Fig 1.
Metformin does not inhibit growth of PDX tumors.
No significant growth inhibition was observed in four different pancreatic cancer PDX tumor lines at any time point during a 28 day treatment course with 200 mg/kg or 400 mg/kg metformin administered by daily oral gavage.
Fig 2.
Activation of AMPK and inhibition of p70S6K phosphorylation in PDX tumors is not sustained after 28 days of metformin treatment.
Phosphorylation of AMPKα and p70S6K in (A) P505 and (B) P710 PDX tumors after 28 day treatment with 400 mg/kg metformin. Phosphorylation of AMPKα and p70S6K in (C) P505 and (D) P710 PDX tumors after 3 day treatment with 400 mg/kg metformin (*p<0.05).
Fig 3.
Metformin inhibits growth and alters AMPK and mTOR signaling in pancreatic cancer cell lines.
(A) Cells were plated in quadruplicate into 96-well plates at a density of 5x103 per well, incubated overnight, and then treated with media containing either PBS as a vehicle control or different concentrations of metformin (0–5 mM). After 48 hours, proliferation indices were determined using the MTT assay and normalized to those of the vehicle-treated cells. All assays were performed in triplicate. (B) Phosphorylation of AMPKα and total AMPKα at various time points after treatment with 5 mM metformin. Treatment began at 0 hours (hrs). (C) Phosphorylation of mTOR, p70S6K, and 4E-BP1 at various time points after treatment with 5 mM metformin. (D) Densitometry of phosphorylated AMPKα, mTOR, p70S6K, and 4E-BP1 relative to total levels shown in (B) and (C).
Fig 4.
AMPK is only partially required for the anti-proliferative effects of metformin.
(A) shRNA knockdown of AMPKα subunits in CFPAC-1 and HPAF-II cell lines. (B) Proliferation of CFPAC-1 and HPAF-II cell lines with stable knockdown of AMPKα subunits after treatment with different concentrations of metformin (0–5 mM). (C) Phosphorylation of mTOR and p70S6K in CFPAC-1 and HPAF-II cell lines with stable knockdown of AMPKα subunits.
Fig 5.
mTOR expression is sufficient to promote resistance to metformin in cell lines, but combinatorial treatment with metformin and mTOR inhibitors does not produce synergy.
(A) Phosphorylation of p70S6K in and (B) proliferation of HPAF-II cells after treatment with 5 mM metformin following transient expression of a transfected myc-mTOR construct. Using the median effect equation calculation for combination index (CI) following 3 day treatment with constant-ratio doses of metformin and either (C) the allosteric mTOR inhibitor rapamycin or (D) the catalytic mTOR inhibitor BEZ235 failed to produce synergy (CI<1) at any dose combination. CI values at 50% growth inhibition: (C) CFPAC-1 1.54, HPAF-II 1.43; (D) CFPAC-1 1.30, HPAF-II 1.29. (*p<0.050, **p<0.005, ***p<0.001).