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BACKGROUND
Metformin, one of the most commonly used medications for treatment of type 2 diabetes, has emerged as a potential anticancer agent. The molecular mechanisms associated with the antitumor effect of metformin are still poorly understood. In this study, we show that metformin represses cancer cells via alternate pathways in N-Cadherin Wild type and N-Cadherin deficient cells.
METHODS
Cell viability and apoptosis were determined by MTT, TUNEL and FACS assays, respectively. Western blot and immunofluoresence staining were performed to evaluate protein expression. Various stable cell lines were generated in vitro and then injected into both flanks of nude mice to generate xenografts. Mice were treated with i.p. doxorubicin (every 5 days × 4 cycles with 4 mg/kg) or p.o.metformin (200 ug/ml, diluted in the drinking water). Xenograft tumor volume was measured every 5 days.
RESULTS
We found that metformin represses N-cadherin, independent of AMPK, in wild-type N-cadherin cancer cells. Metformin down-regulated N-cadherin in a dose and time- dependent manner, comparable to the N-cadherin specific antagonist, GC-4. Ectopic expression of N-cadherin rendered cells resistant to metformin. Silencing or over-expression of AMPK did not alter sensitivity of cancer cells to metformin in N-cadherin wild-type cells. In metformin-resistant cancer cells, N-cadherin level did not change with metformin treatment. Suppression of N-cadherin changed the phenotype of metformin-resistant to metformin-sensitive cells. We also found that NF-kB is a downstream molecule of N-cadherin and metformin regulated NF-kB signaling via suppression of N-cadherin, rather than direct modulation of NF-kB. Silencing of NF-kB in resistant cells sensitized the cells to metformin. We also found that metformin down-regulated N-cadherin and TWIST1 concurrently. Ectopic expression of TWIST1 regulated N-cadherin/NF-kB signaling and made cells more resistant to metformin. Suppression of TWIST1 sensitized resistant cells to metformin. Metformin inhibited growth of subcutaneously implanted xenografts. However, xenografts with high N-cadherin levels were resistant to metformin, while suppression of N-cadherin sensitized tumors to metformin.
In N-cadherin deficient cancer cells, metformin played an antitumor role in a different manner by activation of AMPK. Suppression of AMPK in N-cadherin deficient cells changed the cells to a more metformin-resistant phenotype. In addition, ectopic expression of N-cadherin attenuated AMPK’s ability to suppress NF-kB, leading to a more metformin resistant phenotype. Compound C, a specific AMPK inhibitor, masked the therapeutic effects of metformin. Similar to the in vitro findings, suppression of AMPK in xenografts rendered tumors resistant to metformin.
CONCLUSIONS:
We suggest that metformin’s anti-cancer therapeutic effect in N-cadherin wild-type cells may be mediated through repression of the TWIST/N-cadherin/NF-kB signaling pathway, independent of AMPK. However, in N-cadherin deficient cells, metformin plays an antitumor role via activating AMPK. Elucidating the molecular pathways responsible for metformin’s anti-cancer effect may help delineate its role for cancer therapies.