withanolides and Leukemia

Topics: Adjuvants, Immunologic; Apoptosis; Ceramides; Herbal Medicine; Humans; Leukemia; Medicine, Ayurvedic; Withanolides

Leukemia is a blood or bone marrow cancer with increasing incidence in developed regions of the world. Currently, there is an ongoing need for novel and safe anti-leukemic agents, as no fully effective chemotherapy is available to treat this life-threatening disease. Herein, are reported the isolation, structural elucidation, and anti-leukemic evaluation of twenty-nine withanolide-type steroids (

Topics: Animals; Antineoplastic Agents; Cell Line; Cell Line, Tumor; Chlorocebus aethiops; HL-60 Cells; Humans; Leukemia; Steroids; Structure-Activity Relationship; Vero Cells; Withania; Withanolides

Ceramide is an important second messenger that has diverse cellular and biological effect. It is a specific and potent inducer of apoptosis and suppressor of cell growth. In leukemia, chemoresistance generally developed due to deregulated ceramide metabolism. In combinatorial treatment strategies of leukemia, few components have the capability to increases ceramide production. Manipulation in ceramide production by physiological and pharmacological modulators therefore will give additive effect in leukemia chemotherapy.. Here, we show that Withanolide D (C4β-C5β,C6β-epoxy-1-oxo-,20β, dihydroxy-20S,22R-witha-2,24-dienolide; WithaD), a pure herbal compound isolated from Withania somnifera could effectively induces apoptosis in a dose and time dependant manner both in myeloid (K562) and lymphoid (MOLT-4) cells being nontoxic to normal lymphocytes and control proliferative cells. WithaD potentially augment ceramide production in these cells. Downstream of ceramide, WithaD acted on MKK group of proteins and significantly increased JNK and p38MAPK phosphorylation. Pharmacological inhibition of p38MAPK and JNK proves their cooperative action on WithaD-induced cell death. Dissecting the cause of ceramide production, we found activation of neutral sphingomyelinase and showed neutral-sphingomyelinase 2 (N-SMase 2) is a critical mediator of WithaD-induced apoptosis. Knockdown of N-SMase 2 by siRNA and inhibitor of N-SMase (GW4869) significantly reduced WithaD-induced ceramide generation and phosphorylation of MKK4 and MKK3/6, whereas phosphorylation of MKK7 was moderately regulated in leukemic cells. Also, both by silencing of N-SMase 2 and/or blocking by GW4869 protects these cells from WithaD-mediated death and suppressed apoptosis, whereas Fumonisin B1, an inhibitor of ceramide synthase, did not have any effect. Additionally, WithaD effectively induced apoptosis in freshly isolated lymphoblasts from patients and the potent cell killing activity was through JNK and p38MAPK activation.. Our results demonstrate that WithaD enhance the ceramide accumulation by activating N-SMase 2, modulate phosphorylation of the JNK and p38MAPK and induced apoptosis in both myeloid and lymphoid cells along with primary cells derived from leukemia patients. Taken together, this pure herbal compound (WithaD) may consider as a potential alternative tool with additive effects in conjunction with traditional chemotherapeutic treatment, thereby accelerate the process of conventional drug development.

Topics: Aniline Compounds; Apoptosis; Benzylidene Compounds; Blotting, Western; Cell Line, Tumor; Ceramides; Flow Cytometry; Humans; JNK Mitogen-Activated Protein Kinases; Leukemia; MAP Kinase Kinase 3; MAP Kinase Kinase 4; MAP Kinase Kinase 6; MAP Kinase Kinase 7; Microscopy, Electron, Scanning; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; Signal Transduction; Sphingomyelin Phosphodiesterase; Tumor Cells, Cultured; Withanolides

Withaferin A (WA) is present abundantly in Withania somnifera, a well-known Indian medicinal plant. Here we demonstrate how WA exhibits a strong growth-inhibitory effect on several human leukemic cell lines and on primary cells from patients with lymphoblastic and myeloid leukemia in a dose-dependent manner, showing no toxicity on normal human lymphocytes and primitive hematopoietic progenitor cells. WA-mediated decrease in cell viability was observed through apoptosis as demonstrated by externalization of phosphatidylserine, a time-dependent increase in Bax/Bcl-2 ratio; loss of mitochondrial transmembrane potential, cytochrome c release, caspases 9 and 3 activation; and accumulation of cells in sub-G0 region based on DNA fragmentation. A search for the downstream pathway further reveals that WA-induced apoptosis was mediated by an increase in phosphorylated p38MAPK expression, which further activated downstream signaling by phosphorylating ATF-2 and HSP27 in leukemic cells. The RNA interference of p38MAPK protected these cells from WA-induced apoptosis. The RNAi knockdown of p38MAPK inhibited active phosphorylation of p38MAPK, Bax expression, activation of caspase 3 and increase in Annexin V positivity. Altogether, these findings suggest that p38MAPK in leukemic cells promotes WA-induced apoptosis. WA caused increased levels of Bax in response to MAPK signaling, which resulted in the initiation of mitochondrial death cascade, and therefore it holds promise as a new, alternative, inexpensive chemotherapeutic agent for the treatment of patients with leukemia of both lymphoid and myeloid origin.

Topics: Animals; Apoptosis; bcl-2-Associated X Protein; Caspase Inhibitors; Caspases; Cell Survival; Dose-Response Relationship, Drug; Enzyme Activation; Ergosterol; Humans; Leukemia; MAP Kinase Signaling System; Medicine, Traditional; Membrane Potential, Mitochondrial; Mice; Mitochondria; Molecular Structure; p38 Mitogen-Activated Protein Kinases; Plants, Medicinal; Poly(ADP-ribose) Polymerases; Proto-Oncogene Proteins c-bcl-2; RNA, Small Interfering; Tumor Cells, Cultured; Withanolides

Withaferin A, a major chemical constituent of Withania somnifera, has been reported for its tumor cell growth inhibitory activity, antitumor effects, and impairing metastasis and angiogenesis. The mechanism by which withaferin A initiates apoptosis remains poorly understood. In the present report, we investigated the effect of withaferin A on the apoptotic pathway in U937 human promonocytic cells. We show that withaferin A induces apoptosis in association with the activation of caspase-3. JNK and Akt signal pathways play crucial roles in withaferin A-induced apoptosis in U937 cells. Furthermore, we have shown that overexpression of Bcl-2 and active Akt (myr-Akt) in U937 cells inhibited the induction of apoptosis, activation of caspase-3, and PLC-gamma1 cleavage by withaferin A. Taken together, our results indicated that the JNK and Akt pathways and inhibition of NF-kappaB activity were key regulators of apoptosis in response to withaferin A in human leukemia U937 cells.

Topics: Apoptosis; Caspase 3; Caspase Inhibitors; Cell Line, Tumor; Cell Proliferation; Cytochromes c; Down-Regulation; Enzyme Inhibitors; Ergosterol; Humans; JNK Mitogen-Activated Protein Kinases; Leukemia; Matrix Metalloproteinases; NF-kappa B; Phosphorylation; Proto-Oncogene Proteins c-akt; Proto-Oncogene Proteins c-bcl-2; Withanolides