Gambogic acid inhibits STAT3 phosphorylation through activation of protein tyrosine phosphatase SHP-1: potential role in proliferation and apoptosis

Abstract

The transcription factor, STAT3, is associated with proliferation, survival, and metastasis of cancer cells. We investigated whether gambogic acid (GA), a xanthone derived from the resin of traditional Chinese medicine, Garcinia hanburyi (mangosteen), can regulate the STAT3 pathway, leading to suppression of growth and sensitization of cancer cells. We found that GA induced apoptosis in human multiple myeloma cells that correlated with the inhibition of both constitutive and inducible STAT3 activation. STAT3 phosphorylation at both tyrosine residue 705 and serine residue 727 was inhibited by GA. STAT3 suppression was mediated through the inhibition of activation of the protein tyrosine kinases Janus-activated kinase 1 (JAK1) and JAK2. Treatment with the protein tyrosine phosphatase (PTP) inhibitor pervanadate reversed the GA-induced downregulation of STAT3, suggesting the involvement of a PTP. We also found that GA induced the expression of the PTP SHP-1. Deletion of the SHP-1 gene by siRNA suppressed the ability of GA to inhibit STAT3 activation and to induce apoptosis, suggesting the critical role of SHP-1 in its action. Moreover, GA downregulated the expression of STAT3-regulated antiapoptotic (Bcl-2, Bcl-xL, and Mcl-1), proliferative (cyclin D1), and angiogenic (VEGF) proteins, and this correlated with suppression of proliferation and induction of apoptosis. Overall, these results suggest that GA blocks STAT3 activation, leading to suppression of tumor cell proliferation and induction of apoptosis.

Figure 1

Effects of GA in induction of apoptosis in multiple myeloma cells. (A) Chemical structure of GA. (B) GA-induced apoptosis. U266 cells were treated with GA (2.5 µM) for indicated times, and cell death was determined by fluorescence-activated cell sorting using annexin V/propidium iodide staining (left panel) and propidium iodide staining (right panel). (C) GA (2.5 µM) induced cell death treated for indicated times as determined by the live/dead assay. (D) Cleavage of procaspase-9, -3 and poly(ADP ribose) polymerase as determined by Western blotting in whole-cell extracts of GA-treated cells. The same blot was stripped and reprobed with STAT3 antibody to verify equal protein loading (left panel). MCF-7 and MCF-10A cells were pretreated with indicated concentration of GA for 24 h. Cell viability was then analyzed by the MTT method (right panel).

Figure 2

(A) GA suppresses constitutive expression of phospho-STAT3. U266 (2×10⁶ cells/mL) were treated with the indicated concentrations of GA for 6 h, after which whole-cell extracts were prepared, and 40 µg of protein were resolved on 10% SDS-PAGE gel, electrotransferred onto nitrocellulose membranes, and probed for phospho-STAT3 (Tyr705). The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. (B) GA suppresses STAT3 phosphorylated at tyrosine705 and serine727 in a time-dependent manner. U266 cells (2 ×10⁶/mL) were treated with the 2.5 µM GA for the indicated times, after which Western blotting was performed for phospho-STAT3 (Tyr705) as described previously. The same blots were stripped and reprobed with phospho STAT-3 (Ser727) and then STAT3 to verify equal protein loading. (C) GA causes inhibition of translocation of STAT3 to the nucleus. U266 cells (2×10⁶/mL) were incubated with or without 2.5 µM GA for 6 h and then analyzed for the intracellular distribution of STAT3 by immunocytochemistry. (D) GA suppresses STAT3 DNA binding in a dose-dependent manner. U266 cells (2×10⁶/mL) were treated with the indicated concentrations of GA for 6 h and analyzed for nuclear STAT3 levels by EMSA (left panel). GA suppresses STAT3 DNA binding in a time-dependent manner. U266 cells (2×10⁶/mL) were treated with 2.5 µM GA for the indicated times and analyzed for nuclear STAT3 levels by EMSA (right panel).

