We have found that the K562 clones (Lin et al. 2001) have enhanced expression of NGAL (neutrophil gelatinase-associated lipocalin, human homologue of 24p3) transcripts compared to uncloned K562 cells. We generated additional soft agar K562 clones, each with different expression levels of NGAL transcripts. NOD/scid mice injected with the clone (C5) of K562 cell line expressing a high level of NGAL had severe depression of hematopoiesis and significantly shorter survival time as compared with mice injected with parental K562 cells and a clone (C6) expressing a low level of NGAL. Co-culture studies showed that the C5 K562 clone also induced apoptosis in BCR-ABL negative cells. We detected two glycosylated forms of NGAL/24p3 migrating at 24 kDa and 21 kDa on SDS-PAGE. The 21 kDa form is the major form in CM from mouse BCR-ABL+ cells and K562 clones. Our preliminary data with CML patient samples showed that levels of 21 kDa NGAL protein in bone marrow fluid correlated with BCR-ABL/ABL ratio. Further studies with more patient samples are ongoing to confirm the role of NGAL in suppressing normal hematopoiesis in CML patients and to determine the structural change(s) that leads to the modified form of 24p3/NGAL secreted by CML cells.

VASP Protein, a Binding Partner Of BCR-ABL and FAK, May Be Implicated In The Chronic Myeloid Leukemia Phenotype

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 5162-5162
Author(s):  
Vanessa Aline Bernusso ◽  
João Agostinho Machado-Neto ◽  
Fernando V Pericole ◽  
Karla Priscila Vieira ◽  
Adriana Silva Santos Duarte ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Adhesion ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
K562 Cells ◽  
Fold Increase ◽  
Imatinib Treatment ◽  
Gene Expressions ◽  
Megakaryocytic Differentiation ◽  
Healthy Donors

Abstract Background VASP (vasodilator-stimulated phosphoprotein) and Zyxin are actin regulatory proteins that control cell-cell adhesion. Zyxin directs actin assembly by interacting and recruiting VASP to specific sites of adhesion. The phosphorylation of VASP modifies their activity in cell-cell junctions. PKA phosphorylates VASP at serine 157 regulating VASP cellular functions. VASP is a substrate of BCR-ABL oncoprotein and is tyrosine-phosphorylated in leukemic cells. However, the function of VASP and Zyxin in hematopoietic cells, in the BCR-ABL pathway and its participation in chronic myeloid leukemia (CML) remains unknown. Aims To analyze VASP and Zyxin expression in bone marrow cells from CML patients and healthy donors, as well the involvement of these proteins in hematopoietic cell differentiation and in the BCR-ABL signaling pathway. Materials and Methods VASP and Zyxin expression and phosphorylation were studied in bone marrow samples from 29 individuals (5 healthy donors, 5 CML patients at diagnosis, 16 CML patients responsive to treatment with tyrosine kinase inhibitors (ITK) and 3 CML patients resistant to ITK). One patient was analyzed at diagnosis and after ITK response. VASP or Zyxin silencing was performed by shRNA-lentiviral delivery in K562 cell line, an appropriated shControl was used. ShControl, shVASP and shZyxin K562 cells were induced to megakaryocytic differentiation with 20nM of PMA (phorbol myristate -13 -12 acetate) during 4 days and CD61 expression, a marker for maturing megakaryocytes, was verified by flow cytometry. During megakaryocytic differentiation, VASP and Zyxin gene expressions were evaluated by quantitative PCR; protein expression and activation were determined by Western blotting. Effector proteins of proliferation, apoptosis and adhesion in the BCR-ABL signaling pathway were analyzed in cells silenced for VASP or Zyxin. The interaction of VASP and BCR-ABL or FAK was evaluated by co-immunoprecipiation. Results Healthy donors showed p-VASP ser157 expression, in contrast to CML patients at diagnosis who did not present phospho-VASP ser157. After Imatinib treatment CML patients restored VASP phosphorylation however resistant patients maintained this absence. Zyxin showed the same expression in patients and healthy donors. During Imatinib treatment of K562 cells, phospho-VASP ser157 expression was increased and its interaction with BCR-ABL protein was reduced. VASP and Zyxin gene expressions were upregulated during megakaryocyte differentiation of K562 cells (8.7-fold increase, P=0.0115, and 3.6-fold increase, P=0.015, respectively). VASP and Zyxin protein expressions were increased during megakaryocytic differentiation, including the active form of these proteins (p-VASP ser157 and p-Zyxin ser142). VASP silencing in K562 cells resulted in a 40% decrease of CD61 expression at the end of the megakaryocytic differentiation (P<0.05). In addition, VASP and Zyxin silencing resulted in a decrease of BCL-2 and BCL-XL proteins. VASP binds to FAK, an adhesion effector protein of the BCR-ABL pathway, and it´s silencing resulted in a decreased phosphorylation of FAK y925. Conclusions In BCR-ABL cells, VASP and Zyxin modulated anti-apoptotic proteins and megakaryocytic differentiation. Hence, the altered expression of VASP activity in CML patients may contribute to the pathogenesis of the disease, affecting cellular differentiation or leukemic cell adhesion. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Orlistatand and Rosuvastatin Suppressed Proliferation of K562 Human Myelogenous Leukemia Cell Line through AMPK/Akt/c-Myc Signaling

