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Re-Expression of Bone Marrow Proteoglycan-2 by 5-Azacytidine is associated with STAT3 Inactivation and Sensitivity Response to Imatinib in Resistant CML Cells

Al-Jamal HAN, Johan MF, Mat Jusoh SA, Ismail I, Wan Taib WR

Asian Pac J Cancer Prev, 2018 Jun 25;19(6):1585-1590.
PMID: 29936783

Background: Epigenetic silencing of tumor suppressor genes (TSG) is involved in development and
progression of cancers. Re-expression of TSG is inversely proportionate with STAT3 signaling pathways.
Demethylation of DNA by 5-Azacytidine (5-Aza) results in re-expression of silenced TSG. Forced expression of
PRG2 by 5-Aza induced apoptosis in cancer cells. Imatinib is a tyrosine kinase inhibitor that potently inhibits BCR/
ABL tyrosine kinase resulting in hematological remission in CML patients. However, majority of CML patients treated
with imatinib would develop resistance under prolonged therapy. Methods: CML cells resistant to imatinib were
treated with 5-Aza and cytotoxicity of imatinib and apoptosis were determined by MTS and annexin-V, respectively.
Gene expression analysis was detected by real time-PCR, STATs activity examined using Western blot and methylation
status of PRG2 was determined by pyrosequencing analysis. Result: Expression of PRG2 was significantly higher in
K562-R+5-Aza cells compared to K562 and K562-R (p=0.001). Methylation of PRG2 gene was significantly decreased
in K562-R+5-Aza cells compared to other cells (p=0.021). STAT3 was inactivated in K562-R+5-Aza cells which showed
higher sensitivity to imatinib. Conclusion: PRG2 gene is a TSG and its overexpression might induce sensitivity to
imatinib. However, further studies are required to evaluate the negative regulations of PRG2 on STAT3 signaling.

Matched MeSH terms: K562 Cells
Silencing of suppressor of cytokine signaling-3 due to methylation results in phosphorylation of STAT3 in imatinib resistant BCR-ABL positive chronic myeloid leukemia cells

Al-Jamal HA, Jusoh SA, Yong AC, Asan JM, Hassan R, Johan MF

Asian Pac J Cancer Prev, 2014;15(11):4555-61.
PMID: 24969884

BACKGROUND: Silencing due to methylation of suppressor of cytokine signaling-3 (SOCS-3), a negative regulator gene for the JAK/STAT signaling pathway has been reported to play important roles in leukemogenesis. Imatinib mesylate is a tyrosine kinase inhibitor that specifically targets the BCR-ABL protein and induces hematological remission in patients with chronic myeloid leukemia (CML). Unfortunately, the majority of CML patients treated with imatinib develop resistance under prolonged therapy. We here investigated the methylation profile of SOCS-3 gene and its downstream effects in a BCR-ABL positive CML cells resistant to imatinib.
MATERIALS AND METHODS: BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562 cell lines to the drug. Cytotoxicity was determined by MTS assays and IC50 values calculated. Apoptosis assays were performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profiles were investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Gene expression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1, 2 and 3 were examined by Western blotting.
RESULTS: The IC50 for imatinib on K562 was 362 nM compared to 3,952 nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing the concentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation of the SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed down- regulation of both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but not K562.
CONCLUSIONS: Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells. Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNA methylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying the development of imatinib resistant in CML patients.

Matched MeSH terms: K562 Cells
Fulltext Soluble Flt-1 gene delivery in acute myeloid leukemic cells mediating a nonviral gene carrier

Amini R, Azizi Jalilian F, Veerakumarasivam A, Abdullah S, Abdulamir AS, Nadali F, et al.

Biomed Res Int, 2013;2013:752603.
PMID: 23509773 DOI: 10.1155/2013/752603

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor involved in angiogenesis-mediated progression of acute myeloid leukemia (AML). Studies have reported the role of soluble form of fms-like tyrosine kinase (sFlT-1) delivery as an antitumor agent by inhibiting VEGF. This study investigates the outcome of delivery of a VEGF165 antagonist, soluble vascular endothelial growth factor receptor, namely sFLT-1, mediating lipofectamine 2000 in acute myeloid leukemic cells. A recombinant plasmid expressing sFLT-1 was constructed and transfected into the K562 and HL60 cells using lipofectamine 2000 transfection reagent. sFLT-1 expression/secretion in pVAX-sFLT-1 transfected cells was verified by RT-PCR and western blot. MTS assay was carried out to evaluate the effect of sFLT-1 on human umbilical vein endothelial cells and K562 and HL60 cells in vitro. Treatment with pVAX-sFLT-1 showed no association between sFLT-1 and proliferation of infected K562 and HL60 cells, while it demonstrated a significant inhibitory impact on the proliferation of HUVECs. The results of the current study imply that the combination of nonviral gene carrier and sFLT-1 possesses the potential to provide efficient tool for the antiangiogenic gene therapy of AML.

Matched MeSH terms: K562 Cells
Styryl-lactone goniothalamin inhibits TNF-α-induced NF-κB activation

Orlikova B, Schumacher M, Juncker T, Yan CC, Inayat-Hussain SH, Hajjouli S, et al.

