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

Abstract

Background: 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.

Results: 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.

Conclusion: 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.

Figure 1

Anti-proliferative effect of WithaD. (A) Chemical structure of WithaD. (B) Morphological changes induced by WithaD in K562, MOLT-4 and Vero cells, as demonstrated by phase contrast microscopy. (C) Effect of WithaD (0-5 μM) on the cell viability of K562 and MOLT-4 cells after 24 and 48 hr as demonstrated by MTT assay. (D) Normal lymphocytes were isolated form normal blood, cultured for 48 hr in the presence of WithaD at concentration ranged from 0-20 μM and checked the cell viability by trypan blue dye exclusion assay. (E) Flow cytometric analysis revealed in situ DNA fragmentation by TUNEL assay in WithaD treated MOLT-4 (0.5 μM) and K562 (1.5 μM) cells at indicated time. The plots are representative of three independent experiments. The percentages of TUNEL positive cells were indicated in respective panel.

Figure 2

WithaD induces ceramide production in K562 and MOLT-4 cells. (A) Cells were treated with WithaD for 0-2 hr and the intracellular ceramide content were determined using anti-ceramide antibody by flow cytometer as described in Materials and methods. Representative histogram (top) and the percentages of positive cells (bottom) are shown. Results are the mean ± S.D. in duplicate in three independent experiments. Asterisk indicates statistically significant difference (P < 0.005) in 2 hr treated cells with respect to untreated cells. (B) The intracellular ceramide levels induced by WithaD in K562 and MOLT-4 cells were measured in the cell lipid extract by the diacylglycerol kinase assay as described in Materials and methods. Cells were incubated with 1.5 and 0.5 μM WithaD respectively at the indicated times. * represents statistical significant difference in ceramide level between 15 min and control, where P < 0.05. ** indicates significant difference in ceramide level between 90 min and control. Data are expressed as % of control values and are the mean ± S.D. of three different experiments performed in triplicate. (C) Level of ceramide and SM were determined in K562 and MOLT-4 cells separated in HPTLC Si 60 plates after 1 hr of WithaD treatment, along with standard ceramide and SM. This is a representative profile of three independent experiments. (D) Densitometric quantitation of ceramide and SM in lipid extracts from treated and untreated K562 and MOLT-4 cells. Data are mean ± SD of three independent experiments.

Figure 3

Effect of WithaD on MAPKs in K562 and MOLT-4 cells. (A) K562 and MOLT-4 cells were treated with WithaD at the indicated time and cell lysates were resolved in SDS-PAGE (10%). Western blot analysis showed higher level of phosphorylation of JNK and p38 whereas reduced level of p-ERK was observed in different time duration. (B) MOLT-4 cells were treated with WithaD at the indicated time and p-JNK⁺ and p-p38⁺ cells were analyzed by flow cytometry. (C) K562 and MOLT-4 cells were pretreated with SP600125 (10 μM) and SB203580 (10 μM) for 1 hr, then treated with withaD for another 48 hr and % of annexin V⁺ cells were determined using flow cytometer. Results are the mean ± S.D. in duplicate in two independent experiments. *indicates statistically significant difference (P < 0.005) between SP600125 treated and untreated cells. **indicates statistically significant difference (P < 0.005) between SP600125 + SB203580 treated and untreated cells. # considered not significant difference (P = 0.125) between SB203580 treated and untreated cells.

Figure 4

WithaD activates MKK group of kinases upstream of p38 and JNK. (A) K562 and MOLT-4 cells were treated with WithaD (1.5 and 0.5 μM respectively) for different time points. The level of phosphorylation of MKK4, MKK7 and MKK3/6 was determined in WithaD-treated cells by Western blot analysis using appropriate anti-MKK antibody. β-actin served as loading control. (B) The level of phosphorylation of MKK4, MKK7 and MKK3/6 was further determined after 2 hr treatment with different amount (0-4 μM) of WithaD as described above.