2020 ◽  
Author(s):  
Behnam Mojjarad ◽  
Yaghub Pazhang
Keyword(s):  
Cell Cycle ◽  
Chronic Myeloid Leukemia ◽  
Cell Line ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Mrna Levels ◽  
Hsp 70

Abstract Background: Chronic myeloid leukemia is a myeloproliferative cancer with worldwide incidence, has become as a clinical concern due to chemoresistance in the patients received chemotherapy. Here, we investigated the effect of Orlistat and Rosuvastatin on K562 human myelogenous leukemia cell line in vitro and attempted to illuminate their possible underlying mechanisms. Methods: Cells were exposed to Orlistat and Rosuvastatin, the inhibitors of lipogenesis, then survival and apoptosis rate of K562 cells were examined by MTT assay and flow cytometric analysis respectively. The real time-PCR analysis was used to quantify mRNA levels of Bax, Bcl-2, and Hsp-70 genes. Cell cycle analysis was performed using flow cytometry, whereas the subcellular distribution of c-Myc was measured via immunofluorescence imaging technique. Additionally, the protein level of AMPK, p-AMPK Akt-1, and p-Akt-1 were studied by western blotting. Results: The results showed Orlistat and Rosuvastatin had synergistic anticancer effects on cells and in comparison with the control group, viability and apoptosis rate decreased and increased in treated cells respectively in a dose/time-dependent manner (P<0.05). The mRNA levels of Bax increased while expression of Hsp-70 decreased (P< 0.05). K562 cells treated with Orlistat and Rosuvastatin showed a cell cycle arrest in sub-G1 phase and a decreased level of c-Myc positive cells. Upon outlining the mechanism, it was revealed that AMPK/p-AMPK and p-Akt-1/Akt-1 ratio decreased in treated cells (P< 0.05). Conclusions: Data suggest Orlistat and Rosuvastatin could synergically suppress proliferation of K562 cells through AMPK/Akt/c-Myc axis, proposing a theoretical basis for upcoming application in the treatment of chronic myeloid leukemia

scholarly journals The Role of Hypoxia on ABL Tyrosine Kinase Inhibitor Resistant Chronic Myeloid Leukemia Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5369-5369
Author(s):  
Seiichi Okabe ◽  
Yuko Tanaka ◽  
Mitsuru Moriyama ◽  
Akihiko Gotoh
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
K562 Cells ◽  
Leukemia Cells ◽  
Leukemia Stem Cells ◽  
Transfected Cells ◽  
Resistant Cells

