Combination of guanine arabinoside and Bcl‐2 inhibitor YC137 overcomes the cytarabine resistance in HL‐60 leukemia cell line

Abstract

Cytarabine (ara‐C) is the key agent for treating acute myeloid leukemia. After being transported into leukemic cells, ara‐C is phosphorylated, by several enzymes including deoxycytidine kinase (dCK), to ara‐C triphosphate (ara‐CTP), an active metabolite, and then incorporated into DNA, thereby inhibiting DNA synthesis. Therefore, the cytotoxicity of ara‐C depends on the production of ara‐CTP and the induction of apoptosis. Here, we established a new ara‐C‐resistant acute myeloid leukemia cell line (HL‐60/ara‐C60) with dual resistance characteristics of the anti‐antimetabolic character of decreased ara‐CTP production and an increase in the antiapoptotic factors Bcl‐2 and Bcl‐XL. We further attempted to overcome resistance by augmenting ara‐CTP production and stimulating apoptosis. A relatively new nucleoside analog, 9‐β‐d‐arabinofuranosylguanine (ara‐G), and the small molecule Bcl‐2 antagonist YC137 were used for this purpose. HL‐60/ara‐C60 was 60‐fold more ara‐C‐resistant than the parental HL‐60 cells. HL‐60/ara‐C60 cells exhibited low dCK protein expression, which resulted in decreased ara‐CTP production. HL‐60/ara‐C60 cells were also refractory to ara‐C‐induced apoptosis due to overexpression of Bcl‐2 and Bcl‐XL. Combination treatment of ara‐C with ara‐G augmented the dCK protein level, thereby increasing ara‐CTP production and subsequent cytotoxicity. Moreover, the combination of ara‐C with YC137 produced a greater amount of apoptosis than ara‐C alone. Importantly, the three‐drug combination of ara‐C, ara‐G and YC137 provided greater cytotoxicity than ara‐C+ara‐G or ara‐C+YC137. These findings suggest possible combination strategies for overcoming ara‐C resistance by augmenting ara‐CTP production and reversing refractoriness against the induction of apoptosis in ara‐C resistant leukemic cells.

Venetoclax and alvocidib are both cytotoxic to acute myeloid leukemia cells resistant to cytarabine and clofarabine

“…Our previous study demonstrated that the Bcl-2 inhibitor YC137 is cytotoxic to ara-C-resistant leukemic cells [16]. ABT737, which inhibits Bcl-2 and Bcl-xL, effectively induced death in CAFdA-resistant leukemic cells [17].…”

“…Kinases (dCK, dGK), transporters (hENT1, hENT2, hENT3, hCNT3), and apoptosis-related factors (Bcl-2, Mcl-1, Bad, Bim, Bax, Bak) were determined using Western blot analysis [16]. Mouse monoclonal anti-dCK (Abcam Cambridge, UK), rabbit polyclonal anti-dGK (Abcam), mouse monoclonal anti-hENT1 (Santa Cruz Biotechnology, Dallas, TX, USA), rabbit monoclonal anti-hENT2 (Abcam), rabbit polyclonal anti-hCNT3 (Abcam), rabbit polyclonal anti-Bcl-2 (Cell Signaling Technology, Beverly, MA, USA), rabbit polyclonal anti-Mcl-1 (Cell Signaling Technology), rabbit monoclonal anti-Bad (Cell Signaling Technology), anti-Bim (Cell Signaling Technology), anti-Bax (Cell Signaling Technology), anti-Bak (Cell Signaling Technology), and anti-actin (Sigma-Aldrich, St. Louis, MO, USA) antibodies were used as the primary antibodies.…”

“…2b, c). In addition, we have already produced several nucleoside analog-resistant cell lines, in which protein levels, [7,9,16,17]. The degree of the reduction was greater in HL-60/CAFdA30 cells than in HL-60/CAFdA4 cells.…”

Efficient Biocatalytic Synthesis of Dihalogenated Purine Nucleoside Analogues Applying Thermodynamic Calculations

“…Dihalogenated nucleosides 3a-d were tested for their ability to inhibit the growth of HL-60 cells in culture. Monohalogenated cladribine, which is known to efficiently inhibit HL-60 cells [18,19], was used as positive control. While for cladribine an IC50 value of 0.22 µ M [± 0.11 µ M] was determined, 50% of inhibition by the dihalogenated nucleoside analogues was observed for concentrations between 10 and 100 µ M (Figure 2).…”

“…Chemical shifts are reported in parts per million (ppm) and are referenced to the residual solvent resonance as the internal standard ( 1 H NMR: δ = 2.50 ppm for DMSO-d 5 ; 13 C NMR: δ = 39.52 ppm for DMSO-d 6 ) [37]. 19 F NMR spectra are referenced in compliance with the unified scale for NMR chemical shifts as recommended by the IUPAC stating the chemical shift relative to CCl 3 F [38] . Data are reported as follows: chemical shift, multiplicity (s = singlet, d = doublet, m c = centrosymmetric multiplet, br = broad signal), coupling constants (Hz), and integration.…”