Berbamine (BBM), a Natural STAT3 Inhibitor, Synergistically Enhances the Antigrowth and Proapoptotic Effects of Sorafenib on Hepatocellular Carcinoma Cells

Abstract

Sorafenib (SORA), a multi kinase inhibitor, is the standard first-line targeted therapy approved by the Food and Drug Administration for advanced hepatocellular carcinoma (HCC). However, emerging evidence from clinical practice indicates that SORA alone has only moderate antitumor effects and could not completely inhibit the progression of the disease. Therefore, it is very necessary and urgent to develop novel combination therapy to improve the clinical outcomes of SORA. The pharmacological study on the chemosensitizing effects of natural products has become a hotspot in recent years, which is commonly thought to be a potential way to improve the effectiveness of drugs in clinical use. Berbamine (BBM) has potential sensitizing effects in multiple chemotherapies and target therapy. However, it remains unclarified whether the combination of BBM and SORA as a treatment could exert a synergistic effect on HCC cell lines. In this study, we first investigated whether BBM can increase the sensitivity of HCC cell lines to SORA. The results revealed that the combination of BBM and SORA could synergistically inhibit the growth of two HCC cell lines and promoted their apoptosis. Mechanistically, our results showed that BBM exerted a dose-dependent inhibitory effect on the basal and IL-6-induced STAT3 activation of HCC cell lines. In addition, the combined treatment of BBM and SORA synergistically suppressed STAT3 phosphorylation at Tyr705 and knockdown of STAT3 abolished the sensitization effect of BBM, indicating that BBM's sensitization effect is mainly mediated by its inhibition of STAT3. These findings identify a new type of natural STAT3 inhibitor and provide a novel approach to the enhancement of SORA efficacy by blocking the activation of STAT3.

Figure 1

(A) Chemical structures of berbamine (BBM). (B) Antiproliferative effects of BBM on PRF-PLC-5 and HCC-Lm3 cells. The cells were treated with compounds at different concentrations (50, 25, 12.5, 6.25, and 3.125 μM) for 48 h, and then MTS assay was performed to calculate the survival rate for each group of cells. Data were compiled from three independent experiments.

Figure 2

Combined use of BBM and SORA can act synergistically to inhibit HCC cell growth. (A) Effect of combining SORA with BBM on the cell viability of PRF-PLC-5, HCC-Lm3, and HL-7702 cells. MTS assay was performed to measure cell viability. (B) Effect of combining SORA with BBM on the formation of PRF-PLC-5 and HCC-Lm3 cell clones. Cells in each group were exposed variably for 10–14 days, after which they were photographed. The data were compiled from three independent experiments that were replicated in triplicate. Representative photos are shown. (C) Quantitative results of the colony formation were analyzed through dissolving the crystal violet dye in 33% aqueous acetic acid and reading the absorbance at 570 nm (OD570). **P < 0.01 compared to the negative control.

Figure 3

BBM and SORA synergistically induce HCC cell cycle arrest. (A) Single-agent treatment with BBM (10 μM), SORA (10 μM) alone, or combined treatment with BBM (10 μM) and SORA (10 μM) for 48 h. Cell cycle distribution was assessed using cytometry (Becton Dickinson Fascalibor, BD Biosciences, Franklin Lakes, NJ). For cell cycle distribution, a representative histogram is provided. Data were compiled from three independent experiments and reported in the form of mean ± SD. All of the experiments were performed in triplicate. (B) Western blot analysis of G0/G1 phase-related protein phosphorylated Rb (p-Rb), cyclin D1, Cdk4, and Cdk6. Actin was shown as the control of equal loading. (C) Quantification of the relative expression of cell cycle-related proteins (p-Rb/actin, cyclin D1/actin, Cdk4/actin, and Cdk6/actin) using ImageJ software and analysis with Graphpad prism 7. **P < 0.01 compared to the negative control.

Figure 4

BBM and SORA synergistically induce HCC cell apoptosis. (A) Single-agent treatment with BBM (10 μM), SORA (10 μM) alone, or combined treatment with BBM (10 μM) and SORA (10 μM) for 48 h. Apoptosis was analyzed using Annexin V/propidium iodide (PI) staining. Data were compiled from three independent experiments and reported in the form of mean ± SD. All of the experiments were performed in triplicate. (B) Extraction of total protein and detection of the expression of cleaved PARP, cleaved caspase 3, and Bcl-2 using western blotting. Actin was shown as the control of equal loading. (C) Quantification of the relative expression of apoptosis-related proteins (cleaved PARP/actin, cleaved caspase 3/actin, and Bcl-2/actin) using ImageJ software and analysis with Graphpad prism 7. **P < 0.01 compared to the negative control.

Figure 5

Combined use of BBM and SORA activities in synergy to suppress STAT3 activation in HCC cells, and knockdown of STAT3 abolished the sensitization effect of BBM. (A) HCC-Lm3 cells were pretreated with 2.5, 5, 10, and 20 μM BBM for 2 h, and then stimulated with IL-6 (10 ng/mL) for 30 min. Western blot analysis was performed to determine STAT3 phosphorylation. Relative STAT3-phosphorylated (Y705) expression (p-STAT3/t-STAT3) was quantified. (B) After HCC-Lm3 cells were treated with IL-6 (10 ng/mL) with or without BBM (5 and 10 μM) for 2 h, western blotting was used to determine the levels of STAT3 in the nucleus and the cytoplasm. (C) HCC-Lm3 cells were treated with BBM at an indicated concentration for 24 h, and then western blot analysis was used to determine the phosphorylated and total STAT3 proteins. (D) Treatment of HCC-Lm3 cells with SORA alone (10 μM), BBM alone (10 μM), or in combination with SORA (10 μM) and BBM (10 μM) for 24 h. Western blotting was used to determine the phosphorylated and total STAT3. The relative expression of all bands in (A)–(D) was quantified using ImageJ software and analysis with Graphpad prism 7. **P < 0.01 compared to the negative control. ns means no significance. (E) STAT3 expression level and cell viability of HCC-Lm3 cells with STAT3 knocking down were examined by western blot analysis and MTS assay. **P < 0.01 compared to the negative control. (F) siNC or siSTAT3 transfected cells were treated with SORA (10 μM) or SORA (10 μM) plus BBM (10 μM) for 48 h, and then the cell viability was measured by MTS assay.