Introduction: ABL tyrosine kinase inhibitors (TKIs) improved outcomes for patients with chronic myeloid leukemia (CML) and Philadelphia chromosome (Ph)-positive leukemia, however, some patients are still resistance to ABL TKIs. One of the most common mechanisms involves point mutations in the kinase domain of BCR-ABL1, however, mechanisms of intrinsic resistance without point mutation of ABL kinase domain are not fully understood. Moreover, ABL TKIs cannot cure the Ph-positive leukemia patients because of leukemia stem cells in the bone marrow niche. Therefore, new approach against leukemia stem cells may improve the outcome of Ph-positive leukemia patients. Hypoxia is an important component of the bone marrow microenvironment. Because oxygen tension plays a key role in driving normal hematopoiesis, leukemia stem cells may be maintained in hypoxic areas of the bone marrow. Materials and methods: In this study, we established ABL TKI-resistant in vitro cell line models (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R and Ba/F3 T315I). We investigated gene expression profiles in cultured ABL TKI resistant cells and parental cell line, K562 in normoxia and hypoxia condition by DNA microarray. Results: We first investigated gene expression profiles in cultured K562 cells in hypoxia condition. We found gene expression of insulin-like growth factor 1 (IGF1) was increased K562 cells in hypoxia condition by DNA microarray. We next examined ABL TKI resistant cell lines (K562 imatinib-R, K562 nilotinib-R, K562 dasatinib-R, K562 ponatinib-R) in this study. We could not detect the BCR-ABL point mutation in ABL TKI resistant cells. We found gene expression of insulin-like growth factor 1 (IGF1) receptor (IGF1R) was increased ABL TKI resistant K562 cells. IGF1R gene amplification was confirmed by RT-PCR analysis. IGF is tightly regulated by six related IGF-binding proteins (IGFBPs). One of IGFBP, IGFBP5 is related to imatinib sensitivity and resistant in chronic myeloid leukemia (CML) patients (GSE12211). In hypoxia condition, several IGFBPs were also increased in ABL TKI resistant cells. IGF cause intracellular signaling that ultimately results in cellular growth and proliferation. Thus, we initially examined whether addition of IGF1R inhibition could enhance ABL TKIs sensitivity. One of IGF1R inhibitor, linsitinib was inhibited ABL TKI resistant cells and parental cell line, K562 in hypoxia condition. ABL TKI resistant cell lines were more sensitive against linsitinib. Combined treatment of ABL TKI resistant cells and K562 cells with ABL TKIs and linsitinib caused more cytotoxicity than each drug alone in hypoxia condition. Caspase 3/7 activity and cellular cytotoxicity was also increased after ABL TKIs and linsitnib treatment. In the colony formation method, the number of cell colonies were also reduced in hypoxia condition. Intracellular ATP levels have been implicated in vitro as a determinant of cell death by apoptosis. The concentrations of intracellular ATP were reduced after ABL TKIs and linsitinib. We next blocked IGF1R function by small interfering RNA (siRNA). SiRNA transfected cells were reduced cellular proliferation. We also found drug sensitivity of the cells to the imatinib was increased compared to mock-transfected cells. Apoptotic cells and caspase 3/7 activity were increased after imatinib treatment in siRNA transfected cells. Conclusion: The IGF1 pathway is involved in Ph-positive leukemia cells in hypoxia condition and ABL TKI resistant in CML cells. We also provide the promising clinical relevance as a candidate drug for treatment of residual leukemia cells in bone marrow niche which is in hypoxia condition. Disclosures No relevant conflicts of interest to declare.

Preliminary Study On Influence of Imatinib-Induced Apoptosis in Chronic Myeloid Leukemia Cells Co-Culture with Fibronectin Adhesion.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4264-4264
Author(s):  
Liu Xiaoli ◽  
Na Xu ◽  
Qingfeng Du ◽  
Zhi Liu ◽  
Rong Li ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
Growth Inhibition ◽  
Myeloid Leukemia ◽  
K562 Cells ◽  
Bone Marrow Microenvironment ◽  
Dependent Manner ◽  
Imatinib Resistance ◽  
Significant Difference

Abstract Abstract 4264 The bone marrow microenvironment supports growth, differentiation and proliferation of normal hematopoietic cells and can also contribute to malignant growth. Recently,it is considered that except for the point mutant of BCR-ABL kinase contribute to imatinib-resistant therapy for patients with chronic myeloid leukemia(CML), environment-mediated drug resistance (EM-DR) is a potential factor in imatinib resistance. Our previous studies found that Integrin, focal adhesion kinase(FAK), RhoA(a small GTPase) are important adhesion molecules,and related to imatinib resistance. But how and what they crosstalk with each other is still open to debate. In order to simulated bone marrow microenvironment, we used the major components of bone marrow microenvironment- Fibronectin (Fn) co-cultured with human leukemia K562 cells.and then K562 cells were inoculated with Fn, collagen-coated plate(Co) and suspended cultures as control(mask) group,and then treated with 0.4μM,0.8μM,1.6μM,3.2μM,6.4μM imatinib for 24h,48h and 72h, detected cell apoptosis and proliferation by MTT and AnnexinV-PI assay, examined p-FAK and Rho-GTP by Wersten Blotting and Pull down-Wersten Blotting. The data showed that compared to the Co and mask groups, the cells growth inhibition and apoptosis in Fn co-culture group was significantly reduced. The protein expression of p-FAK and Rho-GTP was higher in the Fn group,and in time-dependent manner. When K562 cells in Fn group were transfected with 150nM siRNA-RhoA for 48h, there was no significant difference compared with the Co and mask groups. Furthermore, the above groups treated with anti-integrin monoclonal antibody (anti-CD29 mAb), we found that p-FAK was significant lower compared with without anti-CD29 mAb in the Fn group; but there was no significant difference of Rho-GTP compard with without anti-CD29 mAb in Fn group. These results indicate that Fn adhesion co-culture could reduce imatinib-induced cell growth inhibition and apoptosis, and this mechanism may be correlated to Rho-GTP activity,and anti-integrin monoclonal antibody could not completely block the integrin binding to Fn on K562 cells, or there was other pathway activated RhoA. The mechanism of EM-DR is complex,and which is well worth us to speculate and study. Disclosures: No relevant conflicts of interest to declare.

