These actions were associated with AMP-activated protein kinase (AMPK) and p38 mitogen-activated protein kinase (MAPK) activation, p53 phosphorylation and acetylation, as well as the modulation of p21, cyclin D1, survivin and Bax.
AMPK-p38MAPK signaling blockade reduced WMJ-S-001-induced p53 phosphorylation.
As such, alteration in Bcl-2 family proteins, which are the fundamental regulators of intrinsic apoptotic pathway, can contribute to aberrantly increased cancer cell survival.
Any impairment of its regulation may promote tumor formation and malignant progression.In summary, the death of HCT116 colorectal cancer cells exposed to WMJ-S-001 may involve AMPK-p38MAPK-p53-survivin cascade.These results support the role of WMJ-S-001 as a potential drug candidate and warrant the clinical development in the treatment of cancer..We next determined whether WMJ-S-001 activates caspase 3. 1d, WMJ-S-001 exposure led to an increase in the cleaved (active) form of caspase 3, and resultant cleavage of PARP, a selective caspase 3 substrate. of at least five independent experiments performed in triplicate (*p Flow-cytometric analysis with PI labeling to determine whether WMJ-S-001 at concentrations of 10 μM or lower affects cell cycle progression. 1e, the percentage of PI-stained cells in the S region was significantly decreased after 24 h treatment with WMJ-S-001. 5e) were reduced in cells transfected with AMPK dominant negative mutant (DN). 5g) phosphorylation in cells exposed to WMJ-S-001.(A−C) Cells were pretreated for 30 min with vehicle or compound c followed by the treatment with 10 μM WMJ-S-001 for another 30 min (A) or 24 h (B, C). A Ch IP experiment was conducted to determine whether p53 or Sp1 is recruited to the endogenous survivin promoter region in response to WMJ-S-001. The full-length blot is presented in Supplementary Fig. However, the inhibitory effect of WMJ-S-001 on HT29 cells was less pronounced (Fig. Moreover, WMJ-S-001 caused increases in AMPK and p38MAPK phosphorylations (Fig. These results further confirm that AMPK-p38MAPK-p53-survivin cascade contributes to WMJ-S-001’s effects on colorectal cancer cell death.(A) HT29 and Colo205 cells were starved in serum-free DMEM (HT29) or RPMI1640 (Colo205) medium for 24 h.Cells were treated with vehicle or WMJ-S-001~005 at indicated concentrations for 24 (A) or 48 h (B). These effects were accompanied by a concomitant increase in the percentage of PI-stained cells in the G1 region (Fig. Brd U labeling analysis was employed to confirm whether WMJ-S-001 inhibits HCT116 cell proliferation. Furthermore, AMPK-DN significantly suppressed p38MAPK (Fig. The phosphorylation status of AMPK (A), the protein levels of p21 (B) and Bax (C) were then determined by immunoblotting. Primers encompassing the survivin promoter region (−264 to −37) containing putative p53 and Sp1 binding sites were used. 5h, the binding of p53 to the survivin promoter region (−264/−37) increased after 2 h of WMJ-S-001 exposure, and this was accompanied by a decrease in Sp1 binding to the promoter region. of three independent experiments performed in duplicate (*p (D), Bax (E), cyclin D1 (F) and survivin (G) were then determined by immunoblotting. 7c) as well as p53 phosphorylation and acetylation (Fig. Furthermore, compound C significantly suppressed the phosphorylation of AMPK, p38MAPK and p53 (Fig. After starvation, cells were treated with indicated concentrations of WMJ-S-001 in the presence of 10% FBS for indicated time periods. Each column represents the mean ± SEM of five independent experiments performed in duplicate (*p , either vehicle or WMJ-S-001 (20 mg/kg/day) was administered intraperitoneally for 20 days. M of three tumors from each group performed in duplicate. M of three tumors from each group performed in duplicate.