Figure 1.
WithaD-induced apoptosis occurs mainly through intrinsic pathway.
(A) Chemical structure of WithaD isolated from leaves of Withania somnifera. MOLT-3 and K562 cells were treated with (0–4 µM) WithaD for 15 hr and subsequently evaluated for the (B) expression of pro-survival (Bcl-xl, Bcl-2) and pro-apoptotic (Bak, Bax) Bcl-2 family members; (C) proteolytic processing of pro-caspase-9, -3 and -7 and the cleavage of PARP and (D) activation of caspase-8 and reduction of total Bid. In each experiment, β-actin served as the loading control. (E) For blocking assay, MOLT-3 and K562 cells were pre-incubated with or without IETD-FMK (20 µM), LEHD-FMK (20 µM) and DEVD-CHO (100 µM) for 1 hr followed by incubation for an additional 48 hr in presence of WithaD (2 µM) and % of apoptotic cells (annexinV+/PI+) were measured by flow cytometry. ‘*’ indicates the difference was statistically significant (P<0.005) in WithaD+z-LEHD-FMK and WithaD treated cells.
Figure 2.
p53 is crucial in WithaD-induced apoptosis.
(A) Cells were treated with WithaD (0–4 µM) for 15 hr and p53 expression was evaluated in the lysate of MOLT-3, HCT116, U87MG and K562 cells by Immunoblot. (B) Status of p53 in HCT116 p53+/+ and stably knockdown HCT116p53−/− cells. (C) WithaD (0–4 µM) treatment led to proteolytic processing of pro-caspase-9, -3 and -7 and the cleavage of PARP as determined by immunoblot analysis after 15 hr in HCT116p53+/+ and HCT116p53−/− cells. (D) Effect of WithaD (0–4 µM) on activation of caspase-8 and reduction of total Bid after 15 hr of treatment in HCT116 p53+/+ and HCT116p53−/− cells. In each immunoblot, β-actin was served as the loading control. (E) Difference in WithaD (0–4 µM) mediated apoptosis induction in HCT116 p53+/+ and HCT116p53−/− cells for 24 hr. (F) Morphological changes as evaluated by phase contrast microscopy in HCT116 p53+/+ and HCT116p53−/− cells after 24 hr WithaD (2 and 4 µM) treatment.
Figure 3.
WithaD induced p53-dependent Bax and p53-independent Bak activation.
(A) Expression of p53 downstream effector molecules including Bcl-xl, Bcl-2, p21, Bak and Bax were evaluated by immunoblot assay after WithaD (0–4 µM) for 15 hr in HCT116p53+/+ and HCT116p53−/− cells. (B) HCT116p53+/+ cells were pre-incubated with pifithrin α (30 µM) for 1 hr followed by 15 hr WithaD (0–2 µM) treatment and the protein level of p53, Bax and Bak were evaluated by Western blot. In each blot, β-actin served as the loading control.
Figure 4.
Cytochrome c was released before mitochondrial depolarization.
(A) To detect cytochrome c release, cytosolic and mitochondrial fraction of HCT116p53+/+ and HCT116p53−/− cells were separated as mentioned in materials and methods and electrophoresed on 15% SDS-PAGE and immunoblotted using anti-cytochrome c antibody. Cytosolic Bax and cytochrome c were evaluated by Western blot analysis and β-actin served as the loading control. Mitochondrial Bak oligomerization, Bax and cytochrome c were detected by Western blot and Cox IV served as mitochondrial loading control. (B) Mean fluorescence intensity (MFI) of FL1 was evaluated in HCT116p53+/+ and HCT116p53−/− after JC1 staining. Dose dependent treatment of WithaD (0–4 µM) revealed no significant changes in MFI value at15 hr. # considered not significant difference (P = 0.125) between untreated and WithaD (3 µM) treated cells.
Figure 5.
Bax and Bak were critical mediator of WithaD-mediated apoptosis.
(A) Expression of p53, Bax, Bak and Cytochrome c were evaluated in the lysate of HCT116 cells proficient for Bax and Bak (Bax+/Bak+), deficient for either Bax (Bax−/Bak+) or Bak (Bax+/Bak−) or deficient for both Bax and Bak (Bax−/Bak−) after WithaD (0–2 µM) treatment for 15 hr by Western blot analysis. Each sub-lines were treated with WithaD (0–4 µM) for (B) 24 and (C) 48 hr and viabilities were determined by MTT assay. (D) Apoptosis induction was assessed by flow cytometric detection of each sub-lines by 7-AAD staining. ‘*’ indicates the difference was statistically significant (P<0.005) between HCT116Bax+/Bak+ and HCT116Bax−/Bak− cells.
Figure 6.
WithaD inhibit in vitro and in vivo K562 xenograft growth.
(A) WithaD could prolong the survival time of nude mice injected with K562. The tumor load of treated mice (10 mg/kg body weight) was visibly lower than untreated control mice. (B) WithaD significantly reduced tumor volume. Each point represents mean of three tumors. (C) Status of PI-positive cells in untreated and WithaD-treated tumor cells. PI-positive cells (▪) were determined with respect to PI unstained cells (□). (D) Tissue section of spleen, lungs and liver of nude mice (control and WithaD treated) determined by H&E staining and observed both in 20× and 40×.
Figure 7.
Probable mechanism of WithaD induced apoptosis.
WithaD treatment induces p53 activation and mitochondrial apoptosis in p53wt cells in Bax and Bak dependent manner. However, in p53-deficient cells, lack of Bax function is complemented with Bak in WithaD-induced mitochondrial apoptosis.