A Critical YAP in Malignancy of HCC Is Regulated by Evodiamine

Abstract

Liver cancer has relatively few early symptoms and is usually diagnosed in the advanced stage. Sorafenib is the only first-line anticancer drug approved by the Food and Drug Administration (FDA) for advanced HCC; however, its use is limited due to resistance. Therefore, the development of new drugs is essential to achieving customized treatment. Many studies have suggested that Yes-associated protein (YAP)/transcriptional co-activator with PDZ-binding motif (TAZ) is associated with metastasis and cancer formation and progression in various cancers. In the present study, YAP was overexpressed in various patient-derived hepatocarcinoma (HCC) tissues. In addition, this study examined whether evodiamine (which has anticancer effects) can inhibit YAP and, if so, modulate HCC. Evodiamine significantly reduced both the YAP level and cell growth of HCC in a dose-dependent manner. Biochemical analysis indicated mitochondria dysfunction-mediated apoptosis to be the cause of the reduction in HCC cell growth by evodiamine. YAP was overexpressed in metastatic HCC tissues as well when compared to primary HCC tissues. Migration and invasion analysis showed that evodiamine has anti-metastatic ability on Hep3B and Huh-7 cells and reduces the level of vimentin, an EMT marker. In conclusion, YAP is a critical target in HCC therapy, and evodiamine can be an effective HCC anticancer drug by reducing the YAP level.

Keywords: AMPK; HCC; YAP; evodiamine; liver cancer.

Figure 1

YAP expression in liver tissues and its counterparts. Cancer patient-derived microarrays containing HCC and normal tissues were examined for YAP expression. Scale bar = 500 μm. YAP expression was scored according to the intensity and proportion of positive cells, and representative immunohistochemical images of YAP are shown; none (-), weak (+), moderate (++), or strong (+++). The staining results show the summarized expression of YAP in normal (n = 9) and tumor tissues (n = 40).

Figure 2

Effects of verteporfin on cell growth and apoptosis of HCC. (A) Huh-7 cells were treated with the indicated verteporfin (VP) concentration. Cell growth was measured by a MTT assay at 24 h and 48 h, with error bars representing S.D. (versus control, ** p < 0.01). (B) Huh-7 cells were treated with the indicated concentration of VP for 48 h, followed by live/dead analysis with calcein-AM (live) and PI (dead). Images were taken by fluorescence microscopy; scale bar = 200 μM. (C) Huh-7 cells were treated with the indicated concentration of VP for 48 h followed by cell cycle analysis by flow cytometry. Sub-G1 is summarized in the histogram, with error bars representing S.D. (versus control, * p < 0.05, ** p < 0.01). (D) Huh-7 cells were treated with 20 μM VP for 48 h, followed by immunoblotting analysis with indicated antibodies. (E) Huh-7 cells were treated with 10 μM VP for 48 h and stained with JC-1, followed by cell cycle analysis by flow cytometry. The numbers under the western blot indicate the intensity of the band. CON, control; VP, verteporfin.

Figure 3

YAP regulation and cell death by evodiamine. (A) Hep3B and Huh-7 cells were treated with the indicated concentration of evodiamine for 48 h, followed by immunoblotting analysis with YAP and β-actin antibodies. The loading control was β-actin. (B) Hep3B and Huh-7 cells were treated with the indicated concentration of evodiamine (EVO). Cell growth was measured using a MTT assay at 24 h and 48 h, with error bars representing S.D. (versus control, ** p < 0.01) (C) Hep3B and Huh-7 cells were treated with the indicated concentration of evodiamine (EVO) for 48 h, which was then changed with fresh media without EVO and incubated for ten days. (D) Hep3B and Huh-7 cells were treated with 5 μM EVO for 48 h, followed by live/dead analysis with calcein-AM (live) and PI (dead). Images were taken by fluorescence microscopy. Scale bar = 200 μM. (E,F) Hep3B (E) and Huh-7 (F) cells were treated with 5 μM EVO for 48 h, followed by cell cycle analysis by flow cytometry. The Sub-G1 is summarized in the histogram, with error bars representing S.D. (versus Control, ** p < 0.01). The numbers under the western blot indicate the intensity of the band. CON, control; EVO, evodiamine.

Figure 4

Apoptosis induction by evodiamine. (A,B) Hep3B and Huh-7 cells were treated with the indicated concentration of evodiamine for 48 h, stained with Annexin V/PI, and analyzed by flow cytometry (A). This was followed by immunoblotting analysis with the indicated antibodies. The loading control was β-actin (B). (C) Huh-7 and Hep3B cells were treated with the indicated concentration of evodiamine for 48 h and stained with JC-1, analyzed by flow cytometry. The numbers under the western blot indicate the relative intensity of the band. CON, control; EVO, evodiamine.

Figure 5

Effects of evodiamine on the metastatic ability of HCC. (A) Cancer patient-derived microarrays containing primary HCC and its metastatic HCC tissues were examined for YAP expression. Staining results were graded according to the intensity and proportion of positive cells as described in ‘Materials and Methods’. Scale bar = 500 μm. (B) Hep3B cells were scrapped and incubated with 0, 2, and 5 μM EVO for 48 h. The scrapped scratches were imaged using an optical microscope. The data are expressed as the percentage of wound width. Scale bar = 1000 μm. (C) Hep3B cells were treated with 5 μM EVO for 24 h and plated on transwell coated with matrigel (invasion) or without matrigel (migration). (D) Hep3B cells were treated with the indicated concentration of evodiamine for 48 h, followed by immunoblotting analysis with vimentin and GAPDH antibodies. The numbers under the western blot indicate the intensity of the band. CON, control; EVO, evodiamine.