The Nitric Oxide-Generating Prodrug Js-K Reverses the Multi-Drug Resistance Phenotype in AML Cells

Nitric oxide (NO) is toxic to acute myeloid leukemia (AML) cells. The NO prodrug O²-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K) is a lead agent of the arylated diazeniumdiolates class. JS-K is active in vitro and in vivo against AML, multiple myeloma, and several solid tumors. JS-K is directly cytotoxic to malignant cells and inhibits angiogenesis in vitro and in vivo. Aiming at its clinical application to treat AML, we have developed a nanoscale micelle formulation for JS-K (P123/JS-K) using Pluronic^®P123 polymers.

A major cause of treatment failure in AML is the multi-drug resistance (MDR) phenotype associated with overexpression of the P-glycoprotein (Pgp) by leukemic cells. Here, we investigated the effect of JS-K and its formulation on the MDR phenotype in AML cells using HL-60 cells and HL-60/RV+ cells as a model. HL-60/RV+ are selected from the parent HL-60 line and express the Pgp. They are maintained in culture under vincristine (VCR) selection. We studied the effect of the formulation alone (P123), JS-K without the formulation (free JS-K), and JS-K in the formulation (P123/JS-K).

Both VCR and free JS-K were cytotoxic towards HL-60 cells with 50% inhibitory concentrations (IC₅₀) of around 0.4 and 0.1 μM, respectively. By contrast, the IC₅₀ for VCR-treated HL-60/RV+ cells, was greater than 1 μM. Free JS-K was cytotoxic to HL-60/RV+ with an IC₅₀ of around 0.2 μM. Pretreatment of HL-60/RV+ cells for 2 hours with free JS-K (0.1 μM), P123/JS-K (0.1 μM), or an equivalent volume of P123 sensitized HL-60/RV+ cells to the effect of VCR: after 3 days of culture, the VCR IC₅₀went from 1.5 μM in control cells to an estimated 1, 0.9, and 0.75 μM for P123, free JS-K, and P123/JS-K pre-treated cells, respectively.

We then conducted cell cycle analyses of HL-60/RV+ cells treated with different combinations of JS-K and VCR using propidium idodide staining. Cells were treated with combinations of VCR (1.75 μM), free JS-K (0.1 μM), P123/JS-K (0.1 μM), or an equivalent volume of P123. After 24 hours, the observed percentage (average of 3 different repeats per variable) of the sub-G₀/G₁ fraction was 7, 6,14, 14, 25, 26, and 43 for untreated controls, free JS-K, P123/JS-K, VCR, free JS-K + VCR, P123 + VCR, and P123/JS-K + VCR, respectively. At 48 hours, the sub-G₀/G₁ fraction was 4, 5, 5, 19, 20, 23, and 28% for the same variables, respectively. At 72 hours, the sub-G₀/G₁fraction was 3, 3, 3, 11, 12, 19, and 19% for the same variables, respectively. Thus, the peak effect on the cell cycle was observed at 24 hours. We also observed an effect of P123 alone.

In order to determine whether JS-K affects the efflux pump itself, we investigated the effect of 0.1 μM free JS-K, 0.1 μM P123/JS-K, or an equivalent volume of P123 on Rho-123 accumulation in HL-60/RV+ cells after 6 hours of treatment. As a percent of untreated controls, Rho-123 accumulation (average of 9 repeats) was 143, 159, and 144 for free JS-K, P123, and P123/JS-K, respectively (P < 0.05 for all differences with untreated controls).

Using flow cytometry, we also determined whether JS-K affects the expression of the Pgp by HL-60/RV+ cells treated with 0.1 μM free JS-K, 0.1 μM P123/JS-K, or an equivalent volume of P123. After 24 hours, the observed (average of 6 different repeats per variable) expression of Pgp as a percent of untreated controls was 90, 79, and 63, for P123, free JS-K, and P123/JS-K, respectively (P < 0.05 for the difference between P123/JS-K and untreated controls). After 48 hours, the observed (average of 6 different repeats per variable) expression of Pgp as a percent of untreated controls was 83, 79, and 57, for the same variables, respectively (P < 0.05 for the difference between P123/JS-K and untreated controls). After 72 hours, there were no significant differences in the expression of Pgp between the different treatments and untreated controls.

We conclude that JS-K in a P123 Pluronic^® formulation can reverse the MDR phenotype. The Pluronic^® polymers themselves could also affect the MDR phenotype. As such, P123/JS-K could constitute a major addition to our armamentarium for the treatment of AML.