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. 2013 Jul;42(5):781-5.
doi: 10.1097/MPA.0b013e31827aec40.

Metformin Inhibits the Growth of Human Pancreatic Cancer Xenografts

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Free PMC article

Metformin Inhibits the Growth of Human Pancreatic Cancer Xenografts

Krisztina Kisfalvi et al. Pancreas. .
Free PMC article

Abstract

Objective: Pancreatic ductal adenocarcinoma is a devastating disease, with an overall 5-year survival rate of only 3% to 5%. As the current therapies offer very limited survival benefits, novel therapeutic strategies are urgently required to treat this disease. Here, we determined whether metformin administration inhibits the growth of PANC-1 and MiaPaCa-2 tumor xenografts in vivo.

Methods: Different xenograft models, including orthotopic implantation, were used to determine whether intraperitoneal or oral administration of metformin inhibits the growth of pancreatic cancer in vivo.

Results: We demonstrate that metformin given once daily intraperitoneally at various doses (50-250 mg/kg) to nude mice inhibited the growth of PANC-1 xenografts in a dose-dependent manner. A significant effect of metformin was obtained at 50 mg/kg and maximal effect at 200 mg/kg. Metformin administration also caused a significant reduction in the phosphorylation of ribosomal S6 protein and ERK in these xenografts. Metformin also inhibited the growth of pancreatic cancer xenografts when administered orally (2.5 mg/mL) either before or after tumor implantation. Importantly, oral administration of metformin also inhibited the growth of MiaPaCa-2 tumors xenografted orthotopically.

Conclusions: The studies presented here provide further evidence indicating that metformin offers a potential novel approach for pancreatic ductal adenocarcinoma prevention and therapy.

Conflict of interest statement

The authors have no conflicts of interest to disclose.

Figures

Figure 1
Figure 1. Metformin dose-dependently inhibits the rate of growth of PANC-1 tumor xenografts
Xenografts were generated by implantation of 2 × 106 cells of PANC-1 cells into the right flanks of male nu/nu mice. When the tumors reached a mean diameter of 2 mm, the animals were randomized into control and treated groups (10 mice per group). Metformin was given once daily intraperitoneally at various doses (50, 125, 200 and 250 mg/kg) for the duration of the experiment. The first day of treatment was designated as day 0. Control animals received saline. Tumor volumes were measured every 4 days as described in Materials and Methods. Insert: Representative subcutaneous tumors of control and metformin-treated (250 mg/kg), as indicated by the arrows.
Figure 2
Figure 2. Metformin decreases the final volume and the phosphorylation of S6 and ERK in the excised tumors
A. Metformin decreases tumor volume at the end of the experiment in a dose-dependent manner. On day 44 the tumors generated in Fig. 1 were removed, measured, and tumor volumes estimated as V = 0.52 (length × width × depth). The results are shown are means ± SE. [Points, mean; bars, SE (*, p < 0.05; **, p < 0.01 versus control, Student's t test]. B. Metformin decreases the phosphorylation of S6K and ERK in extracts of the excised tumors. Tumors samples of were prepared for SDS-PAGE as described in “Materials and Methods”. The samples were analyzed by SDS-PAGE and immunoblotting with the following phospho antibodies: S6 Ser235/236 (pS6), ERK1/2 Thr202 and Tyr204 (pERK). Immunoblotting with total tubulin was used to verify equal loading. Shown here are representative immunoblots (two controls and two metformin-treated mice). Similar results were obtained in 4 additional tumors Quantification was performed using Multi Gauge V3.0. Results are expressed as the ratio of the intensities of the phosphosphorylated S6 or ERK immune-reactive bands to the intensity of the tubulin band. P values were determined using the t-test (Sigma Plot 12) n=6, **, P = 0.01, * P = 0.03.
Figure 3
Figure 3. Orally administered metformin inhibits the growth of Mia PaCa-2 xenografts
Xenografts were generated by implantation of 2 × 106 cells of Mia PaCa-2 cells into the right flanks of male nu/nu mice. When the tumors reached a mean diameter of 2 mm, the animals were randomized into control and treated groups (10 mice per group). Metformin was dissolved in the drinking water in the final concentration of 2.5mg/ml. Animals were allowed to drink ad libidum and the drinking water (with or without metformin) was replenished every other day. The tumor measurements and removal were performed as described in Materials and Methods. In the inset shown are the tumor volumes after the removal of the tumors from the animals on the last day of the experiment. [Points, mean; bars, SE (*, P < 0.05; **, P < 0.01 versus control, Student's t test].
Figure 4
Figure 4. Prior administration of metformin prevents tumor development and growth in nu/nu mice
Animals received drinking water or metformin containing drinking water (2.5mg/ml, as described in Materials and Methods) for 5 days. Then, the animals were divided into groups for implantation of PANC-1 cells (either 4×105 or 106) into the right flanks in nu/nu mice (Day 0). The animals continued receiving control or metformin-treated drinking water for the duration of the experiment. The development and growth of the tumors in the flanks were measured in every 4 days, as described in Materials and Methods. On the last day of the experiment (Day 49) the animals were sacrificed, the tumors removed, measured and weighted (inset). [Points, mean; bars, SE (*, P < 0.05; **, P < 0.01 versus control, Student's t test].
Figure 5
Figure 5. Metformin inhibits the growth of MIA PaCa-2 tumor xenografts implanted in nu/nu mice orthotopically
Small (1mm3) fragments of MiaPaCa-2 tumors (developed in donor nu/nu mice) were implanted into microsurgicaly prepared tissue pockets within the parenchyma of the body/tail of the pancreas in nu/nu mice. Next day the animals were randomly selected into metformin-treated or control groups (10 animals/group). Metformin was administered orally in the drinking water (2.5 mg/kg), as in previous experiments. After 8 weeks of treatment the animals were sacrificed and the abdominal, retroperitoneal and chest cavities explored. The primary tumors in the pancreas were removed and local invasion and macroscopic dissemination into the liver, spleen, lymph nodes, kidneys, small bowel and lungs was assessed.

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