JS-K, an arylating nitric oxide (NO) donor, has synergistic anti-leukemic activity with cytarabine (ARA-C).

We have designed prodrugs that release nitric oxide (NO) on metabolism by glutathione S-transferases (GST). This design exploits the upregulation of GST in acute myeloid leukemia (AML) cells. O(2)-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate (JS-K, a member of this class) has potent anti-leukemic activity. HL-60 myeloid leukemia cells were used for in vitro studies of the combination of JS-K with daunorubicin (DAUNO), cytarabine (ARA-C) or etoposide (ETOP) using the median effect method to determine synergistic, antagonistic, or additive effects. Combinations of JS-K added simultaneously, 2h before or 2h after the other compounds were used. JS-K and DAUNO were antagonistic in all three drug sequences. JS-K and ETOP were also antagonistic but to a lesser degree. JS-K and ARA-C showed strong synergy. The combination index at the 50% fraction affected was 0.37+/-0.23, 0.24+/-0.27, and 0.15+/-0.11 for simultaneous, JS-K first and ARA-C first additions, respectively. JS-K by itself induced DNA strand breaks at relatively high concentrations. However, at submicromolar concentrations, it significantly augmented ARA-C-induced DNA strand breaks. NMR spectroscopy revealed no evidence of chemical interaction between JS-K and the other chemotherapeutic agents. We conclude that ARA-C and JS-K have synergistic anti-leukemic activity and warrant further exploration in combination.

Synthesis and in vitro anti-leukemic activity of structural analogues of JS-K, an anti-cancer lead compound.

Structural analogues of JS-K, an anti-cancer lead compound, were prepared and their in vitro anti-leukemic activity was determined. The rate of nitric oxide release from the corresponding diazeniumdiolate anions did not appear to affect the anti-leukemic activity of the prodrug forms. Two compounds with potent inhibitory activity and a potentially favorable toxicological profile were identified.

Antitumor activity of JS-K [O2-(2,4-dinitrophenyl) 1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate] and related O2-aryl diazeniumdiolates in vitro and in vivo.

The literature provides evidence that metabolic nitric oxide (NO) release mediates the cytotoxic activities (against human leukemia and prostate cancer xenografts in mice) of JS-K, a compound of structure R(2)N-N(O)=NO-Ar for which R(2)N is 4-(ethoxycarbonyl)piperazin-1-yl and Ar is 2,4-dinitrophenyl. Here we present comparative data on the potencies of JS-K and 41 other O(2)-arylated diazeniumdiolates as inhibitors of HL-60 human leukemia cell proliferation, as well as in the NCI 51-cell-line screen for six of them. The data show JS-K to be the most potent of the 42 in both screens and suggest that other features of its structure and metabolism besides NO release may contribute importantly to its activity. Results with control compounds implicate JS-K's arylating ability, and the surprisingly low IC(50) value of the N-(ethoxycarbonyl)piperazine byproduct of NO release suggests a role for the R(2)N moiety. In addition to the above-mentioned in vivo activities, JS-K is shown here to be carcinostatic in a rat liver cancer model.

JS-K, a nitric oxide prodrug, induces cytochrome c release and caspase activation in HL-60 myeloid leukemia cells.

Nitric oxide (NO) induces differentiation and apoptosis in acute myelogenous leukemia (AML) cells. The NO prodrug O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen-1-ium-1,2-diolate, or JS-K, has potent antileukemic activity. JS-K induces apoptosis in HL-60 cells by a caspase-dependent mechanism. The purpose of this study was to determine the pathway through which JS-K induces apoptosis. We show that JS-K alters mitochondrial membrane potential (DeltaPsim) and induces cytochrome c release from mitochondria into the cytoplasm. Treatment with JS-K resulted in activation of Caspase (Casp) 9, Casp 3 and Casp 8. JS-K constitutes a promising lead for a new class of anti-leukemic agents.

Prolonged treatment with prostaglandin E1 increases the rate of lipolysis in rat adipocytes.

