Berberine regulates the Notch1/PTEN/PI3K/AKT/mTOR pathway and acts synergistically with 17-AAG and SAHA in SW480 colon cancer cells

Context: Berberine (BBR) is used to treat diarrhoea and gastroenteritis in the clinic. It was found to have anticolon Cancer effects.

Objective: To study the anticolon Cancer mechanism of BBR by connectivity map (CMAP) analysis.

Materials and methods: CMAP based mechanistic prediction was conducted by comparing gene expression profiles of 10 μM BBR treated MCF-7 cells with that of clinical drugs such as helveticoside, ianatoside C, pyrvinium, gossypol and trifluoperazine. The treatment time was 12 h and two biological replications were performed. The DMSO-treated cells were selected as a control. The interaction between 100 μM BBR and target protein was measured by cellular thermal shift assay. The protein expression of 1-9 μM BBR treated SW480 cells were measured by WB assay. Apoptosis, cell cycle arrest, mitochondrial membrane potential (MMP) of 1-9 μM BBR treated SW480 cells were measured by flow cytometry and Hoechst 33342 staining methods.

Results: CMAP analysis found 14 HSP90, HDAC, PI3K or mTOR protein inhibitors have similar functions with BBR. The experiments showed that BBR inhibited SW480 cells proliferation with IC₅₀ of 3.436 μM, induced Apoptosis, autophage, MMP depolarization and arrested G1 phase of cell cycle at 1.0 μM. BBR dose-dependently up-regulated PTEN, while inhibited Notch1, PI3K, Akt and mTOR proteins at 1.0-9.0 μM (p < 0.05). BBR also acted synergistically with HSP90 and HDAC Inhibitor (0.01 μM) in SW480 cells at 0.5 and 1.0 μM.

Discussion and conclusions: The integrative gene expression-based chemical genomic method using CMAP analysis may be applicable for mechanistic studies of other multi-targets drugs.