Anti-neoplastic Activity Of Metformin In Hypopharyngeal Carcinoma Fadu Cells And Its Effect On Chemotherapy Drugs

Objective: To investigate the role of metformin on the proliferation，cell-cycle and its effects on chechemotherapeutic agents cisplatin,5-fluorouracil or paclitaxel in hypopharyngeal carcinoma Fadu cells.Supplying a new drug for the medication of patients who with diabetescomplicating hypopharynx cancer.Materials and Methods:1Fadu cells were treated with different concentrations of metformin (0,2mmol/L,5mmol/L,10mmol/L,20mmol/L and40mmol/L) for24h,48h,72h.MTT assay was used to evaluate the influence of metformin on theproliferation of Fadu cells and cell-cycle was detected by flow cytometry.2Fadu cells were cultured in the medium containing5mmol/L metforminor10mmol/L for24h and48h, the expression of AMPK and P21were testedby immunocytochemistry.3Cells were treated with cisplatin (0,0.1μg/ml,0.5μg/ml,2μg/ml and4μg/ml),5-fluorouracil (0,4μg/ml,20μg/ml,100μg/ml and500μg/ml) orpaclitaxel (0,1ng/ml,10ng/ml,100ng/ml,1000ng/ml) in vitro together withor without metformin5mmol/L. Cell growth was examined using MTT assayfollowing the manufacturer’s protocol at the time of24,48and72hours aftertreatment.Results:1Metformin induced significant proliferation inhibition on Fadu cells ina dose-and time-dependent manner. Multiple factor ANOVA analysis showedthat both the concentration and the action time of metformin make a differenceto the proliferation of cells (Fc=97.411, P<0.01; Ft=323.251, P<0.01). IC50was15.036mmol/L when Fadu cells treated with metformin for48h. We detected an cell cycle arrest in G1phase after the treatment of metformin byFlow cytometry in Fadu cells, which is also presentation in a dose-andtime-dependent manner (P<0.01).2Immunocytochemical results showed that protein levels of both AMPKand P21in cells intervened with metformin are higher than that without.Correlation analysis showed that there was a positive correlation between theexpression of AMPK and P21.3The MTT results of the proliferation of Fadu cells intervened inchemotherapeutics with or without metformin:(1) The inhibition ofproliferation of cells cultured with different concentration of cisplatincombined with5mmol/L metformin for24h,48h and72h was higher thanwith cisplatin alone (all P<0.01). Calculating the CI of cisplatin andmetformin for48h, we worked out the value was0.43. It indicates that thecombination of cisplatin and metformin possess a high-synergism in Faducells.(2) Compared to the5-fluorouracil alone, the cells’ survival rates at24hand48h showed a slight suppression at the lower concentrations of5-fluorouracil (4μg/ml and20μg/ml) when united with5mmol/L metformin.However, this phenomenon reversed at the point of72h. The highestconcentration of5-fluorouracil (500μg/ml) displayed a similar reverse at anytime in our study. The value of CI was1.15, we deemed that metformin had anantagonism to5-fluorouracil in Fadu cells.(3) In the experiment for thecomparison of paclitaxel combined with metformin, we discovered that thecombination degraded the survival rates of Fadu cells at any time andconcentration except for concentration of1000ng/ml at72h. Multiple factorANOVA analysis showed there has a signification diference between thepaclitaxel alone or to united with metformin (P<0.01). The CI value was0.37,the metformin could potentiate the anti-neoplastic activity of paclitaxel.Conclusions:1Metformin was found to inhibit the proliferation of Fadu cells, whichwas associated with the cell cycle arrest of G1phase. Further more, the rolesplayed, at least, partially through the correlative signal molecules of AMPK and P21.2Metformin potentiates the sensitivity of cisplatin and paclitaxel in Faducells, but antagonized the effective of5-fluorouracil. Our results suggest thatmetformin may be a sensitizer to combined with cisplatin or paclitaxel, but itscombination with5-fluorouracil might not be as valuable in clinical.