Resibufogenin suppresses transforming growth factor-β-activated kinase 1-mediated nuclear factor-κB activity through protein kinase C-dependent inhibition of glycogen synthase kinase 3

Abstract

Resibufogenin (RB), one of the major active compounds of the traditional Chinese medicine Chansu, has received considerable attention for its potency in cancer therapy. However, the anticancer effects and the underlying mechanisms of RB on pancreatic cancer remain elusive. Here, we found that RB inhibited the viability and induces caspase-dependent apoptosis in human pancreatic cancer cells Panc-1 and Aspc. Resibufogenin-induced apoptosis was through inhibition of constitutive nuclear factor-κB (NF-κB) activity and its target genes' expression, which was caused by downregulation of transforming growth factor-β-activated kinase 1 (TAK1) levels and suppression of IκB kinase activity in Panc-1 and Aspc cells. This induction of TAK1-mediated NF-κB inactivation by RB was associated with increased glycogen synthase kinase-3 (GSK-3) phosphorylation and subsequent suppression of its activity. Moreover, RB-induced GSK-3 phosphorylation/inactivation acted through activation of protein kinase C but not Akt. Finally, RB suppressed human pancreatic tumor xenograft growth in athymic nude mice. Thus, our findings reveal a novel mechanism by which RB suppresses TAK1-mediated NF-κB activity through protein kinase C-dependent inhibition of GSK-3. Our findings provide a rationale for the potential application of RB in pancreatic cancer therapy.

Keywords: glycogen synthase kinase-3; nuclear factor-κB; protein kinase C; resibufogenin; transforming growth factor-β-activated kinase 1.

Figure 1

Resibufogenin (RB) inhibits cell viability and changes cell morphology. A, Chemical structure of RB. B, Cell viabilities in normal HPDE, Panc‐1, and Aspc cells treated with various concentrations of RB for the indicated times, determined by MTT. C, Cell morphology changes in HPDE, Panc‐1, and Aspc cells treatment with RB (5 μmol/L) for 48 hours were observed by microscope. Total numbers of live cells were counted and quantified as mean ± SD of three samples of each group. D, Colony formation showed the proliferation of Panc‐1 and Aspc cells treated with the indicated concentrations of RB. **P < .005, ***P < .0005; ^## P < .005, ^### P < .0005

Figure 2

Resibufogenin (RB) induces caspase‐dependent apoptosis in pancreatic cancer cells. A, Hoechst 33342 staining was used to analyze apoptotic cells in Panc‐1 and Aspc cells treated with RB (5 μmol/L) for 24 hours. B, Apoptosis of Panc‐1 and Aspc cells treated with RB for 24 hours, measured by flow cytometry. C, D, Levels of cleaved poly(ADP‐ribose) polymerase (PARP)1, caspase 9, and caspase 3 in Panc‐1 and Aspc cells treated with RB for 24 hours in the presence or absence of Z‐VAD‐FMK (20 μmol/L) were detected by immunoblotting. E, MTT was used to determine the cell viability in Panc‐1 and Aspc cells treated with RB (5 μmol/L) for 24 or 48 hours in the presence or absence of necrostatin‐1 (50 μmol/L). *P < .05, **P < 0.005; ^# P < .05, ^## P < .005

Figure 3

Resibufogenin (RB) inhibits canonical and noncanonical nuclear factor‐κB (NF‐κB) activity. A, Dual luciferase assay verified the NF‐κB activity in RB‐treated Panc‐1 and Aspc cells. B, C, Quantitative RT‐PCR and immunoblotting (IB) analysis of the mRNA and protein expression of c‐FLIP_L and Bcl‐2 in Panc‐1 cells treated with 5 μmol/L RB for indicated the times. *P < .05, **P < .005; ^# P < .05, ^## P < .005. D, E, Levels of phosphorylated (p‐)p65 and p65, p100, and p52 in Panc‐1 and Aspc cells treated with RB (5 μmol/L) for the indicated times, detected by IB. F, IB analysis of the nuclear (Nuc) and cytoplasmic (Cyt) fraction of p100 and p52 in Panc‐1 cells treated with 5 μmol/L RB for 24 hours

Figure 4

Resibufogenin (RB) treatment led to changes in nuclear factor‐κB (NF‐κB) target gene expression. A, Microarray analysis of genes differentially expressed in Panc‐1 cells treated with DMSO or RB (5 μmol/L). B, Differential changes in known NF‐κB target genes are shown in the heat map. C, NF‐κB target genes that were downregulated more than 3.5‐fold are listed with their known function in pancreatic cancer

Figure 5

Resibufogenin (RB) suppresses transforming growth factor‐β‐activated kinase 1 (TAK1) and IκB kinase (IKK) activities in pancreatic cancer cells. A, B, Immunoblotting determined the levels of TAK1, TAB 1, and TAB 2 (A) and p‐IKKα/β, IKKα, p‐IκBα, and IκBα (B) in Panc‐1 and Aspc cells treated with 5 μmol/L RB for the indicated times. C, Flow cytometry analysis of apoptosis in Panc‐1 cells transfected with Flag‐TAK1 and TAB 1 following 5 μmol/L RB treatment for 24 hours. ^# P < .05, **P < .005

Figure 6

Resibufogenin (RB) inhibits glycogen synthase kinase‐3 (GSK‐3) activity in pancreatic cancer cells. A, B, Immunoblotting determined the levels of p‐GSK‐3α, GSK‐3α, p‐GSK‐3β, and GSK‐3β (A), glycogen synthase (GS), p‐GS, and Notch1 (B) in Panc‐1 and Aspc cells incubated with 5 μmol/L RB for the indicated times. C, Dual luciferase assay verified the TOPFlash and FOPFlash activity in RB‐treated Panc‐1 and Aspc cells. D, Viability in Panc‐1 cells transfected with GSK‐3β WT, S9A, and K85A following RB (5 μmol/L) treatment for 24 hours was measured by MTT. *P < .05, **P < .005; ^# P < .05

Figure 7

Resibufogenin (RB) induces protein kinase C (PKC)‐mediated glycogen synthase kinase‐3 (GSK‐3) phosphorylation. A, B, Immunoblotting (IB) analyzing the levels of p‐Akt, p‐GSK‐3α, and p‐GSK‐3β in Panc‐1 cells pretreated with LY294002 (A) or Go6983 (B) for 1 hour following RB (5 μmol/L) treatment for 6 hours. C, IB determined the levels of p‐GSK‐3α and p‐GSK‐3β in Panc‐1 cells pretreated with indicated PKC inhibitors for 1 hour following RB (5 μmol/L) treatment for 6 hours. D, p‐PKCα/β levels in Panc‐1 cells treated with 5 μmol/L RB for the indicated times were determined by IB. E, IB analysis of p‐GSK‐3α, p‐GSK‐3β, p‐IKKα/β, and transforming growth factor‐β‐activated kinase 1 (TAK1) expression post‐siRNA knockdown of PKCβ in Panc‐1 cells

Figure 8

Resibufogenin (RB) inhibits Aspc xenograft tumor growth in nude mice as a single‐agent therapy. A, Mean body weight of RB‐treated mice measured at the indicated number of days. B‐D, Gross morphology of tumors, average xenograft tumor weight, and tumor volume were measured over 20 days (n = 6). E, Immunohistochemical staining was carried out for the determination of phosphorylated protein kinase C (p‐PKC)α/β, glycogen synthase kinase (p‐GSK)3β, and p‐p65 in mice tumor samples. Columns are expressed as mean ± SD of 6 samples in each group. *P < .05, **P < .005, ***P < .0005