The natural product berberine synergizes with osimertinib preferentially against MET-amplified osimertinib-resistant lung cancer via direct MET inhibition

Abstract

Berberine is a natural product that has long been used in traditional Chinese medicine due to its antimicrobial, anti-inflammatory and metabolism-regulatory properties. Osimertinib is the first third-generation EGFR-tyrosine kinase inhibitor (TKI) approved for the treatment of non-small cell lung cancer (NSCLC) with activating EGFR mutations and those resistant to earlier generation EGFR-TKIs due to a T790M mutation. However, emergence of acquired resistance to osimertinib limits its long-term efficacy in the clinic. One known mechanism of acquired resistance to osimertinib and other EGFR-TKIs is MET (c-MET) gene amplification. Here, we report that berberine, when combined with osimertinib, synergistically and selectively decreased the survival of several MET-amplified osimertinib-resistant EGFR mutant NSCLC cell lines with enhanced induction of apoptosis likely through Bim elevation and Mcl-1 reduction. Importantly, this combination effectively enhanced suppressive effect on the growth of MET-amplified osimertinib-resistant xenografts in nude mice and was well tolerated. Molecular modeling showed that berberine was able to bind to the kinase domain of non-phosphorylated MET, occupy the front of the binding pocket, and interact with the activation loop, in a similar way as other known MET inhibitors do. MET kinase assay showed clear concentration-dependent inhibitory effects of berberine against MET activity, confirming its kinase inhibitory activity. These findings collectively suggest that berberine can act as a naturally-existing MET inhibitor to synergize with osimertinib in overcoming osimertinib acquired resistance caused by MET amplification.

Keywords: Berberine; EGFR; Lung cancer; MET; Osimertinib; Resistance.

Fig. 1.. Berberine and osimertinib combination synergistically decreases the survival of osimertinib-resistant NSCLC cell lines (A-C) with MET amplification (D-F).

A and B, The indicated cell lines were seeded in 96-well plates and treated on the second day with varied concentrations of osimertinib (Osim) alone, berberine (BBR) alone and their respective combinations. After 3 days, cell numbers were determined with the SRB assay. The data are means ± SDs of four replicate determinations. The numbers inside the graphs are CIs. C, The indicated cell lines seeded in 12-well plates were treated with DMSO, 100 nM osimertinib, 5 μM berberine or the combination of osimertinib and berberine; these treatments were repeated with fresh medium every 3 days. After 10 days, the cells were then fixed and stained with crystal violet dye. Columns are means ± SDs of triplicate determinations. D and E, The indicated cell lines were harvested for preparation of whole-cell protein lysates and then used for Western blotting to detect the proteins of interest. SE, shorter exposure. F, qPCR was used to detect MET gene copy numbers in the given cell lines. The data are means ± SEs of two experiments. *, P < 0.05, **, P < 0.01 and ***, P < 0.001 compared with parental cell line.

Fig. 2.. The combination of berberine and osimertinib augments induction of apoptosis in MET-amplified osimertinib-resistant cells.

The indicated cell lines were treated with DMSO, 200 nM osimertinib (Osim), 100 μM berberine (BBR) or their combination (Comb) for 48 h. Apoptosis was detected with Western blotting for protein cleavage (A) and with flow cytometry for annexin V-positive cells (B). Data in each group are means ± SDs of duplicate determinations.

Fig. 3.. The combination of berberine and osimertinib augments Bim elevation and Mcl-1 reduction (A) through modulating their degradation rates (B and C), which critically mediates the enhanced apoptosis by the combination (D-G), in MET-amplified osimertinib-resistant cells.

A, HCC827/AR were treated with DMSO, 200 nM osimertinib (Osim), 100 μM berberine (BBR) or their combination (Comb) for varied times as indicated. Western blotting was used to detect the proteins of interest. B and C, HCC827/AR cells were exposed to DMSO, 200 nM Osimertinib, 100 μM berberine or the combination of osimertinib and berberine for 12 h followed by the addition of 10 μg/ml CHX. The cells were then harvested at the indicated times for preparation of whole cell-protein lysates and subsequent detection of the tested proteins by Western blotting. NIH image J software was used to quantify band intensities. Bim and Mcl-1 levels are shown as percentage of levels at 0 time post CHX treatments. D-G, The indicated cell lines with Bim knockout (D and E) and Mcl-1 overexpression (F and G) were exposed to DMSO, 200 nM osimertinib, 100 μM berberine or osimertinib plus berberine for 48 h. The cells were then harvested for preparation of whole-cell lysates and subsequent Western blotting to detect the indicated proteins (D and F) and for detection of apoptotic cells with annexin V/flow cytometry (E and G). The data are means ± SDs of duplicate determinations.

Fig. 4.. Berberine and osimertinib combination exerts enhanced inhibitory activity against the growth of MET-amplified osimertinib-resistant tumors in vivo (A-C) with good tolerability (D) and altered levels of cleaved PARP, Bim and Mcl-1 (E).

HCC827/AR cells grown in NU/NU mice as xenografted tumors were treated with vehicle, osimertinib (Osim) alone (5 mg/kg/day, og), berberine (BBR) alone (25 mg/kg/day, ip) or the combination (Comb) of osimertinib with berberine. Tumor sizes and mouse body weights were measured at the indicated time points (A and D). At the end of treatments, tumors in each group were also collected, weighed (B) and photographed (C). The data in each group are means ± SEs of 5 tumors from 5 mice. E, IHC staining of Bim, Mcl-1 and cleaved PARP in sections of indicated tumor xenografts. Scale bars: 50 μm.

Fig. 5.. Berberine and osimertinib combination enhances suppression of MET and its downstream signaling in MET-amplified osimertinib-resistant cells (A), which are relatively more sensitive to berberine (B).

A, HCC827/AR cells were treated with DMSO, 200 nM osimertinib (Osim), 100 μM berberine (BBR) or their combination for varied times as indicated, then harvested for preparation of whole-cell protein lysates and subsequent Western blotting to detect the indicated proteins. B, The indicated cell lines seeded in 96-well plates were exposed to varied concentrations of berberine for 72 h. Cell numbers were determined with the SRB assay. The data are means ± SDs of four replicate determinations.

Fig. 6.. Berberine can potentially bind to MET in a docking analysis.

A, Chemical structures of berberine and other known MET inhibitors. B, Berberine docking into MET kinase domain. H-bond: purple dashed line; π-π and π-cation: blue dashed lines; Berberine: magenta sticks; Water molecules: red spheres. C, Interaction diagram of Berberine docking pose. H-bond: purple line; π-π: green line; π-cation: red line. D, Binding site of the MET inhibitor, PF-04217903. H-bond: purple dashed line; π-π: blue dashed lines; PF-04217903: green sticks; Water molecules: red spheres. E, Interaction diagram of PF-04217903. F. Overlay of Berberine docking pose with MET protein co-crystal structures (PDB ID: 3ZXZ). Berberine: magenta; PF-04217903: green. G, Berberine docking pose and PF-04217903 overlay at the binding pocket of MET kinase domain in surface representation. Berberine: magenta; PF-04217903: green.

Fig. 7.. Berberine inhibits MET kinase activity (A) and HGF-induced activation of MET and its downstream signaling (B).

A, MET kinase assay was determined under the conditions indicated in the absence and presence of different concentrations of berberine and given MET inhibitors. B, HCC827 and HCC827/AR cell lines were pre-treated with 10 ng HGF for 2 h and then co-treated with DMSO or 100 μM berberine for another 8 h. Whole cell-protein lysates were prepared, and Western blot analysis was used to detect the indicated proteins.