ITR‑284 modulates cell differentiation in human chronic myelogenous leukemia K562 cells

Abstract

ITR‑284 is a carboxamide analog that can inhibit proliferation in human promyelocytic leukemia HL-60 cells. To understand the effects and molecular mechanisms of ITR‑284 in human erythromyeloblastoid leukemia, we treated K562 cells with different concentrations of ITR‑284 (0, 2, 4, 6, 8 and 10 nM) and all-trans retinoic acid (ATRA) (0, 0.1, 0.5, 1, 5 and 10 µM) for 24 h. The IC50 of ITR‑284 was ~10 nM in K562 cells treated for 24 h as determined by MTT assay. May-Grünwald-Giemsa staining and nitro blue tetrazolium (NBT) assays were used to determine cell morphology changes and differentiation after ITR‑284 and ATRA treatment. In addition, mRNA expression levels of hematopoietic factors, including GATA‑1, NF-E2 and GATA‑2, were elevated, while expression levels of BCR‑ABL were downregulated in K562 cells after 24 h of treatment with ITR‑284 as determined by quantitative reverse transcription polymerase chain reaction. In addition, western blot analyses showed that FOXM1, GLI 1 and c-MYC protein levels were decreased by ITR‑284. Taken together, our data show that ITR‑284 induced K562 cell differentiation, which led to decreased tumorigenesis. Our findings suggest that ITR‑284 could be a potential candidate for treating chronic myelogenous leukemia.

Tetrandrine Inhibits Epithelial-Mesenchymal Transition in IL-6-Induced HCT116 Human Colorectal Cancer Cells

“…Total RNA was extracted from HCT116 cells using REzol TM C&T reagent (PROtech Technologies, Inc.) and RT-qPCR was performed as previously described. 34 Briefly, RNA samples were adjusted to a concentration of 50 ng/mL and mixed with 10 μM forward primer, 10 μM reverse primer and reagents contained within the One Step SYBR ® PrimeScript™ RT-PCR Kit II; the total volume of the positive and negative primers and the master mix was 15 μL. The following primer sequences were used for the qPCR: β-actin forward, 5ʹ-CCAACCGCGAGAAGATGA-3ʹ and reverse, 5ʹ-TCCATCACGATGCCAGTG-3ʹ; twist1 forward, 5ʹ-ACGCTGCCCTCGGACAA-3ʹ and reverse, 5ʹ-TCGCTCTGGAGGACCTGGTA-3ʹ; SNAIL forward, 5ʹ-CCCAATCGGAAGCCTAACTACA-3ʹ and reverse, 5ʹ-GGGCTGCTGGAAGGTAAACTCT-3ʹ; ZEB1 forward, 5ʹ-CACCATCCCCATCACCTCTAAA-3ʹ and reverse, 5ʹ- GCACCCTCAGCTGTGTACAAGTAA-3ʹ; MMP-2 forward, 5ʹ-GCGGCGGTCACAGCTACTT-3ʹ and reverse, 5ʹ-TTCACGCTCTTCAGACTTTGGTT-3ʹ; heat shock protein 90 (HSP90) β family member 1 (HSP90B1) forward, 5ʹ-CAGTTTGGTGTCGGTTTCTATTCC-3ʹ and reverse, 5ʹ-CTGGGTATCGTTGTTGTGTTTTGA-3ʹ; forkhead box A1 (FOXA1) forward, 5ʹ-CTCTAGGCAGCGCCTCGGTGA-3ʹ and reverse, 5ʹ-CAGCATGGCTATGCCAGACAAACCC-3ʹ; and E-cadherin forward, 5ʹ-ACGCATTGCCACATACACTCTCT-3ʹ and reverse, 5ʹ- CCATTGGATCCTCAACTGCATT-3ʹ.…”

“…Total RNA was extracted from HCT116 cells using REzol TM C&T reagent (PROtech Technologies, Inc.) and RT-qPCR was performed as previously described. 34 Briefly, RNA samples were adjusted to a concentration of 50 ng/mL and mixed with 10 μM forward primer, 10 μM reverse primer and reagents contained within the One…”

Curcumin Derivative MTH-3 Regulates Palmitate-induced Insulin Resistance in Mouse Myoblast C2C12Cells

“…RNA extract. Total RNA was isolated using TRIzol ® reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol, as described previously (25). Briefly, 2×10 6 cells for each treatment were harvested and mixed with 1 ml TRIzol ® reagent at room temperature for 5 min.…”

“…Quantitative (q)PCR. qPCR was performed using a TaKaRa One Step SYBR Ex Taq™ qRT-PCR kit (Takara Bio, Inc., Shiga, Japan) as described previously (25) in a QuantStudio™ 3 Real-Time PCR system (Thermo Fisher Scientific, Inc.). Relative expression levels of target genes were normalized to β-actin.…”