Metformin as an Anticancer Agent in Oral Cancer Therapy by Regulating Tumor Associated Macrophage

Objectives: Growing evidence suggests that metformin possesses anticarcinogenic. In oral cancer, the use of metformin in clinical implication and biologic significance is rare. Recent studies indicated that the cancer microenvironment is important for tumor progression. Among this microenvironment, tumor-associated macrophages (TAMs) are the major component in tumor. Previously, our study had demonstrated that Axl signal involved in the crosstalk between oral cancer cells and TAMs. Interestingly, metformin also been reported to regulate Axl expression. However little is known about the effect of metformin on the Axl expression in the crosstalk between tumor and TAMs..The aim of this study is to investigate the role of metformin in the determination of M1 versus M2 polarization by Axl signal pathway.
Methods: Cell Culture
THP-1 and oral cancer cells were maintained in RPMI-1640 medium supplemented with 10% FBS under 5% CO 2 at 37°C .
Flow Cytometry
The THP-1 cells were incubation with medium or oral cancer CM solution. For surface markers, cells were incubated with
FITC-CD16/PE-CD206 antibody. Labeled cells were analyzed by flow cytometry.
q-PCR
Total RNA was isolated from THP-1 cells using Trizol. Complementary DNA synthesis was performed from 2 μg
of the extracted total RNA. Then we performed real-time PCR with gene specific primers and SYBR green dye
ELISA
The THP-1 cells were incubation with medium or oral cancer CM solution. After incubation, the supernatants were
collected, followed by the determination of human IL-10, IL-12, TNF-α, TGF-β by ELISA kit.
Results: Metformin treatment could promote M2 polarization of macrophage (IL-10, TGF-β, CD206 were increased and IL-12, TNF-α, CD16 were decreased) (P<0.05). Notably, inhibition of Axl signaling markedly reduced M2 polarization of macrophage (IL-10, TGF-β, CD206 were decreased and IL-12, TNF-α, CD16 were increased) (P<0.05). These data indicated that Metformin treatment might induce M2 polarization by Axl signaling signaling.
Conclusions: Through above mentioned experiments, we will clarify the function of metformin for the use in oral cancer prevention and treatment, and then illustrate metformin in modulating inflammatory process and the relevance between macrophage polarization and the antitumor effect.