Sorafenib inhibits STAT3 activation to enhance TRAIL-mediated apoptosis in human pancreatic cancer cells

Abstract

Signal transducers and activators of transcription 3 (STAT3) is constitutively active in human pancreatic cancer cells and can promote cell growth and apoptosis resistance that contribute to tumorigenesis. We determined if sorafenib, a multikinase inhibitor, can induce apoptosis by targeting STAT3 signaling to enhance apoptosis induction by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). Human pancreatic cancer cell lines (PANC-1 and BxPC-3) were preincubated with sorafenib (Nexavar) alone or followed by TRAIL. Apoptosis was determined by Annexin V labeling, caspase cleavage, and Bax/Bak activation. Protein expression was analyzed by immunoblotting. Knockdown of STAT3, Mcl-1, and Bim were achieved by lentiviral small hairpin RNA. Adenoviral dominant-negative or retroviral constitutively active (CA) STAT3 were also used. Sorafenib inhibited constitutive STAT3 phosphorylation (Tyr(705)) and suppressed Mcl-1 and Bcl-x(L) proteins in a dose- and time-dependent manner. CA-STAT3 overexpression was shown to attenuate caspase-3 cleavage and suppression of Mcl-1 by sorafenib. STAT3 knockdown or a DN STAT3 was shown to downregulate Mcl-1 and Bcl-x(L) and to sensitize cells to TRAIL-mediated apoptosis. Treatment with sorafenib enhanced TRAIL-induced Annexin V staining and release of mitochondrial cytochrome c and AIF. Because the BH3-only Bim protein is a potent inducer of mitochondrial apoptosis, Bim knockdown was shown to attenuate caspase-3, caspase-9 cleavage, and Bax/Bak activation by sorafenib plus TRAIL. The suppression of STAT3 by genetic means or using sorafenib was shown to downregulate Mcl-1 and Bcl-x(L) and to sensitize cells to TRAIL-mediated apoptosis. These data indicate that targeting STAT3 may enhance treatment efficacy against pancreatic cancer.

Figure 1

Sorafenib inhibits constitutive STAT3 phosphorylation at Tyr⁷⁰⁵ and down-regulates Mcl-1 and Bcl-x_L expression in PANC-1 (A) or BxPC-3 (B) cell lines. Cells were incubated with sorafenib at the indicated doses for 6, 12, or 24 h, and the whole cell lysate (WCL) was analyzed by Western blotting using highly specific antibodies. The level of p-ERK1/2 was normalized to total ERK1/2 in the same blots and the relative ratio is shown (see Methods). C, BxPC-3 cells were stably transduced with a retroviral constitutively active STAT3-FLAG or an empty vector. Overexpression of STAT3 was confirmed by detecting STAT3 and the FLAG tag using Western blotting. Cells were treated with sorafenib (0, 16 µM) for 12h and Mcl-1 and cleaved caspase-3 were probed. The relative intensity of the Mcl-1 protein bands was determined by densitometry. D, PANC-1 cells containing a lentiviral STAT3 shRNA or control were probed for Mcl-1, Bcl-x_L, VEGF, or cleaved caspase-3 by Western blotting (left panel). Cleaved caspase-3 was detected after a prolonged exposure. Alternatively, PANC-1 cells were transduced with an adenoviral dominant negative STAT3 containing a 2HA tag at the N-terminus at an increasing multiplicity of infection (MOI). The WCL was prepared 48 h post-transduction and subjected to Western blotting for Stat-3, Mcl-1, Bcl-x_L or HA tag (* refers to a non-specific band).

Figure 2

Sorafenib enhances TRAIL-mediated apoptosis. PANC-1 (A) or BxPC-3 (B) cells were pre-treated with sorafenib for 24 h or 12 h, respectively, followed by TRAIL for 24 h. Apoptosis was analyzed by annexin V labeling and FACS analysis. Annexin V(+) cells were quantified (see Methods) and shown in a bar graph. Experiments were conducted in triplicate and mean values ± SD (bars) are shown.

Figure 3

Sorafenib enhances TRAIL-mediated apoptotic signaling. PANC-1 (A) and BxPC-3 cells (B) cells were pre-treated with sorafenib (16 µM) for 24 h or 12 h, respectively, followed by TRAIL (10 ng/ml for PANC-1; 2.5 ng/ml for BxPC-3) for 5 h. Then, the WCLs (left panels) were analyzed by Western blotting for caspase-8, -9, -3 and Bid. The cytosolic fractions (right panels) were analyzed for mitochondrial cytochrome c and AIF release. COX IV was utilized as a mitochondrial marker and β-tubulin served as a loading control.

Figure 4

STAT3 shRNA or dominant negative (DN) STAT3 sensitizes cells to TRAIL-mediated apoptosis whereas constitutively active (CA)-STAT3 attenuates apoptosis induction by sorafenib and TRAIL. A, PANC-1 cells containing lentiviral STAT3 shRNA or control were treated with TRAIL for 5 h and caspase-3 cleavage in WCLs was determined by Western blotting. B, PANC-1 cells were transduced with an adenoviral DN STAT3 (MOI = 2000 pfu/cell) for 48 h, and then treated with TRAIL for 5 h. Cleavage of caspase-8, -9, -3 and expression of Bid were analyzed by Western blotting in the WCL. C, Retroviral CA-STAT3-FLAG overexpression or control BxPC-3 cells were treated with sorafenib (0, 16 µM) for 12h followed by TRAIL (0, 2.5 ng/ml) for 3h. Cleaved caspase-3 was then probed. D, PANC-1 cells were transduced with Mcl-1 shRNA (si1 and si8 constructs) or control shRNA and the efficiency of Mcl-1 knockdown was analyzed in cell lysates by Western blotting (inset). Knockdown efficiency was higher in the si8 construct that was used for subsequent experiments. Cells were treated with TRAIL (0, 5, 10 ng/ml) for 48 h and cell viability was analyzed using the MTS assay. Experiments were conducted in triplicate and mean values ± SD (bars) are shown. Next, PANC-1 cells containing Mcl-1 shRNA (si8) or control shRNA were treated in the absence or presence of TRAIL (10 ng/ml, 5 h) and Mcl-1 expression and caspase-3 cleavage (CL) were analyzed in cell lysates by Western blotting (right panel). β-tubulin was utilized as a control for protein loading.

Figure 5

BH3-only Bim mediates sorafenib and/or TRAIL-induced apoptosis. A, BxPC-3 cells were treated with vehicle or sorafenib (16 µM) for 24h and WCLs were probed for the indicated proteins by Western blotting. B, BxPC-3 cells were transduced with shRNA to Bim or control and the WCL was analyzed for Bim proteins by Western blotting (inset). Cells with Bim knockdown or control shRNA were then incubated with TRAIL for 48 h, and cell viability was determined using the MTS assay. Experiments were conducted in triplicate and mean values ± SD (bars) are shown. C, Bim knockdown and shRNA control BxPC-3 cells were pre-treated with sorafenib (0, 8 µM; 4 h) followed by incubation with TRAIL (2.5 ng/ml; 2 h), and DNA fragmentation (see Methods) was analyzed. Experiments were conducted in triplicate and mean values ± SD (bars) are shown. D, Bim knockdown or control BxPC-3 cells were pretreated with vehicle or sorafenib (8µM; 12 h) alone and in combination with TRAIL (2.5 ng/ml; 2 h). Caspase activation was analyzed in WCL by Western blotting. In addition, a treatment-induced Bak or Bax conformational change was analyzed by immunoprecipitation (IP) using conformation-specific antibodies. β-tubulin was used as a control for protein loading.