RFX1-dependent activation of SHP-1 induces autophagy by a novel obatoclax derivative in hepatocellular carcinoma cells

Abstract

Obatoclax is a small molecule which targets the Bcl-2 family, and is to treat leukemia, lymphoma and lung carcinoma. Previously, an obatoclax analogue, SC-2001, was found to disrupt the protein-protein interactions of the Bcl-2 family and also repress Bcl-XL and Mcl-1 expression via STAT3 inactivation. Here, we report a novel mechanism of autophagy induction by SC-2001 in liver cancer cells. The findings indicate that SC-2001 induced the autophagy marker LC3-II in four hepatocellular carcinoma (HCC) cells. Autophagosomes induced by SC-2001-treated cells were confirmed by electron microscopy. SC-2001 activated SHP-1, dephosphorylated STAT3 and Mcl-1, and subsequently released free beclin 1. Overexpression of STAT3 and Mcl-1 in PLC5 cells attenuated the induction of SC-2001 on autophagy. Abolishment of SHP-1 by a specific inhibitor aboragated the autophagic effects induced by SC-2001. In addition, it was further revealed that RFX-1, a transcription factor of SHP-1, is a critical regulator in SC-2001-mediated autophagy. Downregulation of RFX-1 by si-RNA protected cells from SC-2001-induced autophagy. Importantly, Huh7 tumor-bearing nude mice treated with SC-2001 showed downregulation of Mcl-1 and p-STAT3 protein expression and upregulation of SHP-1, LC3II, and RFX-1 protein expression. In summary, our data suggest that SC-2001 induces autophagic cell death in a RFX1/SHP-1/STAT3/Mcl-1 signaling cascade.

Figure 1. SC-2001-induced autophagy in HCC cells

A, SC-2001 induced the generation of LC3-II in a dose- and time-dependent manner. HCC cells were exposed to obatocalx or SC-2001 at various doses for various periods of time. Total lysates were subjected to LC3 protein analysis by Western blot. β-actin was used as a loading control. WL2 was used as a negative control. B, Left, autophagy inhibitor, 3-MA, reduced the SC-2001-induced LC3 protein expression in HCC cells. Cells were pretreated with or without 3-MA for 2 h, then treated with 2 μM of SC-2001 for 48 h. Total cell lysates were analyzed by western blot to determine LC3 protein expression. Right, SC-2001 activates autophagic flux in HCC cells. Cells were treated with or without 2 μM of SC-2001 in the presence of or absence of bafilomycin for 48 h. Total cell lysates were analyzed by western blot to determine LC3 protein expression. C, PLC5 and Huh7 cells expressing GFP-LC3 were treated with 2 μM of SC-2001 for 48 h. The cells were fixed with paraformaldehyde and visualized with epifluorescence. The punctate pattern of GFP-LC3, representative of autophagosomes, and nuclei were visualized using DAPI staining. Scale bar represents 50 μm. D, PLC5 and SK-Hep-1 cells were treated with 2 μM of SC-2001 alone or SC-2001+3-MA. Images showing acridine orange (AO) staining were taken 48 h post-treatment using fluorescence microscopy. Average numbers of acidic vacuolar organelles (AVOs) per cell were counted in three fields for each condition. Scale bar represents 50 μm. E, Transmission electron microscopy (TEM) images showing autophagosome (arrow) formation in PLC5 and Huh7 cells treated with 2 μM of SC-2001 for 48 h. Scale bar represents 0.5 or 1 μm.

Figure 2. SC-2001 induces autophagic cell death and disrupts the MCL-1beclin 1 complex

