piplartine and Leukemia--Myeloid--Acute

The development of strategies to eradicate primary human acute myelogenous leukemia (AML) cells is a major challenge to the leukemia research field. In particular, primitive leukemia cells, often termed leukemia stem cells, are typically refractory to many forms of therapy. To investigate improved strategies for targeting of human AML cells we compared the molecular mechanisms regulating oxidative state in primitive (CD34(+)) leukemic versus normal specimens. Our data indicate that CD34(+) AML cells have elevated expression of multiple glutathione pathway regulatory proteins, presumably as a mechanism to compensate for increased oxidative stress in leukemic cells. Consistent with this observation, CD34(+) AML cells have lower levels of reduced glutathione and increased levels of oxidized glutathione compared with normal CD34(+) cells. These findings led us to hypothesize that AML cells will be hypersensitive to inhibition of glutathione metabolism. To test this premise, we identified compounds such as parthenolide (PTL) or piperlongumine that induce almost complete glutathione depletion and severe cell death in CD34(+) AML cells. Importantly, these compounds only induce limited and transient glutathione depletion as well as significantly less toxicity in normal CD34(+) cells. We further determined that PTL perturbs glutathione homeostasis by a multifactorial mechanism, which includes inhibiting key glutathione metabolic enzymes (GCLC and GPX1), as well as direct depletion of glutathione. These findings demonstrate that primitive leukemia cells are uniquely sensitive to agents that target aberrant glutathione metabolism, an intrinsic property of primary human AML cells.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Antigens, CD34; Dioxolanes; Female; Glutamate-Cysteine Ligase; Glutathione; Glutathione Peroxidase; Glutathione Peroxidase GPX1; Humans; Leukemia, Myeloid, Acute; Male; Oxidation-Reduction; Oxidative Stress; Sesquiterpenes; Tumor Cells, Cultured

Synergistic-to-additive antileukemic interactions of piperlongumine (PL) and HDAC inhibitor (HDACi) SAHA (Vorinostat) provide a compelling rationale to construct PL-HDACi hybrids, such as 1-58, which recapitulated the synergism between the parental compounds in high-risk and chemoresistant AML cells. Both PL and HDACi components, either in combination or in hybrid molecules, are essential for inducing significant DNA damage and apoptosis. Introducing C2-chloro substituent to 1-58 yielded 3-35 with increased cytotoxicity but decreased selectivity in noncancerous MCF-10A cells; eliminating C7-C8 olefin of PL obtained 3-31/3-98 scaffolds which were still more active than PL or SAHA in AML and were well-tolerated by MCF-10A cells. The HDACi function was crucial for modulating expression of DNA repair and apoptosis-related proteins. Collectively, PL and SAHA hybrids are potent, multifunctional anti-AML agents, acting in part, by interfering cellular GSH defense, suppressing expression of DNA repair and pro-survival proteins, and inducing expression of pro-apoptotic proteins.

Topics: Antineoplastic Agents; Apoptosis Regulatory Proteins; Cell Line, Tumor; Dioxolanes; DNA Repair; Drug Screening Assays, Antitumor; Glutathione; Histone Deacetylase Inhibitors; Humans; Hydroxamic Acids; Leukemia, Myeloid, Acute; Structure-Activity Relationship; Vorinostat

To investigate the effects of piperlongumine (PL), an anticancer alkaloid from long pepper plants, on the primary myeloid leukemia cells from patients and the mechanisms of action.. Human BM samples were obtained from 9 patients with acute or chronic myeloid leukemias and 2 patients with myelodysplastic syndrome (MDS). Bone marrow mononuclear cells (BMMNCs) were isolated and cultured. Cell viability was determined using MTT assay, and apoptosis was examined with PI staining or flow cytometry. ROS levels in the cells were determined using DCFH-DA staining and flow cytometry. Expression of apoptotic and autophagic signaling proteins was analyzed using Western blotting.. PL inhibited the viability of BMMNCs from the patients with myeloid leukemias (with IC50 less than 20 μmol/L), but not that of BMMNCs from a patient with MDS. Furthermore, PL (10 and 20 μmol/L) induced apoptosis of BMMNCs from the patients with myeloid leukemias in a dose-dependent manner. PL markedly increased ROS levels in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the antioxidant N-acetyl-L-cysteine abolished PL-induced ROS accumulation and effectively reduced PL-induced cytotoxicity. Moreover, PL markedly increased the expression of the apoptotic proteins (Bax, Bcl-2 and caspase-3) and autophagic proteins (Beclin-1 and LC3B), and phosphorylation of p38 and JNK in BMMNCs from the patients with myeloid leukemias, whereas pretreatment with the specific p38 inhibitor SB203580 or the specific JNK inhibitor SP600125 partially reversed PL-induced ROS production, apoptotic/autophagic signaling activation and cytotoxicity.. Piperlongumine induces apoptotic and autophagic death of the primary myeloid leukemia cells from patients via activation of ROS-p38/JNK pathways.

Topics: Antineoplastic Agents, Phytogenic; Antioxidants; Apoptosis; Apoptosis Regulatory Proteins; Autophagy; Cell Survival; Dioxolanes; Dose-Response Relationship, Drug; Enzyme Activation; Humans; Inhibitory Concentration 50; JNK Mitogen-Activated Protein Kinases; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Leukemia, Myeloid, Acute; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Primary Cell Culture; Protein Kinase Inhibitors; Reactive Oxygen Species; Signal Transduction; Tumor Cells, Cultured