Figure 1.
Triptolide induces cancer cell apoptosis dependent on intracellular tRXRα expression.
(A) The chemical structure of triptolide. (B) Growth inhibitory effect. MCF-7 and SW480 cells were treated with various concentrations of triptolide as indicated. Cell viability was measured by the MTT colorimetric assay. *, P<0.05; **, P<0.01 (vs respective controls). (C) The effect of triptolide on tRXRα expression and PARP cleavage was examined in MCF-7 cells. The cells were treated with vehicle or increasing concentrations of triptolide for 9 h. (D) Triptolide induced proteasome-mediated tRXRα degradation. MCF-7 cells were treated with 50 nM triptolide with or without 10 µM MG132, a specific proteasome inhibitor. The impact of MG132 on tRXRα turnover was evaluated. (E) tRXRα expression was determined in various cancer cells as indicated. The apoptotic effects of triptolide in different cells were compared. The cells were treated with vehicle or 50 nM triptolide for 9 h. (F) HeLa and MCF-7 cells were transfected with scramble or RXRα siRNA and incubated with vehicle or 50 nM triptolide for 12 h. Triptolide-induced PARP cleavage was compared between control and RXRα siRNA transfections. (G) MCF-7 cells transfected with scramble or RXRα siRNA were treated with 50 nM triptolide for 12 h and subjected to DAPI staining. The apoptotic cells induced by triptolide were quantified and expressed as percentage of the counted cells.
Figure 2.
Triptolide induces tumor growth inhibition and tRXRα degradation in vivo.
(A) Nude mice with HepG2 heptoma xenografts were intraperitoneally injected (i.p.) daily with saline or 0.2 mg/kg triptolide for 12 days. Tumor sizes and weights in control and triptolide-treated mice were compared. **, P<0.01 (vs control). (B) Tumor sections were stained for TUNEL by immunohistochemistry to show the apoptotic effect of triptolide. (C) The whole lysates prepared form HepG2 xenografts treated with triptolide or vehicle were subjected to Western blotting assays for detecting tRXRα expression. **, P<0.01 (vs control).
Figure 3.
Triptolide inhibits tRXRα-dependent AKT activity and induces cancer cell apoptosis.
(A) HepG2 cells were treated with vehicle or 50 nM triptolide for various time intervals as indicated. Time-dependent effects of triptolide on AKT activity, tRXRα degradation and PARP cleavage were examined. (B) Effect of RXRα siRNA. HepG2 cells transfected scramble or RXRα siRNA were treated with vehicle or 50 nM triptolide for 9 h. The effect of siRNA-mediated knocking down tRXRα expression on triptolide-inducing AKT dephosphorylation was studied. (C) Effect of CA-AKT. HepG2 cells were transiently transfected with active form of AKT expression vector (GFP-CA-AKT) and treated with 80 nM triptolide for 12 h. Apoptotic cells (condensed and fragmentated) induced by triptolide were recognized by DAPI staining, while Bax activation was detected by conformation-sensitive Bax/6A7 antibody.
Figure 4.
Triptolide inhibits TNFα-induced AKT activation.
(A, B) The effect of triptolide on TNFα-induced AKT phosphorylation was determined in MCF-7 cells (A) and A549 cells (B). Cells were treated with vehicle or 10 ng/ml TNFα in the absence or presence of increasing concentrations of triptolide for 12 h. (C) Co-immunoprecipitate assays were carried out in MCF-7 cells to determine tRXRα interaction with p85α. The cells were treated with vehicle or 50 nM triptolide in the absence or presence of 10 ng/ml TNFα for 6 h. Cell lysates were immunoprecipitated with ΔN197 anti-RXRα antibody. The coimmunoprecipitates were then subjected to Western blotting analysis for tRXRα expression and its co-precipitated p85α by ▵N197 anti-RXRα and anti-p85α antibodies respectively.
Figure 5.
Triptolide enhances the apoptotic effect of TNFα and other chemotherapies.
(A) MCF-7 cells were transfected with caspase 8 siRNA to evaluate whether triptolide could activate TNFα-dependent death effect. Untransfected and transfected cells were treated with vehicle or 50 nM triptolide with or without 10 ng/ml TNFα for 12 h. Expression and cleavages of caspase 8, 9 and PARP were analyzed. (B, C) Triptolide-enhanced the apoptotic effect of 5-Fu and camptothecin was examined in HepG2 (B) and MCF-7 cells (C) respectively. Cells were treated with 50 nM triptolide alone or in combination with 10 µM 5-Fu or 10 µM camptothecin for 9 h.
Figure 6.
Triptolide induces tRXRα degradation and AKT inactivation through activation of p38.
(A) Triptolide induced activation of several MAPK pathways. HepG2 cells were treated with vehicle or 50 nM triptolide for various time intervals as indicated. Triptolide-induced time-dependent phosphorylation of p38, JNK and Erk1/2 was compared to its effect on decreasing AKT phosphorylation and PARP cleavage. (B) HepG2 cells were treated with 50 nM triptolide for 9 h with or without p38 inhibitor SB203580 (10 µM), JNK inhibitor SP600125 (10 µM) or Erk1/2 MAPK inhibitor PD98059 (10 µM). The impact of inhibition of the individual pathways on tRXRα degradation and PARP cleavage was determined. (C) HepG2 cells transfected with scramble or p38 siRNAs were treated with vehicle or 50 nM triptolide for 9 h. The effect of siRNA-mediated p38 inhibition on triptolide inactivation of AKT and tRXRα degradation was assayed. (D) HepG2 cells were treated with vehicle or 50 nM triptolide for 9 h in the presence or absence of SB203580. The lysates were immunoprecipitated with ΔN197 anti-RXRα antibody and analyzed for its co-immunoprecipitated with p85α.