A, SC-2001 induced the disassociation of beclin 1 and Mcl-1. Upper, Mcl-1 was immunoprecipitated from PLC5 or HepG2 cells treated with 2 μM of SC-2001 or obatocax for 48 h and analyzed for the presence of beclin 1. WL-2 was used as a negative control. Lower, Beclin 1 was immunoprecipitated from PLC5 or HepG2 cells treated with 2 μM of SC-2001 for 48 h and analyzed for the presence of Mcl-1 and Bcl-2. B, Effects of SC-2001 on autophagy-related proteins in HCC cells. Cells were treated with SC-2001 at the indicated dose for 48 h. C, PLC5 cells were transfected with control siRNA or beclin 1 siRNA for 48 h then treated with SC-2001 at 2 μM for 48 h. Left, silencing beclin 1 by siRNA reduces the effects of SC-2001 on cell proliferation in HCC cells. The off-target effect of the si-RNA can be ruled out by comparison between the group of no-transfection and si-scramble. Right, silencing beclin 1 by siRNA reduces the effects of SC-2001 on LC3II protein expression. The off-target effect of the si-RNA can be ruled out by no-transfection group. D, SC-2001 treatment inhibits clonogenic survival of SK-Hep1 cells. 3-MA rescued SK-Hep1 cells from autophagic cell death induced by SC-2001 treatment. Cells were treated with SC-2001 for 48 h, and clonogenic survival in crystal violet was assessed after incubation for two weeks.

Figure 3. SHP-1-STAT3 signaling is involved in SC-2001-induced autophagy

A, SK-Hep-1 and PLC5 cells were treated with SC-2001 for 48 h. Total cell lysate of SK-Hep-1 and PLC5 were subjected to pSTAT3, SHP-1, STAT3, Mcl-1, and LC3 protein analysis by Western blot. β-actin was used as a loading control. B, PLC5 cells were transfected with Mcl-1 overexpression plasmid. After transfection for 48 h, cells were treated with 2 μM of SC-2001 for 48 h. Total cell lysates were subjected to Mcl-1 and LC3 protein analysis by Western blot technique. β-actin was used as a loading control. C, PLC5 cells were transfected with STAT3 overexpression plasmid and stable clones were selected by G418. Cells were treated with 2 μM of SC-2001 for 48 h. Total cell lysates were subjected to STAT3 and LC3 protein analysis by Western blot. β-actin was used as a loading control. D, PLC5 cells were transfected with SHP-1 overexpression plasmid and stable clones were selected by G418. Cells were treated with 2 μM of SC-2001 for 48 h. Total cell lysates were subjected to SHP-1 and LC3 protein analysis by Western blot. β-actin was used as a loading control. E, PLC5 cells were treated with SHP-1 inhibitor. After treatment for 2 h, cells were treated with 2 μM of SC-2001 for 48 h. Total cell lysates were subjected to pSTAT3, SHP-1 and LC3 protein analysis by Western blot. β-actin was used as a loading control. Each band of LC3II was normalized to their actin after quantification by densitometer.

Figure 4. RFX-1 expression is related to LC3II protein level

A, PLC5 cells were treated with 2 uM of SC-2001 for 48 h. DNA from control and SC-2001-treated PLC5 cells was immunoprecipitated with RFX-1 or rabbit IgG antibody and captured by protein A-agarose beads. RFX-1 binding site fragment in SHP-1 promoter was detected by PCR in CHIP samples. B, PLC5 cells were transfected with RFX-1 si-RNA for 48 h and treated with 2 μM of SC-2001 for 48 h. Total lysates of control and SC-2001-treated groups were subjected to RFX-1, SHP-1 and LC3 protein analysis by Western blot. β-actin was used as a loading control. C, PLC5 cells were transfected with RFX-1 overexpression plasmid for 48 h and treated with 2 μM of SC-2001 for 48 h. Total lysates of control and SC-2001-treated groups were subjected to RFX-1, SHP-1 and LC3 protein analysis by Western blot. β-actin was used as a loading control. Each band of LC3II was normalized to their actin after quantification by densitometer.

Figure 5. SC-2001 inhibits tumor growth via RFX-1/SHP-1/STAT3 dependent autophagic cell death

A, Huh7 tumor-bearing mice were treated with or without SC-2001 at 20 mg/kg orally every other day. Tumor size was measured and mice were sacrificed after 21 days. B, Body weights of control and SC-2001-treated mice were measured. C, Western blot analysis of p-STAT3, STAT3, SHP-1, RFX-1, Mcl-1 and LC3 in Huh7 tumors. D, SHP-1 activity in control and SC-2001-treated Huh7 tumors. E, Immunohistochemistry of RFX-1, SHP-1, p-STAT3, and LC3B in control and SC-2001-treated Huh7 tumors.