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. 2017 May 11;8(34):56546-56557.
doi: 10.18632/oncotarget.17798. eCollection 2017 Aug 22.

Metformin increases chemo-sensitivity via gene downregulation encoding DNA replication proteins in 5-Fu resistant colorectal cancer cells

Sung-Hee Kim  1 , Soon-Chan Kim  2 , Ja-Lok Ku  1   2
Affiliations

Metformin increases chemo-sensitivity via gene downregulation encoding DNA replication proteins in 5-Fu resistant colorectal cancer cells

Sung-Hee Kim et al. Oncotarget. .

Abstract

Metformin is most widely prescribed for type 2 diabetes. Recently, evidences have shown that metformin has anticancer effects on pancreatic-, colorectal-, ovarian-, and other cancers. Because metformin has less adverse effects and is inexpensive, it could be a useful chemo-therapeutic agent with anticancer effects. In this study, we demonstrated metformin inhibited by cell proliferation, cell migration ability, clonogenic ability, and cancer stem cell population. Metformin also induced cell cycle arrest in parental-(SNU-C5), and 5-Fu resistant-colorectal cancer cell line (SNU-C5_5FuR). Moreover, a treatment that combines 5-Fu and metformin was found to have a synergistic effect on the cell proliferation rate, especially in SNU-C5_5FuR, which was mediated by the activation of AMPK pathway and NF-ƙB pathway, well-known metformin mechanisms. In this study, we suggested novel anticancer mechanism of metformin that inhibited DNA replication machinery, such as the MCM family in SNU-C5_5FuR. In conclusion, we provided that how metformin acts as not only a chemo-sensitizer, but also as a synergistic effector of 5-Fu in the 5-Fu resistant-cell line. We speculate that metformin used for adjuvant therapy is effective on 5-Fu resistant cancer cells.

Keywords: 5-Fu resistant-cell line; DNA replication; cancer stem cell (CSC); colorectal cancer; metformin.

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Conflict of interest statement

CONFLICTS OF INTEREST We confirm that all authors have no conflict of interest. All authors have seen and approved the manuscript.

Figures

Figure 1
Figure 1. The relative cell proliferation rate as treated with 5-Fu, metformin, and combination 5-Fu with metformin
Metformin only treated with serial dilution from 100 mM (A) 5-Fu only or both 5-Fu and 10 mM Metformin (B). 5-Fu was treated with serial dilution from 10 μg/mL. The cell proliferation rate was confirmed after a 72 h treatment of drugs using Ez-Cytox, which assessed NADH-dehydrogenase in live cells. (C) represents the combination index (CI) that is calculated using Compusyn (http://www.combosyn.com/). CI < 1 means the synergistic effect of combination 5-Fu and metformin treatment, CI = 1 is an additive effect, and CI > 1 is the antagonistic effect. The assay was performed three times. P is the parental cell line, SNU-C5 and R are the resistant cell lines, SNU-C5_5FuR and 5-Fu are the serial dilution of 5-Fu, and Met is the metformin.
Figure 2
Figure 2
The expression levels of apoptotic pathway proteins as treated with various doses of metformin (A) and combination 1 μg/mL of 5-Fu and 50 mM of metformin treatment (B) by western blotting. The arrow head indicates the active form of PARP or caspase-3. Apoptotic cell death detected by Annexin V/PE staining as treated with 0.5 μg/mL of 5-Fu and 10 mM of metformin (C). (D) represents the percentage of Annexin V-positive population (*p < 0.05).
Figure 3
Figure 3. Cell cycle analysis of SNU-C5 and SNU-C5_5FuR when treated with 1 μg/mL of 5-Fu and 50 mM of metformin as well as combination 5-Fu and metformin treatment
The bar graphs indicate the changes in the cell cycle progression (A) and raw data of cell cycle distribution in SNU-C5_5FuR cell lines (B). The assay was performed three times.
Figure 4
Figure 4. Metformin affected wound healing capacity and clonogenicity
The wound healing assay and clonogenic assay were performed by 0.5 μg/mL of 5-Fu and 10 mM of metformin as well as combination 5-Fu and metformin treatment. For the migration assay, 5000 cells/well were seeded, wounded, and then treated with PBS (as control), 5-Fu, and metformin. The wound was observed at 0, 6, 24, 48, and 72 h. (A) represents the taken phase-contrast picture images at 0 and 48 h. (B) shows the calculated cell migration where the black closed circle is control, open circle is 5-Fu treatment, closed square is metformin, and open square is combination treatment. For clonogenic assay, 0.5 × 103 cells are pre-treated by 5-Fu w/ or w/o metformin and seeded in a 60 mm dish. After 14 days, the colonies are counted by staining with crystal violet. The experiments are performed three times (*p < 0.05). (C and D) represent the number of SNU-C5 and SNU-C5_5FuR coloines, respectively (*p < 0.05). (E) shows the picture images of those colonies. The assay was performed three times.
Figure 5
Figure 5. Metformin on AMPKα/mTOR axis pathway and NF-ƙB pathway effector proteins
AMPK/mTOR signaling pathway is well-known mechanism of metformin. Total AMPK and mTOR, and their phosphorylation form were determined by western blot analysis when treated with 5-Fu and/or metformin. Also, NF-ƙB and angiogenesis related related proteins, HIF-1α, VEGF were confirmed. β-tubulin was used an a internal control.
Figure 6
Figure 6
Cancer stem cell marker (CD133) expression levels treated with 0.5 μg/mL of 5-Fu and 10 mM of metformin by RT-PCR (A) quantitative-RT-PCR (B) western blot analysis (C) and FACs analysis (D) (*p < 0.05). Quantitative RT-PCR data was normalized by β-actin expression level and then calculated ddCt value using 7300 System SDS v1.4. Software. Western blot band intensities were calculated by ImageJ (the graph) in the lower bar graph. After 3D culture for tumor sphere formation with RGF-BME, microscopical analysis (magnification × 250, Scale Bar = 50 μm) (E). (F and G) represent relative diameter of tumor sphere measured using Image J (*p < 0.05). All of the experiments were performed three times.
Figure 7
Figure 7. Metformin reduced the expression level of DNA replication machinery and mitotic cell cycle genes
As a result of RNA seq., DNA replication (KEGG: 03030) (black arrows in A and B), and mitotic cell cycle (KEGG: 04110) (white arrows in A and B) genes were downregulated when treated with metformin. A is the gene correction by False Discovery Rate (FDR) and B is by Bonferroni correction. All the genes from DNA replication pathway (C) and mitotic cell cycle pathway (D) were represented in terms of their measured fold change (y-axis) and accumulation (x-axis). The related protein level was detected by western blot analysis (E).
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