Secalonic Acid-F, a Novel Mycotoxin, Represses the Progression of Hepatocellular Carcinoma via MARCH1 Regulation of the PI3K/AKT/β-catenin Signaling Pathway

Abstract

Liver cancer is a very common and significant health problem. Therefore, powerful molecular targeting agents are urgently needed. Previously, we demonstrated that secalonic acid-F (SAF) suppresses the growth of hepatocellular carcinoma (HCC) cells (HepG2), but the other anticancer biological functions and the underlying mechanism of SAF on HCC are unknown. In this study, we found that SAF, which was isolated from a fungal strain in our lab identified as Aspergillus aculeatus, could inhibit the progression of hepatocellular carcinoma by targeting MARCH1, which regulates the PI3K/AKT/β-catenin and antiapoptotic Mcl-1/Bcl-2 signaling cascades. First, we confirmed that SAF reduced the proliferation and colony formation of HCC cell lines (HepG2 and Hep3B), promoted cell apoptosis, and inhibited the cell cycle in HepG2 and Hep3B cells in a dose-dependent manner. In addition, the migration and invasion of HepG2 and Hep3B cells treated with SAF were significantly suppressed. Western blot analysis showed that the level of MARCH1 was downregulated by pretreatment with SAF through the regulation of the PI3K/AKT/β-catenin signaling pathways. Moreover, knockdown of MARCH1 by small interfering RNAs (siRNAs) targeting MARCH1 also suppressed the proliferation, colony formation, migration, and invasion as well as increased the apoptotic rate of HepG2 and Hep3B cells. These data confirmed that the downregulation of MARCH1 could inhibit the progression of hepatocellular carcinoma and that the mechanism may be via PI3K/AKT/β-catenin inactivation as well as the downregulation of the antiapoptotic Mcl-1/Bcl-2. In vivo, the downregulation of MARCH1 by treatment with SAF markedly inhibited tumor growth, suggesting that SAF partly blocks MARCH1 and further regulates the PI3K/AKT/β-catenin and antiapoptosis Mcl-1/Bcl-2 signaling cascade in the HCC nude mouse model. Additionally, the apparent diffusion coefficient (ADC) values, derived from magnetic resonance imaging (MRI), were increased in tumors after SAF treatment in a mouse model. Taken together, our findings suggest that MARCH1 is a potential molecular target for HCC treatment and that SAF is a promising agent targeting MARCH1 to treat liver cancer patients.

Keywords: MARCH1; PI3K/AKT/β-catenin; hepatocellular carcinoma; magnetic resonance imaging; proliferation; secalonic acid-F.

Figure 1

Effect of SAF on HCC cell proliferation. (A) Representative images of human HepG2 and Hep3B HCC cells treated with 0, 1.25, 2.5, 5.0, and 10 μM SAF for 24 h and 48 h, respectively. (B) Cell viability assay of SAF-treated HepG2 and Hep3B cells for 24 h and 48 h, respectively (0, 1.25, 2.5, 5.0, and 10 μM). The cell growth ratio was determined relative to the untreated control (0 μΜ). (C) The expression of the MARCH1 response to SAF in HepG2 and Hep3B cells for 24 h and 48 h was determined by western blotting. (D) The MARCH1 mRNA expressions in HepG2 cells with 0, 5,0 μΜ SAF were measured directly by qRT-PCR, GAPDH as an internal control. (E) The HepG2 cells were pretreated for 5 h MG 132 which is a proteasome inhibitor. Then, the MARCH1 protein expressions in HepG2 cells treated with 0 μΜ, 5,0 μΜ SAF, and 2.5 μΜ MG 132 were measured by immunoblotting, GAPDH was used as an internal control. (F) CCK-8 assays of transfected and nontransfected MARCH1 siRNA of HepG2 and Hep3B cells for 48 h, negative siRNA as control. The knockdown of MARCH1 protein was confirmed by western blot. All data in this figure are presented as means ± SD of three independent experiments. ** p < 0.01.

Figure 2

Effect of SAF on HCC cell apoptosis. (A) Colonies were stained with crystal violet solution as described in the Materials and Methods. Colony formation analysis of HepG2 and Hep3B cells treated with 0, 1.25, 2.5, and 5.0 μM SAF for 24 h and 48 h, 0 μM as control. (B) Flow cytometric analysis of apoptosis in HepG2 and Hep3B cells treated with 0, 1.25, 2.5, and 5.0 μM SAF for 24 h and 48 h. The quantification of apoptotic cells was determined, 0 μM as control. (C) Colony formation analysis of HepG2 and Hep3B cells treated with two sets of MARCH1 siRNA, negative siRNA, and non transfected for 48 h, negative siRNA as control. Western blotting was used to confirm the MARCH1 siRNA knockdown in HepG2 and Hep3B cells. (D) Flow cytometry showed the apoptosis rate of HepG2 and Hep3B cells treated with MARCH1 siRNA, negative siRNA, and nontransfected for 48 h, negative siRNA as control. Western blotting was used to confirm the MARCH1 silencing efficiency in HepG2 and Hep3B cells. All data in this figure are presented as means ± SD. ** p < 0.01, * p < 0.05. These data represent three independent experiments.

