Induction of Apoptosis, Inhibition of MCL-1, and VEGF-A Expression Are Associated with the Anti-Cancer Efficacy of Magnolol Combined with Regorafenib in Hepatocellular Carcinoma

Abstract

While regorafenib was approved for the treatment of advanced HCC in 2017, with a partial response and survival benefit; other combination agents to facilitate the efficacy of regorafenib still need to be explored. Magnolol is a potential natural anti-tumor compound for many types of cancers. Combination indexes calculated on the basis of both in vitro and in vivo models have indicated a synergistic effect of the combination of regorafenib and magnolol. The overexpression of the VEGF-A protein significantly diminished regorafenib's inhibition of cell viability, while the transient knockdown of VEGF-A by siRNA effectively sensitized HCC cells to regorafenib. In addition, the inhibition of MCL-1 by siRNA combined with regorafenib allowed for a significantly greater inhibition of cell growth, compared to regorafenib alone. A lower protein expression level for VEGF-A and MCL-1 was found for the combination treatment of HCC in vitro and in vivo. A superior metastasis inhibition was also found in the combination group, as compared to the single-treatment groups, using a transwell assay, wound healing assay, and Western blotting. The caspase-dependent and -independent and DNA damage effects, as determined by flow cytometry and a comet assay, were increased by the combination therapy. Taken together, magnolol sensitized HCC to regorafenib, which was correlated with the reduction of VEGF-A and MCL-1 and the induction of apoptosis.

Keywords: MCL-1; VEGF-A; apoptosis; hepatocellular carcinoma; magnolol; regorafenib.

Figure 1

Cytotoxicity of regorafenib was triggered by magnolol. Hep3B and SK-Hep1 cells were treated with (A) 0–20 μM regorafenib, (B) 0–100 μM magnolol, or (C,D) 50 μM magnolol combined with 0–20 μM regorafenib for 48 h and assayed by the MTT assay. The combination index value for 50 μM magnolol combined with 0–20 μM regorafenib in (E) Hep3B and (F) SK-Hep1 cells is presented. The protein expression of CyclinD1, XIAP, C-FLIP, MCL-1, MMP-9, VEGF-A, and MDC-1 is presented after the treatments with magnolol, regorafenib, and a combination in (G) Hep3B and (H) SK-Hep1 cells. (a¹ p < 0.05 and a² p < 0.01 vs. 0 μM regorafenib or magnolol).

Figure 1

Cytotoxicity of regorafenib was triggered by magnolol. Hep3B and SK-Hep1 cells were treated with (A) 0–20 μM regorafenib, (B) 0–100 μM magnolol, or (C,D) 50 μM magnolol combined with 0–20 μM regorafenib for 48 h and assayed by the MTT assay. The combination index value for 50 μM magnolol combined with 0–20 μM regorafenib in (E) Hep3B and (F) SK-Hep1 cells is presented. The protein expression of CyclinD1, XIAP, C-FLIP, MCL-1, MMP-9, VEGF-A, and MDC-1 is presented after the treatments with magnolol, regorafenib, and a combination in (G) Hep3B and (H) SK-Hep1 cells. (a¹ p < 0.05 and a² p < 0.01 vs. 0 μM regorafenib or magnolol).

Figure 2

Inhibition of VEGF-A and MCL-1 may enhance the cytotoxicity of regorafenib. (A) Hep3B and (B) SK-Hep1 cells were treated with 0–20 μM regorafenib combined with 30 ng/mL of VEGF-A recombinant protein (or not) for 48 h, and the cell viability was measured via the MTT assay. (C) The Hep3B and (D) SK-Hep1 cells were transfected with siVEGF-A combined with 0–20 μM regorafenib (or not) for 48 h, and the cell viability was determined via the MTT assay. (E) The Hep3B and (F) SK-Hep1 cells were treated with 0–20 μM regorafenib, 50 ng/mL of VEGF-A recombinant protein, and 50 μM magnolol for 48 h, and the cell viability was determined via the MTT assay. (G) The VEGF-A Western blotting results in Hep3B and SK-Hep1 cells after being transfected with siVEGF-A combined with 0–20 μM regorafenib (or not) for 48 h. (H) The Hep3B and (I) SK-Hep1 cells were transfected with siMCL-1 combined with 0–20 μM regorafenib (or not) for 48 h, and the cell viability was determined via the MTT assay. (J) The MCL-1 Western blotting results in Hep3B and SK-Hep1 cells after transfection with siMCL-1 combined with 0–20 μM regorafenib (or not) for 48 h. (a¹ p < 0.05 and a² p < 0.01 vs. 0 μM regorafenib; b¹ p < 0.05 and b² p < 0.01 vs. 30 ng/mL VEGF-A; c¹ p < 0.05 and c² p < 0.01 vs. scramble siRNA; d² p < 0.01 vs. 0 μM MAG).

Figure 3

The metastasis inhibition effect of regorafenib was enhanced by magnolol. Hep3B and SK-Hep1 cells were treated with 10 μM regorafenib, 50 μM magnolol, and a combination for 48 h and subjected to a transwell invasion assay and wound healing assay. (A) A photograph of the invasion transwell and (B) the quantification results of the invasion cells. (C). A photograph of the migration cell pattern and the quantification results of the migrated gap area in (D) the Hep3B and (E) SK-Hep1 cells. (a² p < 0.01 vs. CTRL; b² p < 0.01 vs. 10 μM REG or 50 μM MAG; CTRL: Control, MAG: magnolol; REG: regorafenib).

