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. 2020 May 25;25(10):2454.
doi: 10.3390/molecules25102454.

The Synergistic Anti-Cancer Effects of NVP-BEZ235 and Regorafenib in Hepatocellular Carcinoma

Cheng-Chan Yu  1 , Sung-Ying Huang  2 , Shu-Fang Chang  3 , Kuan-Fu Liao  4 , Sheng-Chun Chiu  3   5   6
Affiliations

The Synergistic Anti-Cancer Effects of NVP-BEZ235 and Regorafenib in Hepatocellular Carcinoma

Cheng-Chan Yu et al. Molecules. .

Abstract

Hepatocellular carcinoma (HCC) is the most common type of liver cancer worldwide. Regorafenib is a multi-kinase inhibitor and the second-line treatment for HCC. Since the PI3K/Akt/mTOR signaling pathway is dysregulated in HCC, we evaluated the therapeutic effects of regorafenib combined with a dual PI3K/mTOR inhibitor BEZ235 in the human HCC cell lines (n = 3). The combined treatment with BEZ235 and regorafenib enhanced the inhibition of cell proliferation and increased the expression of cleaved caspase-3 and cleaved PARP in HCC cells. Moreover, the combined treatment suppressed HCC cell migration and invasion in the transwell assay. Further, the Western blot analyses confirmed the involvement of epithelial-mesenchymal transition (EMT)-related genes such as slug, vimentin, and matrix metalloproteinase (MMP)-9/-2. Additionally, the proteinase activity of MMP-9/-2 was analyzed using gelatin zymography. Furthermore, the inhibition of phosphorylation of the Akt, mTOR, p70S6K, and 4EBP1 after combined treatment was validated using Western blot analysis. Therefore, these results suggest that the combined treatment with BEZ235 and regorafenib benefits patients with HCC.

Keywords: BEZ235; PI3K/Akt/mTOR pathway; combination therapy; hepatocellular carcinoma; metastasis; regorafenib.

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Conflict of interest statement

The authors declare no conflict interests.

Figures

Figure 1
Figure 1
BEZ235 enhances the anti-proliferation effect of regorafenib in HCC cells. The cell viability analysis for HCC cells treated for 48 h with (A) BEZ235 or (B) regorafenib. (C) Cell viability analysis for the HCC cells treated with various combinations of BEZ235 and regorafenib for 48 h. Scale bar: 50 μm. Data are represented as mean ± S.D. * p < 0.05, and ** p < 0.01 versus untreated control, and BEZ235 or regorafenib alone group.
Figure 2
Figure 2
The regorafenib-induced apoptosis in HCC cells increases upon treatment with BEZ235. The flow cytometry analysis of cell cycle profiles in HCC cells treated with BEZ235 and regorafenib for 48 h (A). The Western blot analysis indicating the expression pattern of cleaved caspase-3 and cleaved PARP (B). Data are represented as mean ± S.D. * p < 0.05, and ** p < 0.01 versus untreated control, and BEZ235 or regorafenib alone group.
Figure 3
Figure 3
Treatment with BEZ235 enhances the anti-migration and -invasion effect of regorafenib in HCC cells. The analysis of transwell migration and invasion assay upon treatment of Hep3B (A), HepG2 (B) and Huh7 (C) cells with BEZ235 and regorafenib for 24 h or 48 h. Data are represented as means ± S.D. * p < 0.05, and ** p < 0.01 versus untreated control, BEZ235 or regorafenib alone group.
Figure 4
Figure 4
EMT-associated proteins are down-regulated upon combinatorial treatment in HCC cells. The expression pattern of the EMT-associated proteins was analyzed using Western blot (A), while the enzymatic activity of MMP-9/-2 were checked using zymography (B), following combined treatment of the HCC cells.
Figure 5
Figure 5
The combined treatment inhibits the activation of Akt/mTOR signaling pathway in the HCC cells. The panels indicate Western blot analysis for phosphorylation and total protein levels of the members of the Akt/mTOR pathway in the HCC cells treated with BEZ235 and regorafenib for 48 h.
Figure 6
Figure 6
Model depicting the anti-tumor effect of the combined treatment with BEZ235 and regorafenib in HCC cells. The combined treatment suppressed HCC cell migration and invasion through inhibition of the Akt/mTOR signaling pathway.
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