Enhancement of arabinocytosine (AraC) toxicity to AML cells by a differentiation agent combination

Abstract

Arabinocytosine (AraC, also known as Cytarabine) is one of the mainstays of AML therapy, but like other DNA damaging therapeutic agents it is rarely curative by itself. There is an emerging realization that the therapeutic outcomes may be improved by combining AraC with other compounds. Here we report that the addition of a differentiating agent combination immediately following AraC damage to AML blasts, selectively increases the cell kill. The experiments were performed using cultured cells from established cell lines of AML (HL60 and U937). The cells were exposed to 100 nM AraC, a concentration which produced approximately 25–50% cell kill, followed by a combination of 100 nM 1alpha-hydroxyvitamin D2 (1-D2) and 10 uM carnosic acid (CA), which together can serve as a powerful differentiating agent combination for AML cells, but are not toxic alone. AraC-induced cell death, measured by Annexin V/Propidium Iodide, was significantly (p<0.01) increased by the 1-D2/CA combination in both cell lines, but not by 1-D2 or CA alone. The enhancement of cell death occurred by both apoptosis and necrosis, was associated with increased DNA damage and with higher levels of DNA damage response (DDR) activated marker Chk1, but the expression of p27, a cell cycle inhibitor protein, was not enhanced by 1-D2/CA. The principal finding is that a vitamin D analog 1-D2 combined with a plant-derived antioxidant CA can markedly augment the cytotoxic action of AraC, an anti-leukemia therapeutic agent.

BRAF signals to pro-apoptotic BIM to enhance AraC cytotoxicity induced in AML cells by Vitamin D-based differentiation agents

“…T wo AML cell lines, HL60 (cultured from a patient with acute promyeloblastic leukemia [24], and U937 (monocytes from histiocytic lymphoma) [25], were cultured as previously described [21]. …”

“…Briefly, isolated mononuclear cells were divided into a group exposed for 72 h to 100 nM AraC or the vehicle (0.1% DMSO). The cells were washed with control medium after this 72 h pretreatment, and each of these groups was further divided for addition of D2 (100 nM), or CA (10 μM), or both, for 96 h. Cell viability was determined by Trypan blue (TB) exclusion using a Neubauer hemocytometer as described before [21], and apoptosis, and necrosis by Annexin V as described below.…”

“…Total RNA was extracted by using Trizol (Invitrogen, CA) according to manufacturer's protocol, as described before [21]. The Human Apoptosis RT 2 Profiler PCR Array profiles (PAHS-012Z, Sabiosciences,) which contains 84 key genes reported to be involved in programmed cell death, were performed according to the manufacturer's instructions.…”

“…Thus, numerous attempts have been made to combine VDDs such as Calcitriol (1,25D), or analogs, with either toxic or non-toxic compounds, but to date these combinations have not resulted in clinically demonstrated advances of the field [5, 14]. Recently, we presented in vitro studies of human AML blasts, which demonstrated a selective increase in cytotoxicity of Arabinocytosine (AraC, Cytarabine), the key therapeutic for AML [18, 19], when the exposure of the AML cells to AraC was followed by a combination of differentiating agents [20, 21]. The combination consisted of the vitamin D2 analog Doxercalciferol (D2) already approved for human administration, together with a plant-derived anti-oxidant Carnosic Acid (CA), currently used as a food flavoring agent [22], and previously reported to enhance1,25-dihydroxyvitamin D3-induced differentiation of AML cells [23].…”

The role of VDR and BIM in potentiation of cytarabine-induced cell death in human AML blasts

“…Isolated mononuclear cells were divided into a group exposed for 72 h to 100 nM AraC or the vehicle (0.1% DMSO). The cells were washed with control medium after this 72 h pretreatment, and each of these groups was further divided to add D2 (100 nM), or CA (10 μM), or both, for 96 h. Cell viability was determined by Trypan blue (TB) exclusion using a Neubauer hemocytometer as described before [53]. …”