Table 1 The summary of preclinical (in vitro) use of metformin in CRC models

HT29 cells

Concentration-dependent anti-proliferative of metformin (2.5–20 mM, 72 h) that inhibits HT-29 growth by activating the AMPK (phospho-AMPKα; Thr172).

Metformin (10, 25, and 50 mM) inhibits cell growth in concentration- and time-(24 and 48 h) dependent manner by inducing apoptosis and autophagy (increased expression of APAF-1, caspase-3, PARP, and Map-LC3) through oxidative stress (inactivation NRF-2 and activation NF-κB in HT29 cells.

SW620 cells

Metformin (1–10 mmol/L, 72 h) suppresses proliferation in both concentration- and time-dependent manner via arresting the G₀/G₁ phase.

Metformin (5 mM, 2 h) induces apoptosis in hypoxic SW620 cells and enhanced with co-treatment of (E)-4-((2-(3-oxopop-1-enyl)phenoxy)methyl) pyridinium malonic acid

Metformin in combination with 5-FU significantly enhances antiproliferative, apoptosis, and cell-cycle arrestment in SW620 cells.

Organoid models from peritoneal metastases of CRC patients

Combination of metformin (5 mM for 120 h) with 4-IPP (100 μM, 24 h) synergistically promotes apoptosis by activating AMPK that reduces ribosomal protein S6 and p4EBP-1 activity that depolarizes mitochondrial respiratory chain complex I.

CaCo2 cells

Metformin (5–100 mM, 48 h) significantly decreases cell viability (up to 96% reduction) and edits the methylation status of RASSF1A which causes cellular apoptosis, cell cycle arrestment, and cell migration.

Human LoVo and mouse MCA38 cells

Metformin (10 μg/mL) alone and in combination with adinopectin (20 μg/mL) for 24 h suppresses IL-1β-induced malignant potential via STAT3 and AMPK/LKB1 signaling pathways. Co-administration with IL-1β increases the Sub-G1 population and decreases the G1 and/or S population by modulating cyclin E2, p21, and p27 expression.

COLO 205 cells

Combination of metformin (10 mM) with silibinin (100 mM) demonstrates a better antiproliferative activity as compared to either metformin (20 mM) or silibinin (200 mM) alone without any cytotoxic effects on the normal HCoEpiC.

HCT116 cells

Low concentration (60 μM) in combination with genistein (2 μM) and lunasin (2 μM) increases PTEN expression, inhibits cancer stem cell-likecells CD133⁺CD44⁺ subpopulation, and reduces FASN expression.

Metformin (5–20 mM) synergistically (with 5-FU and oxaliplatin); known as FuOx; 200 μM 5-FU and 5 μM oxaliplatin) induces cell death, inhibits colonospheres formation, enhances colonospheres disintegration, and suppresses CRC cell migration. FuOx combination inactivates Akt with increased miRNA145 (tumor suppressive) and reduction in miRNA 21 (oncogenic) expression. Additionally Wnt/β-catenin signaling pathway and transcriptional activity of TCF/LEF, β-catenin as well as c-myc expression were inhibited in HCT-116 cells.

Metformin (5 mM) and 5-FU (25 μM) enhances antiproliferative and migration through the silencing of miR-21 expression that increases the Sprouty2.

Metformin (1–10 mM, 24–48 h) induces clonogenic cell death in both wild-type p53 HCT-116 (HCT116 p53⁺/⁺) and p53-deficient HCT-116 cells (HCT116 p53⁻/⁻) and augments radio-sensitization towards IR in HCT116 p53⁻/⁻ cells.

Metformin (10 mM) suppresses LCA (30 μM)-oxidative stress by inactivating NF-κB and downregulating IL-8. Metformin-treated conditioned media inhibits of HUVECendothelial cell proliferation and tube-like formation. .

Metformin (1–4 mM, 24–72 h) reduces EMT in HCT116 sphere cells via inactivation of Wnt3α/β-catenin signaling (with reduction of Vimentin and increased epithelial marker). Consequently, metformin promotes sensitization of HCT116 sphere cells towards 5-FU treatment (25 μg/mL).

Caco-2 and HCT116 cells

Addition of metformin to 5-ASA (48 h) inhibits the Caco-2 (13 mM of metformin and 2.5 mM of 5-ASA) and HCT-116 cells proliferation (13 mM of metformin and 2.5 mM of 5-ASA) and induces apoptosis by inducing oxidative stress and NF-κB inflammatory responses.

DLD-1, HT-29, Colo205 and HCT116

Metformin (2.5–10 mM) did not decrease the cell viability but sensitizes the cells towards TRAIL (50 ng/mL) that is followed with induction of extrinsic and intrinsic apoptosis through the suppression of Mcl-1 by promoting the dissociation of Noxa from Mcl-1 that activates E3 ligase Mule.

HT-29, SW620, and HCT116 cells

Metformin addition to sirolimus synergistically promotes the reduction cell viability (48 h) via downregulation of p-mTOR, p-70S6K, p-4EBP1, livin, survivin, E-cadherin, TGF-β, and pSmad3.

HT-29 and HCT116 cells

Single exposure (24 h) either 1,25D3 (10–1000 nM) or metformin (1–20 mM) reduces the cell viability in HCT116 (p53 wild-type), HCT116 (p53⁻/⁻), and HT-29 (p53 mutant). Both 1,25D3 and metformin synergistically promotes apoptosis, and autophagy irrespective of the p53 status in all of the cells tested via AMPK, intracellular ROS, Bcl-2, and increasing LC3II:LC3I ratio. Additionally, metformin addition in the combination treatment arrests cell cycle in G₂/M phase (HCT116 p53⁻/⁻) and S phase (HT-29 cells).

In a different report, metformin at 1 mM (24 h) increases the sensitization of HT29 cells to oxaliplatin (R = 2.66, P < 0.01) but no in HCT116 cells

DLD-1 cells

Metformin (5 mM, 24 h) synergistically promotes oxaliplatin (12.5 μM) cytotoxic and anti-proliferative b increasing HMGB1 expression via Akt and ERK1/2.

Metformin activates AMPK signaling at lower concentration and short time exposure (0.5–2 μM, 1 h) prior to radiation leads to radioresistance.

SW-480 and HT-29

Pretreatment with metformin (2 mM, 16 h) activates AMPK signaling that inhibits the phosphorylation of β-catenin and Akt (Ser473) induced by insulin (10 ng/mL)or IGF-1 (10 ng/mL).

HCT116, RKO and HT-29 cells

Metformin (1 and 5 mM, 24 h) did not inhibit the proliferation and daily treatment (5 mM, 2 weeks) did not suppress the anchorage-independent growth, apoptosis, autophagy, and cell cycle arrest.