Figure 5

WithaD induces N-SMase activation. (A) RT-PCR analysis of N-SMases, ceramide synthase, and A-SMase. K562 and MOLT-4 cells were treated with WithaD (1.5 and 0.5 μM respectively) for 0-120 min. RNA was extracted from total cell lysate and RT-PCR was performed. The band intensity was measured. This is one representative of three independent experiments. *indicates statistically significant difference (P < 0.005) with respect to untreated cells. (B) Measurement of N-SMase activity using Amplex Red sphingomyelinase assay kit using manufacturer's protocol in K562 and MOLT-4 cells after WithaD (1.5 and 0.5 μM respectively) treatment. A-SMase activity was determined using the same kit by two-step SMase assay as described in Materials and methods. *indicates statistically significant difference (P < 0.005) between 45 min activity and 0 min in K562 cells whereas ** indicates significant difference (P < 0.005) in activity in MOLT-4 cells.

Figure 6

Effect of Fumonisin B1, (GW4869) and N-SMase silencing on ceramide production, activation of MKK group of kinases and regulation of WithaD-mediated apoptosis. (A) Effect of Fumonisin B1 (10 μM) and GW4869 (10 μM) on ceramide level induced by WithaD. Cells were pretreated with inhibitors for 1 hr; incubated further 1 hr with WithaD and ceramide levels were measured by FACS. Each column represented the mean ± S.D. in duplicate in three independent experiments. *indicates statistically significant difference (P < 0.005). (B) Effect of SMPD3 silencing in WithaD mediated ceramide accumulation. Cells were transfected with SMPD3 specific siRNAs as described in materials and methods. Ceramide level were measured by DAG kinase assay as described in Materials and methods. Results are the mean ± S.D. of two independent experiments. * indicates statistical significant difference P < 0.05. (C) and (D) Consequence of N-SMase inhibition by GW4869 and N-SMase silencing by siRNA1 on phosphorylation of MKK4, MKK3/6 and MKK7. K562 and MOLT-4 cells were pretreated with GW4869 (10 μM) for 1 hr and further incubated for 2 hr with 3 μM and 1 μM WithaD respectively. (E) Effect of Fumonisin B1 (10 μM) and GW4869 (10 μM) on cell death. Cells were pretreated separately with the inhibitors for 1 hr and after 48 hr apoptosis were measured. Each column represented the mean ± S.D. in duplicate in three independent experiments. * indicates statistically significant difference (P < 0.005). ** indicates difference (p = 0.1250). Etoposide was used as positive control. (F) Effect of SMPD3 silencing in WithaD treated cells. After 48 hr of transfection, apoptosis was measured. Results are representative of three independent experiments. * indicates statistical significant difference, where P < 0.05.

Figure 7

WithaD induced apoptosis in primary cells from both myeloid and lymphoid patients. (A) WithaD reduced the viability of the cells from pediatric myeloid, T- and B-ALL patients in 24 and 48 hr in a dose dependent manner as assessed by trypan blue dye exclusion test. (B) WithaD potentially induced apoptosis in primary cells from patients. WithaD treated primary cells from representative lymphoid and myeloid patients showed increased annexin V+/PI- (down right quadrant) and annexin V+/PI+ (upper right quadrant) cells by flow cytometry. (C) A representative SEM micrograph showing typical blebbing in cell membrane in untreated (×10000) and treated (×10000) lymphoblast. (D) Cell lysates of primary cells from patients were resolved in 10% SDS-PAGE. Western blot analysis showed the cleaved caspase 3, changes in Bcl-2 and Bax expression, after WithaD treatment in a time dependent manner. b-actin was used as loading control. (E) Cell lysates of primary cells from patients were resolved in 10% SDS-PAGE. Western blot analysis showed enhanced phosphorylation of JNK and p38 MAPK in response to WithaD treatment in a time dependent manner. β-actin was used as loading control.

Figure 8

Probable mechanism of WithaD induced apoptosis of K562 and MOLT-4.