Figure 4

The apoptosis and DNA damage effect of regorafenib was enhanced by magnolol. Hep3B and SK-Hep1 cells were treated with 10 μM regorafenib, 50 μM magnolol, and a combination of both for 48 h, combined with ZVAD for 30 min, and assayed by flow cytometry with (A,B) annexin-V/PI staining and (C) cleaved caspase-3. After 48 h, the magnolol and regorafenib treatments were also performed with (D) PI staining and (E) cleaved PARP-1 staining, respectively. H₂O₂ was used as a positive control in the comet assay. The tail movement pattern of the Hep3B and SK-Hep1 cells were (F) photographed and (G,H) quantified. (a² p < 0.01 vs. CTRL; b² p < 0.01 vs. 10 μM REG or 50 μM MAG; c² p < 0.01 vs. treatment groups without ZVAD; scale bar = 200 μm).

Figure 4

The apoptosis and DNA damage effect of regorafenib was enhanced by magnolol. Hep3B and SK-Hep1 cells were treated with 10 μM regorafenib, 50 μM magnolol, and a combination of both for 48 h, combined with ZVAD for 30 min, and assayed by flow cytometry with (A,B) annexin-V/PI staining and (C) cleaved caspase-3. After 48 h, the magnolol and regorafenib treatments were also performed with (D) PI staining and (E) cleaved PARP-1 staining, respectively. H₂O₂ was used as a positive control in the comet assay. The tail movement pattern of the Hep3B and SK-Hep1 cells were (F) photographed and (G,H) quantified. (a² p < 0.01 vs. CTRL; b² p < 0.01 vs. 10 μM REG or 50 μM MAG; c² p < 0.01 vs. treatment groups without ZVAD; scale bar = 200 μm).

Figure 5

The caspase-dependent and -independent apoptotic effects of regorafenib were enhanced by magnolol. Hep3B and SK-Hep1 cells were treated with 10 μM regorafenib, 50 μM magnolol, and a combination for 48 h and assayed by flow cytometry using (A) FAS staining, (B). FAS-L staining, (C) cleaved caspase-8 staining, (D) DCFH-DA staining for ROS, (E) DIOC₆ staining for ∆ψm, (F) Fluo-3/AM for Ca²⁺, and (G) cleaved caspase-9. The Western blotting of BAX, BAK, FAS, FAS-L, cleaved caspase-3 (cleaved C3), -8 (cleaved C8), -9 (cleaved C9), and cleaved PARP-1 in (H) the Hep3B and (I) SK-Hep1 cells. The IF staining of AIF in (J) Hep3B and (K) SK-Hep1 cells. (a² p < 0.01 vs. CTRL; b² p < 0.01 vs. 10 μM REG or 50 μM MAG; scale bar = 100 μm).

Figure 5

The caspase-dependent and -independent apoptotic effects of regorafenib were enhanced by magnolol. Hep3B and SK-Hep1 cells were treated with 10 μM regorafenib, 50 μM magnolol, and a combination for 48 h and assayed by flow cytometry using (A) FAS staining, (B). FAS-L staining, (C) cleaved caspase-8 staining, (D) DCFH-DA staining for ROS, (E) DIOC₆ staining for ∆ψm, (F) Fluo-3/AM for Ca²⁺, and (G) cleaved caspase-9. The Western blotting of BAX, BAK, FAS, FAS-L, cleaved caspase-3 (cleaved C3), -8 (cleaved C8), -9 (cleaved C9), and cleaved PARP-1 in (H) the Hep3B and (I) SK-Hep1 cells. The IF staining of AIF in (J) Hep3B and (K) SK-Hep1 cells. (a² p < 0.01 vs. CTRL; b² p < 0.01 vs. 10 μM REG or 50 μM MAG; scale bar = 100 μm).

Figure 6

The therapeutic efficacy of regorafenib in Hep3B-bearing mice was enhanced by magnolol. (A) The tumor volume and (B) photographs and (C) weight are displayed after various treatments. (D) Micro-CT scan images, (E) mice body weight, and (F) an H&E stain of the liver, kidneys, and spleen are presented. IHC stain against (G) tumor progression, (H) apoptosis, (I) metastasis- and DNA repair-related antibodies are shown in the tumor tissue of each group. (a¹ p < 0.05 and a² p < 0.01 vs. CTRL; b² p < 0.01 vs. 10 μM REG or 50 μM MAG; scale bar = 100 μm).

Figure 6

The therapeutic efficacy of regorafenib in Hep3B-bearing mice was enhanced by magnolol. (A) The tumor volume and (B) photographs and (C) weight are displayed after various treatments. (D) Micro-CT scan images, (E) mice body weight, and (F) an H&E stain of the liver, kidneys, and spleen are presented. IHC stain against (G) tumor progression, (H) apoptosis, (I) metastasis- and DNA repair-related antibodies are shown in the tumor tissue of each group. (a¹ p < 0.05 and a² p < 0.01 vs. CTRL; b² p < 0.01 vs. 10 μM REG or 50 μM MAG; scale bar = 100 μm).