Food Chem Toxicol, 2013 Sep;59:572-8.
PMID: 23845509 DOI: 10.1016/j.fct.2013.06.051

(R)-(+)-Goniothalamin (GTN), a styryl-lactone isolated from the medicinal plant Goniothalamus macrophyllus, exhibits pharmacological activities including cytotoxic and anti-inflammatory effects. In this study, GTN modulated TNF-α induced NF-κB activation. GTN concentrations up to 20 μM showed low cytotoxic effects in K562 chronic myelogenous leukemia and in Jurkat T cells. Importantly, at these concentrations, no cytotoxicity was observed in healthy peripheral blood mononuclear cells. Our results confirmed that GTN inhibited tumor necrosis factor-α (TNF-α)-induced NF-κB activation in Jurkat and K562 leukemia cells at concentrations as low as 5 μM as shown by reporter gene assays and western blots. Moreover, GTN down-regulated translocation of the p50/p65 heterodimer to the nucleus, prevented binding of NF-κB to its DNA response element and reduced TNF-α-activated interleukin-8 (IL-8) expression. In conclusion, GTN inhibits TNF-α-induced NF-κB activation at non-apoptogenic concentrations in different leukemia cell models without presenting toxicity towards healthy blood cells underlining the anti-leukemic potential of this natural compound.

Matched MeSH terms: K562 Cells
Synthesis, Structure, and Anticancer Activity of Symmetrical and Non-symmetrical Silver(I)-N-Heterocyclic Carbene Complexes

Atif M, Bhatti HN, Haque RA, Iqbal MA, Ahamed Khadeer MB, Majid AMSA

Appl Biochem Biotechnol, 2020 Jul;191(3):1171-1189.
PMID: 32002729 DOI: 10.1007/s12010-019-03186-9

Synthesis and anticancer studies of three symmetrically and non-symmetrically substituted silver(I)-N-Heterocyclic carbene complexes of type [(NHC)2-Ag]PF6 (7-9) and their respective (ligands) benzimidazolium salts (4-6) are described herein. Compound 5 and Ag-NHC-complex 7 were characterized by the single crystal X-ray diffraction technique. Structural studies for 7 showed that the silver(I) center has linear C-Ag-C coordination geometry (180.00(10)o). Other azolium and Ag-NHC analogues were confirmed by H1 and C13-NMR spectroscopy. The synthesized analogues were biologically characterized for in vitro anticancer activity against three cancer cell lines including human colorectal cancer (HCT 116), breast cancer (MCF-7), and erythromyeloblastoid leukemia (K-562) cell lines and in terms of in vivo acute oral toxicity (IAOT) in view of agility and body weight of female rats. In vitro anticancer activity showed the values of IC50 in range 0.31-17.9 μM in case of K-562 and HCT-116 cancer cell lines and 15.1-35.2 μM in case of MCF-7 while taking commercially known anticancer agents 5-fluorouracil, tamoxifen, and betulinic acid which have IC50 values 5.2, 5.5, and 17.0 μM, respectively. In vivo study revealed vigor and agility of all test animals which explores the biocompatibility and non-toxicity of the test analogues.

Matched MeSH terms: K562 Cells
Fulltext Thymoquinone Induces Downregulation of BCR-ABL/JAK/STAT Pathway and Apoptosis in K562 Leukemia Cells

Al-Rawashde FA, Wan Taib WR, Ismail I, Johan MF, Al-Wajeeh AS, Al-Jamal HAN

Asian Pac J Cancer Prev, 2021 Dec 01;22(12):3959-3965.
PMID: 34967577 DOI: 10.31557/APJCP.2021.22.12.3959

OBJECTIVE: BCR ABL oncogene encodes the BCR-ABL chimeric protein, which is a constitutively activated non-receptor tyrosine kinase. The BCR-ABL oncoprotein is a key molecular basis for the pathogenesis of chronic myeloid leukemia (CML) via activation of several downstream signaling pathways including JAK/STAT pathway. Development of leukemia involves constitutive activation of signaling molecules including, JAK2, STAT3, STAT5A and STAT5B. Thymoquinone (TQ) is a bioactive constituent of Nigella sativa that has shown anticancer properties in various cancers. The present study aimed to evaluate the effect of TQ on the expression of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes and their consequences on the cell proliferation and apoptosis in K562 CML cells.
METHODS: BCR-ABL positive K562 CML cells were treated with TQ. Cytotoxicity was determined by Trypan blue exclusion assay. Apoptosis assay was performed by annexin V-FITC/PI staining assay and analyzed by flow cytometry. Transcription levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes were evaluated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Protein levels of JAK2 and STAT5 were determined by Jess Assay analysis.
RESULTS: TQ markedly decreased the cell proliferation and induced apoptosis in K562 cells (P < 0.001) in a concentration dependent manner. TQ caused a significant decrease in the transcriptional levels of BCR ABL, JAK2, STAT3, STAT5A and STAT5B genes (P < 0.001). TQ induced a significant decrease in JAK2 and STAT5 protein levels (P < 0.001).
CONCLUSION: our results indicated that TQ inhibited cell growth of K562 cells via downregulation of BCR ABL/ JAK2/STAT3 and STAT5 signaling and reducing JAK2 and STAT5 protein levels.

Matched MeSH terms: K562 Cells