Imidazo[1,2-b]pyrazole-7-Carboxamide Derivative Induces Differentiation-Coupled Apoptosis of Immature Myeloid Cells Such as Acute Myeloid Leukemia and Myeloid-Derived Suppressor Cells

Abstract

Chemotherapy-induced differentiation of immature myeloid progenitors, such as acute myeloid leukemia (AML) cells or myeloid-derived suppressor cells (MDSCs), has remained a challenge for the clinicians. Testing our imidazo[1,2-b]pyrazole-7-carboxamide derivative on HL-60 cells, we obtained ERK phosphorylation as an early survival response to treatment followed by the increase of the percentage of the Bcl-xlbright and pAktbright cells. Following the induction of Vav1 and the AP-1 complex, a driver of cellular differentiation, FOS, JUN, JUNB, and JUND were elevated on a concentration and time-dependent manner. As a proof of granulocytic differentiation, the cells remained non-adherent, the expression of CD33 decreased; the granularity, CD11b expression, and MPO activity of HL-60 cells increased upon treatment. Finally, viability of HL-60 cells was hampered shown by the depolarization of mitochondria, activation of caspase-3, cleavage of Z-DEVD-aLUC, appearance of the sub-G1 population, and the leakage of the lactate-dehydrogenase into the supernatant. We confirmed the differentiating effect of our drug candidate on human patient-derived AML cells shown by the increase of CD11b and decrease of CD33+, CD7+, CD206+, and CD38bright cells followed apoptosis (IC50: 80 nM) after treatment ex vivo. Our compound reduced both CD11b+/Ly6C+ and CD11b+/Ly6G+ splenic MDSCs from the murine 4T1 breast cancer model ex vivo.

Analysis of the Single-Cell Heterogeneity of Adenocarcinoma Cell Lines and the Investigation of Intratumor Heterogeneity Reveals the Expression of Transmembrane Protein 45A (TMEM45A) in Lung Adenocarcinoma Cancer Patients

“…After 24 h treatment, cells were washed with PBS and harvested in Accuzo (Bioneer, Daedeok-gu, Daejeon, Korea) and stored at −80 °C. The RNA was isolated, as described previously [ 79 ]. Briefly, RNA was isolated with the Direct-zol RNA Miniprep Kit (ZymoResearch, Irvine, CA, USA), according to the manufacturer’s instructions.…”

Analysis of Ionizing Radiation Induced DNA Damage by Superresolution dSTORM Microscopy

“…The cell cycle distribution and pγ-H2AX content were assayed simultaneously within one tube. The cell cycle and the Sub-G1 fraction were assayed as described previously with some modifications [ 29 ]. Briefly, the U251 cells (2 × 10 5 ) were plated in 6-well dishes (Corning Life Sciences) and were irradiated with 2 Gy, 5 Gy or were left untreated.…”

Cardiac glycoside ouabain efficiently targets leukemic stem cell apoptotic machinery independent of cell differentiation status

“…Primitive AML cells with sustained self-renewal generally include the early stem/progenitors CD34 + CD38 − LSCs and CD34 + CD38 + LPCs. Having demonstrated the potential of ouabain in targeting primitive AML cells, we next established two study models to specifically determine the effects of ouabain on LSC and LPC death and differentiation, as previous studies reported that the differentiation of AML cells by certain stimuli could induce apoptosis [ 36 , 37 ]. In study model 1, LSCs and LPCs from AML KG-1 cells, that reached a ratio of 1:1 as described in methods section, were cultured in regular AML medium to allow the change in dynamics of LSCs and LPCs.…”

“…Differentiation therapy, i.e., all-trans retinoic acid (ATRA) and arsenic trioxide, has been shown to be an effective treatment for acute promyelocytic leukemia (APL), a distinct AML subtype characterized by the expression of the PML/RARA fusion protein [ 23 ]. Previous studies reported that the induction of AML differentiation could sometimes be accompanied by apoptosis [ 36 , 37 , 48 ]. Herein, we established two study models to specifically determine the effects of ouabain on LSC and LPC death and differentiation using AML KG-1 cells.…”