Z. Bostanci2, E. S. Han1, J. Yan2, Q. Xing2, W. Wen2, J. H. Yim2 1City Of Hope National Medical Center,Department Of Surgery, Division Of Gynecologic Oncology,Duarte, CA, USA 2City Of Hope National Medical Center,Department Of Surgery, Division Of Surgical Oncology,Duarte, CA, USA
Introduction: Baicalein is a flavone with anti-cancer properties found naturally in thyme that upregulates DNA-Damage-Inducible-Transcript 4 (DDIT4) which in turn inhibits the mTOR pathway by acting on the TSC1/2 complex as we have previously demonstrated. Metformin activates AMP-activated protein kinase (AMPK) which also acts on the TSC1/2 complex, resulting in similar anti-cancer properties found with mTOR inhibition. This is the purported mechanism for metformin anti-cancer activity for which there are numerous clinical trials in multiple cancers, including breast cancer, to assess its clinical utility. Because baicalein and metformin appear to target the TSC1/2 complex by different mechanisms we hypothesized that baicelein and metformin will have synergistic anti-proliferative effects which may allow for enhanced anti-cancer effects in patients.
Methods: MDA468 (ER-/PR-/Her2-), MDA231(ER-/PR-/Her2-), SKBR3 (ER-/PR-, Her2+) cell lines were treated with vehicle or baicalein only (2.5 µM -40 µM), metformin only (0.625 mM -10 mM) and combinations of baicalein and metformin. Cell proliferation was measured with MTT assay and Chou Talalay Plots were performed depicting synergism at a combination index (CI) below 1. Western Blot was used to assess protein expression of DDIT4, pAMPK and pS6 as direct and downstream targets of baicalein and metformin on the mTOR pathway.
Results: Cell proliferation decreased in all cell lines when baicalein and metformin were used separately and in combination in a dose dependent manner. The combination of baicalein and metformin had a synergistic (CI<1) anti-proliferative effect on MDA231 and SKBR3 cells at 72 hours at every dose combination. In MDA468 cells, synergistic effect (CI<1) was present at higher doses at 72 hours. Western blotting in separate experiments with MDA468 cells showed that there was an increase in DDIT4 by baicalein and an increase in pAMPK by metformin, for which the mTOR end target pS6 was markedly more suppressed by the combination than either alone.
Conclusion: Baicalein is present in the herb thyme and is an active component of the Traditional Asian Medicine (TAM) extract Huang Qin and has been tolerated well. Metformin is an inexpensive medication that has been commonly used to treat diabetes and is being studied in numerous clinical trials as an anti-cancer agent. Our study shows that targeting both AMPK and DDIT4 results in synergistic suppression of breast cancer cell growth and enhanced mTOR inhibition. The combination of baicalein and metformin or other compounds that activate AMPK may offer a therapeutic alternative or an adjunct to current anti-cancer agents in aggressive breast cancers.