Prolonged treatment of adipocytes with certain inhibitors of lipolysis, including N(6)-phenylisopropyl adenosine (PIA) and prostaglandin E(1) (PGE(1)) leads to down-regulation of G(i). Prolonged treatment with PIA increases the rate of lipolysis, and we have reported that tumor necrosis factor-alpha (TNF alpha) stimulates lipolysis by down-regulating G(i). To determine the relationship between G(i) concentration and lipolysis, we have investigated the effect of two other acute inhibitors of lipolysis; PGE(1), which down-regulates G(i), and nicotinic acid (NA), which does not down-regulate G(i). Rat adipocytes were incubated with PIA (300 nM), PGE(1) (3 microM) or nicotinic acid (1 mM) for 24 h. The rate of lipolysis (glycerol release) was increased approximately 2 to 3-fold in PIA-treated cells, and in PGE(1)-treated cells. Conversely, the rate of lipolysis was not altered by the prolonged nicotinic acid treatment. These findings support the hypothesis that the rate of lipolysis in adipocytes is determined, at least partly, by the cellular concentration of G(i) proteins.

Differences in the lipolytic function of adipose tissue preparations from Black American and Caucasian women.

The purpose of this study was to determine the potential causes of the lower lipolytic rates in obese Black American women compared to obese Caucasian women. Subcutaneous and omental adipose tissue were obtained from subjects during abdominal surgery, and hormone-sensitive lipase (HSL) mass, mRNA, and activity were determined. HSL mRNA levels did not differ between the Black American and Caucasian women in either subcutaneous or omental adipose tissue. However, HSL mass was approximately 35% lower (P <.05) in both subcutaneous and omental adipose tissue of the Black Americans. Because of these differences, we measured HSL activity in frozen subcutaneous and omental adipose tissue, and also measured basal and isoproterenol-stimulated lipolytic rates in tissue fragments. No racial differences were found in the activity of HSL in either subcutaneous or omental adipose tissue. However, basal lipolytic rates in the Black Americans were 53% and 44% lower (P <.05) in the subcutaneous and omental fat, respectively, compared to the Caucasian women, despite a lack of difference in cell size between the 2 groups. Interestingly, the degree of stimulation by isoproterenol was higher in both the subcutaneous and omental adipose tissue of the Black American than those of the Caucasian women, resulting in equal stimulation by isoproterenol in the 2 groups. These results indicate that despite the lower mass and lower basal HSL activity in the obese Black American women, stimulation of HSL results in equal activity of the enzyme in the 2 races. This suggests that the signaling pathway of HSL stimulation is more efficient in the Black American women.

Lipolysis in adipocytes isolated from deep and superficial subcutaneous adipose tissue.

OBJECTIVE: Abdominal subcutaneous adipose tissue (SAT) occurs in two depots separated by a fascial plane: deep SAT and superficial SAT. In a recent study it was demonstrated that the amount of deep SAT has a much stronger relationship to insulin resistance than does superficial SAT. Because insulin resistance may be related to fatty acid release from adipose tissue, we hypothesized that the two SAT depots may have a different lipolytic activity. RESEARCH METHODS AND PROCEDURES: To test this hypothesis, we obtained samples of deep and superficial SAT from patients undergoing elective abdominal surgery. The rate of lipolysis was determined in the collagenase-digested adipocytes obtained from the two fat depots by measuring glycerol release in the presence and absence of isoproterenol. In addition, the relative concentration of hormone-sensitive lipase was determined in both SAT depots by Western blot analysis. RESULTS: Our results showed that the rate of isoproterenol-stimulated lipolysis was approximately 20% higher in cells from deep SAT compared with those from superficial SAT, indicating that the deep SAT is more lipolytically active. The concentration of hormone-sensitive lipase did not differ between the two adipose tissue depots. DISCUSSION: These findings suggest that the higher lipolytic activity of deep SAT may account for its stronger association with insulin resistance. The mechanism seems to be independent of differences in hormone-sensitive lipase concentration.