Induction of Apoptosis in K562 Human Chronic Myeloid Leukemia Cells by Tetra-Arsenic Tetra-Sulfide.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4375-4375
Author(s):  
Qidong Ye ◽  
Long-Jun Gu ◽  
Yanxia Zhao ◽  
Jincai Zhao ◽  
Wengao Chen ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Morphological Changes ◽  
K562 Cells ◽  
Morphologic Change ◽  
Mrna Levels ◽  
Protein Levels ◽  
Reverse Transcription Pcr ◽  
M Phase

Abstract Realgar has been used as a traditional medicine in China for more than 1500 years. Some studies found that tetra-arsenic tetra-sulfide (As4S4), the main ingredient of realgar, used alone was highly effective and safe for all stages of acute promyelocytic leukemia. To explore the effects of As4S4 in treatment of human chronic myeloid leukemia K562 cells, we used microculture MTS assay to measure the growth inhibition of K562 cells. The morphologic change was determined by Wright’s staining and Hoechst33342 assay. Cell apoptosis was evaluated by DNA agarose gel electrophoresis. The apoptotic rate and cell cycle were measured by flow cytometry. The changes of transcript and protein levels were determined by real-time reverse transcription-PCR and Western blot analysis, respectively. As4S4 had signigicant cytotoxicity on K-562 cells. At the concentration of 2.0μmol/L, the cell viability decreased significantly after 24 hours cultured with the reagent. When the concentration was lower than 0.5μmol/L, As4S4 had little effect on K562 cells. The effect of As4S4 on K562 was time and concentration dependent. After cultured with As4S4 at the concentration of 2.0μmol/L for 24 to 48 hours, K562 cells appeared typical morphological changes of apoptosis. At a concentration greater than or equal to 2.0μmol/L, As4S4 could induce apoptosis significantly. After 12 hours of incubation with 2.0μmol/L As4S4, the apoptosis rate increased from 2.05% to 12.03%. At the same time, the percentage of cells in G1 phase decreased from 69.65% to 50.53%, whereas the percentage of G2/M phase increased from 9.56% to 25.91%. The mRNA levels of BCL-2, BCL-XL, BAD and BAX, and the protein levels of Akt and pAkt down-regulated after the inhibition of As4S4. The transcript and protein levels of BCR-ABL had no change after incubation with As4S4. These results indicated that As4S4 can inhibit the growth of K562 cells efficiently through inducing apoptosis and cell cycle arrest. It seems that As4S4 interferes with Akt pathway and down-regulate BCL-2, BCL-XL, BAD and BAX, which may be involved in the response of K562 to this agent. As4S4 could be beneficial for treatment of CML in combination with conventional drugs.

scholarly journals Inhibition of cystathionine β-synthase promotes apoptosis and reduces cell proliferation in chronic myeloid leukemia

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Dan Wang ◽  
Huan Yang ◽  
Yun Zhang ◽  
Rong Hu ◽  
Dongjie Hu ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Mononuclear Cells ◽  
K562 Cells ◽  
Bone Marrow Mononuclear Cells ◽  
Cycle Arrest ◽  
Primary Bone ◽  
And Migration

AbstractIncreased endogenous hydrogen sulfide (H2S) level by cystathionine β-synthase (CBS) has been shown to closely relate tumorigenesis. H2S promotes angiogenesis, stimulates bioenergy metabolism and inhibits selective phosphatases. However, the role of CBS and H2S in chronic myeloid leukemia (CML) remains elusive. In this study, we found that CBS and H2S levels were increased in the bone marrow mononuclear cells of pediatric CML patients, as well as in the CML-derived K562 cells and CBS expression levels were correlated with different disease phases. Inhibition of CBS reduced the proliferation of the CML primary bone marrow mononuclear cells and induced growth inhibition, apoptosis, cell cycle arrest, and migration suppression in K562 cells and tumor xenografts. The knockdown of CBS expression by shRNA and inhibiting CBS activity by AOAA decreased the endogenous H2S levels, promoted mitochondrial-related apoptosis and inhibited the NF-κB-mediated gene expression. Our study suggests that inhibition of CBS induces cell apoptosis, as well as limits cell proliferation and migration, a potential target for the treatment of chronic myeloid leukemia.

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