Figure 3

(A) GA down-regulates IL-6–induced phospho-STAT3. MM1.S cells (2 × 10⁶) were treated with IL-6 (10 ng/mL) for the indicated times. Whole-cell extracts were prepared, and phospho-STAT3 level was detected by Western blot. The same blot was stripped and reprobed with STAT3 antibody to verify equal protein loading. (B) MM1.S cells (2 × 10⁶) were treated with 2.5 µM GA for the indicated times and then stimulated with IL-6 (10 ng/mL) for 15 min. Whole-cell extracts were then prepared and analyzed for phospho-STAT3 by Western blotting. The same blot was stripped and reprobed with STAT3 antibody to verify equal protein loading (lower panel).

Figure 4

(A) GA suppresses phospho-JAK1 expression. U266 cells (2×10⁶/ml) were treated with 2.5 µM GA for indicated time, after which whole-cell extracts were prepared and 40 µg protein were resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed for phospho-JAK1 antibody. The same blots were stripped and reprobed with JAK1 antibody to verify equal protein loading. (B) GA suppresses expression of phospho-JAK2. U266 cells (2×10⁶/ml) were treated with 2.5 µM GA for indicated time, after which whole cell extracts were prepared. Western blotting for phospho-JAK2 was performed. The same blots were stripped and reprobed with JAK2 antibody to verify equal protein loading. (C) To determine the activity of JAK2, a kinase assay was performed using GA (2.5 µM) treated whole-cell extract as described in ‘Materials and Methods'. Total protein level was determined by western bloting.

Figure 5

(A) Pervanadate reverses the phospho-STAT3 inhibitory effect of GA. U266 cells (2×10⁶/ml) were treated with pervanadate and 2.5 µM GA for 6 h, after which whole-cell extracts were prepared, and 40 µg protein extracts were resolved on 7.5% SDS-PAGE gel, electrotransferred onto nitrocellulose membranes, and probed for phospho-STAT3 and STAT3. (B) GA induces the expression of SHP-1 protein. U266 cells (2 × 10⁶/mL) were treated with indicated concentrations of GA for 6 h, after which whole-cell extracts were prepared and 40 µg proteins were resolved on 10% SDS-PAGE, electrotransferred onto nitrocellulose membranes, and probed with SHP-1 antibody. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (C) Effect of SHP-1 knockdown on GA–induced expression of SHP-1. SCC4 cells (2 × 10⁶/mL) were transfected with either SHP-1 siRNA or scrambled siRNA (50 nM). After 48 h, cells were treated with 2.5 µM GA for 6 h, and whole-cell extracts were subjected to Western blot analysis for SHP-1. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading (left panel). Transfection with SHP-1 siRNA reverses GA-induced suppression of STAT3 activation. SCC4 cells (2 × 10⁶/mL) were transfected with either SHP-1 siRNA or scrambled siRNA (50 nM). After 48 h, cells were treated with 2.5 µM GA for 6 h, and whole-cell extracts were subjected to Western blot analysis for phosphorylated STAT3. The same blots were stripped and reprobed with STAT3 antibody (right panel). (D) Knockdown of SHP-1 inhibited the apoptotic effect of GA. SCC4 cells (1 × 10⁵/mL) were transfected with either scrambled or SHP-1–specific siRNA (50 nM). After 48 h, cells were treated with 50 µM GA for 24 h, and the percentage of apoptosis was analyzed by the live/dead assay.

Figure 6

GA suppresses STAT3-regulated proliferative, survival, and angiogenic gene products. U266 cells (2 × 10⁶/mL) were treated with 2.5 µM GA for the indicated time intervals, after which whole cell extracts were prepared and 40 µg proteins resolved on 10% SDS-PAGE and probed against (A) c-IAP, survivin, Mcl-1, bcl-2, bcl-xl (B) cyclin D1, VEGF, COX-2, and ICAM-1 antibody. The same blots were stripped and reprobed with β-actin antibody to verify equal protein loading. (C) Schematic representation of mechanism by which GA inhibits STAT3 activation and induces apoptosis.