Berberine and cinnamaldehyde together prevent lung carcinogenesis

Abstract

Starving tumor cells by restricting nutrient sources is a promising strategy for combating cancer. Because both berberine and cinnamaldehyde can activate AMP-activated protein kinase (AMPK, a sensor of cellular energy status), we investigated whether the combination of berberine and cinnamaldehyde could synergistically prevent lung carcinogenesis through tumor cell starvation. Urethane treatment induced lung carcinogenesis in mice, downregulated AMPK and mammalian target of rapamycin (mTOR) while upregulating aquaporin-1 (AQP-1) and nuclear factor kappa B (NF-κB). Together, berberine and cinnamaldehyde reduced mouse susceptibility to urethane-induced lung carcinogenesis, and reversed the urethane-induced AMPK, mTOR, AQP-1, and NF-κB expression patterns. In vitro, berberine and cinnamaldehyde together induced A549 cell apoptosis, prevented cell proliferation, autophagy, and wound healing, upregulated AMPK, and downregulated AQP-1. The effects of the combined treatment were reduced by rapamycin (a mTOR inhibitor) or HgCL₂ (an AQP inhibitor), but not Z-VAD-FMK (a caspase inhibitor). The berberine/cinnamaldehyde combination also prevented A549 cell substance permeability and decreased intracellular ATP concentrations. These results suggest the combination of berberine and cinnamaldehyde limited both primary and adaptive nutrient acquisition by lung tumors via AMPK-reduced AQP-1 expression, which ultimately starved the tumor cells.

Keywords: AMPK; AQP-1; berberine; cinnamaldehyde; lung cancer.

Figure 1. Berberine and cinnamaldehyde together prevented urethane-induced lung carcinogenesis

Body weight at the beginning (d 1) and end (26 weeks) of the experiment (A). Lung carcinoma incidence and number (B) and multiplicity (average neoplastic nodules per lung) (C). The normal group included non-carcinogenic mice fed in parallel, and the UC group included untreated carcinogenic mice. *P < 0.05, **P < 0.01 vs UC.

Figure 2. Berberine and cinnamaldehyde together decreased inflammation during carcinogenesis

Berberine and cinnamaldehyde together or JTW decreased inflammation as indicated by increased serum IL-1 (A), IL-6 (B), hs-CRP (C), and TNF-α (D) levels (n = 5). *P < 0.05, **P < 0.01 vs UC.

Figure 3. Berberine and cinnamaldehyde together regulated immunity during carcinogenesis

Berberine and cinnamaldehyde together or JTW regulated immunity as indicated by serum levels of the type 1 immunostimulatory cytokines, IFN-γ (A) and IL-2 (B), and type 2 immunoinhibitory cytokines, IL-4 (C) and IL-10 (D) (n = 5). *P < 0.05, **P < 0.01 vs UC.

Figure 4. Berberine and cinnamaldehyde together decreased oxidative stress and lung epithelium integrity

Berberine and cinnamaldehyde together decreased oxidative stress as indicated by increased serum 8-OHdG (A) and ROS (B) levels, and lung epithelium integrity as indicated by evans blue permeability (C) and lung water content (D) (n = 5). *P < 0.05, **P < 0.01 vs UC.

Figure 5. Berberine and cinnamaldehyde together regulated lung signaling protein expression during carcinogenesis

Berberine and cinnamaldehyde together regulated AMPK, mTOR, NF-κB, and AQP-1 expression as indicated by western blotting (A), and promoted lung epithelial cell proliferation as indicated PCNA, EMT, and N-cadherin upregulation and E-cadherin downregulation as shown by IHC (B and C). Assays were performed in triplicate. *P < 0.05, **P < 0.01 vs UC.

Figure 6. Berberine and cinnamaldehyde together suppressed A549 cell proliferation and promoted apoptosis (n = 3) (B) and (C) in a time-dependent manner (n = 6) (A) *P < 0.05, **P < 0.01 vs control

Figure 7. Berberine and cinnamaldehyde together suppressed A549 cell autophagy

Berberine and cinnamaldehyde together aborted berberine- or cinnamaldehyde-induced cell autophagy under 10% serum culture conditions (n = 3) (A). Combination therapy suppressed autophagy under 2% serum culture condition (n = 3) (B). Cells in the AO staining group were in the early stage of culture medium replacement. Cells in the LC3-B staining group were harvested at a late cell culture stage. *P < 0.05, **P < 0.01 vs control.

Figure 8. Berberine and cinnamaldehyde together suppressed wound healing in A549 cells

*P < 0.05, **P < 0.01 vs control.

Figure 9. Berberine and cinnamaldehyde together synergistically regulated AMPK/AQP-1 protein levels and prevented substance permeability in A549 cells

Berberine and cinnamaldehyde together upregulated AMPK and downregulated AQP-1 (n = 3) (A), prevented substance permeability as indicated by increased intracellular rhodamine B (n = 3) (B), and decreased intracellular ATP concentrations (n = 3) (C) *P < 0.05, **P < 0.01 vs control.