Figure 2

Effect of SAF on HCC cell apoptosis. (A) Colonies were stained with crystal violet solution as described in the Materials and Methods. Colony formation analysis of HepG2 and Hep3B cells treated with 0, 1.25, 2.5, and 5.0 μM SAF for 24 h and 48 h, 0 μM as control. (B) Flow cytometric analysis of apoptosis in HepG2 and Hep3B cells treated with 0, 1.25, 2.5, and 5.0 μM SAF for 24 h and 48 h. The quantification of apoptotic cells was determined, 0 μM as control. (C) Colony formation analysis of HepG2 and Hep3B cells treated with two sets of MARCH1 siRNA, negative siRNA, and non transfected for 48 h, negative siRNA as control. Western blotting was used to confirm the MARCH1 siRNA knockdown in HepG2 and Hep3B cells. (D) Flow cytometry showed the apoptosis rate of HepG2 and Hep3B cells treated with MARCH1 siRNA, negative siRNA, and nontransfected for 48 h, negative siRNA as control. Western blotting was used to confirm the MARCH1 silencing efficiency in HepG2 and Hep3B cells. All data in this figure are presented as means ± SD. ** p < 0.01, * p < 0.05. These data represent three independent experiments.

Figure 3

Effect of SAF on HCC cell migration and invasion. (A) Wound healing assay for HepG2 and Hep3B cells after SAF treatment, 0 μM as control. (B) Wound healing assay in HepG2 and Hep3B cells with MARCH1 interference, negative siRNA as control. (C) Knock down of MARCH1 protein with siRNAs in HepG2 and Hep3B cells. (D) Transwell migration assay for HepG2 and Hep3B cells after SAF therapy, 0 μM as control. (E) Transwell invasion assay for HepG2 and Hep3B cells in response to SAF, 0 μM as control. (F) In vitro Transwell migration of HepG2 and Hep3B cells transfected with MARCH1 siRNA, negative siRNA as control. Western blotting was used to assess the silencing efficiency of siRNA against MARCH1 in HepG2 and Hep3B cells. (G) In vitro Transwell invasion assay in HepG2 and Hep3B cells after MARCH1 interference, negative siRNA as control. The efficiency of MARCH1 silencing was assessed by western blot. All data in this figure are presented as means ± SD. ** p < 0.01. These data represent three independent experiments.

Figure 4

Effect of SAF on the cell cycle. (A) Cells were stained with propidium iodide as described in the Materials and Methods and detected by flow cytometry. Cell cycle distribution of SAF-treated or untreated HepG2 cells for 24 h. (B) Cell cycle distribution in SAF-treated or untreated Hep3B cells for 48 h. All experiments were carried out in triplicate. The data in this figure are presented as the means ± SD. 0 μM as control, ** p < 0.01, * p < 0.05.

Figure 4

Effect of SAF on the cell cycle. (A) Cells were stained with propidium iodide as described in the Materials and Methods and detected by flow cytometry. Cell cycle distribution of SAF-treated or untreated HepG2 cells for 24 h. (B) Cell cycle distribution in SAF-treated or untreated Hep3B cells for 48 h. All experiments were carried out in triplicate. The data in this figure are presented as the means ± SD. 0 μM as control, ** p < 0.01, * p < 0.05.

Figure 5

PI3K/AKT/β-catenin involved SAF-mediated suppression of MARCH1 expression in HCC. (A,B) Western blotting showed the expression of MARCH1, PI3K, P-AKT, β-catenin, Mcl-1, Bcl-2, cleaved caspase-3, and cleaved caspase-7 in HepG2 after 24 h and in Hep3B cells after 48 h of SAF treatment (0, 1.25, 2.5, and 5.0 μM). (C,D) Protein expression of MARCH1, PI3K, P-AKT, β-catenin, Mcl-1, Bcl-2, cleaved caspase-3, and cleaved caspase-7 in HepG2 and Hep3B cells transfected with MARCH1 siRNA (siRNA-1 and siRNA-2), as measured by western blotting. All data were carried out at least three times. All data in this figure are presented as means ± SD.

Figure 6

SAF significantly reduces HCC xenograft tumor growth via the MARCH1-mediated inhibition of the downstream PI3K/AKT/β-catenin pathway in vivo. (A) Tumor growth curves of the three SAF therapy groups (0 mg/kg/ig, 12.5 mg/kg/ig, 25 mg/kg/ig). (B–C) Representative images of mice and tumors in the three SAF treatment groups. (D) Tumor weight in the untreated and treated mouse groups. (E) Body weight changes of mice in the untreated and treated groups. (F) T2-weighted MR images and ADC maps of the SAF-treated mice. (G) Average ADC values of tumors from untreated and treated mice. (H) Average volume of tumors from untreated and treated group mice measured on T2WI. (I) Negative correlation between ADC value and tumor volume. (J) Representative images of H-E staining in HCC xenograft tumor sections, magnification 40x. (K) Protein expression of MARCH1, PI3K, P-AKT, β-catenin, MCL-1, BCL-2, cleaved caspase-3 and cleaved caspase-7 in xenografts of SAF untreated and treated mice. (L) A schematic diagram illustrating the underlying anticancer mechanism of SAF on hepatocellular carcinoma, as measured by western blotting. All data in this figure are presented as means ± SD. ** p < 0.01, * p < 0.05.