Berberine induces non-small cell lung cancer apoptosis via the activation of the ROS/ASK1/JNK pathway

Abstract

Background: Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. Berberine (BBR), an isoquinoline alkaloid, is commonly used in traditional Chinese medicine. Previous studies have shown that BBR has a potential anti-tumor effect. However, the mechanisms of BBR on mitochondrial function in anti-lung cancer remain unknown. The aim of this study was to explore mitochondrial function in anti-tumor mechanisms of BBR in NSCLC.

Methods: The NSCLCs were cultured and treated with various doses (40, 80, 120 µg/mL) of BBR for 24 and 48 h. Cell viability was evaluated using Cell Counting Kit-8 (CCK-8). Cell apoptosis, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were detected by flow cytometry. Relative protein expression was examined by western blot and immunohistochemical (IHC) analysis.

Results: BBR potently suppressed NSCLC cells growth by inducing apoptosis in a dose-and time-dependent manner. BBR induced apoptosis in NSCLC cells as evidenced by caspase-3 cleavage, cytochrome c release, and mitochondrial membrane depolarization. BBR-induced, dose-dependent induction of apoptosis was accompanied by sustained phosphorylation of c-jun-NH2-kinase (JNK) and the JNK inhibitor (SP600125) significantly suppressed BBR-induced apoptosis, N-acetyl cysteine (NAC), a ROS scavenger, was sufficient to both suppress apoptosis signal-regulating kinase 1 (ASK1) and JNK activation and disrupt apoptotic induction.

Conclusions: The results suggest that BBR induces apoptosis of NSCLC cells via ROS-mediated ASK1/JNK activation and the mitochondrial pathway.

Keywords: Berberine (BBR); apoptosis; mitochondria; non-small cell lung cancer (NSCLC); reactive oxygen species (ROS).

Figure 1

BBR inhibited proliferation and induced apoptosis in NSCLC cells. (A) The A549 and PC9 cells were treated with various concentrations of BBR for 24, 48, and 72 h. Cell viability was analyzed by CCK-8 assays. (B) Morphological changes were assessed via microscopy after a 48-h treatment (20×). (C) The A549 and PC9 cells were treated with a range of BBR concentrations (0, 40, and 80 μM) for 48 h, after which Annexin V/PI staining was used to evaluate apoptotic death by flow cytometry. (D) The apoptotic rates were quantified. (E) After the same treatment described in (C), CL-caspase-3, Bax, and Bcl-2 in the NSCLC cells were measured by western blotting. (F) The protein levels from (E) were quantified. The data are presented as the mean ± standard deviation for the three different experiments with triplicate sets in each assay. *P<0.05, ***P<0.001, and ^nsP>0.05. BBR, berberine; NSCLC, non-small cell lung cancer; CCK-8, Cell Counting Kit-8.

Figure 2

BBR activated the ASK1/JNK and mitochondrial apoptotic pathway in NSCLC cells. (A) The A549 and PC9 cells were treated with BBR (0, 40, and 80 μM) for 48 h, after which the ASK1, p-ASK1, JNK, and p-JNK levels were assessed by western blotting, (B) The protein levels from (A) were quantified. (C) After the same treatment described in (A), changes in MMP were measured in the NSCLC cells. (D) Cells with low MMP are shown in Q2, and were quantified. (E) After the same treatment described in (A), the A549 and PC9 cells were separated into mitochondrial and cytosolic fractions, and the cytochrome c levels in these fractions were measured by Western blotting. (F) The protein levels from (E) were quantified. The data are presented as the mean ± standard deviation for the three different experiments with triplicate sets in each assay. *P<0.05, **P<0.01, and ***P<0.001. BBR, berberine; ASK1, apoptosis signal-regulating kinase 1; JNK, c-jun-NH2-kinase; NSCLC, non-small cell lung cancer; MMP, mitochondrial membrane potential.

Figure 3

BBR induced NSCLC cell apoptosis via the activation of the ASK1/JNK pathway. (A) The A549 and PC9 cells were treated with or without 40 μM of SP600125 for 1 h before treatment with 80 μM of BBR for 48 h. The protein levels of p-JNK, JNK, CL-caspase-3, Bax, and Bcl-2 in the NSCLC cells were measured by western blotting. (B) The protein levels were quantified. (C) Cell viability was measured by CCK-8 assays. The data are presented as the mean ± standard deviation for the three different experiments with triplicate sets in each assay. *P<0.05, **P<0.01, and ***P<0.001. +, added with indicated agent; −, none. BBR, berberine; NSCLC, non-small cell lung cancer; ASK1, apoptosis signal-regulating kinase 1; JNK, c-jun-NH2-kinase; CCK-8, Cell Counting Kit-8.

Figure 4

BBR induced NSCLC cell apoptosis via the activation of the ROS/ASK1/JNK pathway in vitro. (A) The A549 and PC9 cells were then treated with BBR (0, 40, and 80 μM) for 48 h, and the ROS levels in the A549 and PC9 cells were measured by flow cytometry. (B) The data from (A) were quantified. (C) Following a 1-h pretreatment with NAC (500 μM), the cells were treated for 48 h with 80 μM of BBR, and the ROS levels were then assessed by flow cytometry. (D) The data from (C) were quantified. (E) The cells were treated as described in (C), and the protein levels were then assessed by western blotting. (F) The data are presented as the mean ± standard deviation for the three different experiments with triplicate sets in each assay. *P<0.05, and ***P<0.001. +, added with indicated agent; −, none. BBR, berberine; ROS, reactive oxygen species; NAC, N-acetyl cysteine; ASK1, apoptosis signal-regulating kinase 1; JNK, c-jun-NH2-kinase; NSCLC, non-small cell lung cancer.

Figure 5

BBR induced NSCLC cell apoptosis via the activation of the ASK1/JNK pathway in vivo. (A) IHC analysis of the expression of phosphorylation-ASK1, phosphorylation-JNK, CL-caspase-3, and Bax in the tumor tissues treated with BBR (500 mg/kg). (B) The protein levels from (A) were quantified. ***P<0.001. BBR, berberine; ASK1, apoptosis signal-regulating kinase 1; JNK, c-jun-NH2-kinase; NSCLC, non-small cell lung cancer; IHC, immunohistochemical.

Figure 6

An overview of the BBR-induced apoptotic death of NSCLC cells. BBR-induced ROS generation promotes ASK1/JNK phosphorylation, which in turn suppresses Bcl-2 family proteins, driving a loss of MMP, the release of mitochondrial cytochrome c, and the consequent caspase-dependent apoptosis pathway. NAC, N-acetyl cysteine; ROS, reactive oxygen species; ASK1, apoptosis signal-regulating kinase 1; JNK, c-jun-NH2-kinase; BBR, berberine; NSCLC, non-small cell lung cancer; MMP, mitochondrial membrane potential.