Withanolide D induces apoptosis in leukemia by targeting the activation of neutral sphingomyelinase-ceramide cascade mediated by synergistic activation of c-Jun N-terminal kinase and p38 mitogen-activated protein kinase

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.