Metformin Decouples Phospholipid Metabolism in Breast Cancer Cells

Abstract

Introduction: The antidiabetic drug metformin, currently undergoing trials for cancer treatment, modulates lipid and glucose metabolism both crucial in phospholipid synthesis. Here the effect of treatment of breast tumour cells with metformin on phosphatidylcholine (PtdCho) metabolism which plays a key role in membrane synthesis and intracellular signalling has been examined.

Methods: MDA-MB-468, BT474 and SKBr3 breast cancer cell lines were treated with metformin and [3H-methyl]choline and [14C(U)]glucose incorporation and lipid accumulation determined in the presence and absence of lipase inhibitors. Activities of choline kinase (CK), CTP:phosphocholine cytidylyl transferase (CCT) and PtdCho-phospholipase C (PLC) were also measured. [3H] Radiolabelled metabolites were determined using thin layer chromatography.

Results: Metformin-treated cells exhibited decreased formation of [3H]phosphocholine but increased accumulation of [3H]choline by PtdCho. CK and PLC activities were decreased and CCT activity increased by metformin-treatment. [14C] incorporation into fatty acids was decreased and into glycerol was increased in breast cancer cells treated with metformin incubated with [14C(U)]glucose.

Conclusion: This is the first study to show that treatment of breast cancer cells with metformin induces profound changes in phospholipid metabolism.

Fig 1

Total [³H-methy]choline uptake (A), cellular phosphorylated [³H-methy]choline (B) in untreated (solid) and metformin-treated (white) breast tumour cells incubated with [³H-methyl]choline for 15min (Units: Radioactive counts normalised to protein content and expressed relative to untreated cells). Choline kinase activity in lysates of untreated (black) and metformin-treated (white) breast tumour cells (C) (Units: [³H]PCho formed in 1h normalised to protein content and expressed relative to untreated cells).

Fig 2. Proportion of [3H-methyl]choline accumulated in the lipid fraction by untreated (black) and metformin-treated (white) breast cancer cells during a 15minute incubation with [³H-methyl]choline followed by a 1h chase (medium replaced with non-radioactive medium) expressed as a percentage of total cell [³H-methyl]choline uptake.

(A) BT474, MDA-MB-468 and SKBr3 cells treated with Metformin for 72 h and (B) MDA-MB-468 cells treated with metformin for 24, 72 and 120h.

Fig 3

Total (A) and membrane (B) CTP:PCho cytidylyltransferase (CCT) and PtdCho-specific phospholipase C (C) activity of untreated (black) and metformin-treated (white) breast tumour cells. (units: CCT- [³H]CDP-choline (CDP-chol) formed after 1h incubation with [³H]PCho normalised to protein content and expressed relative to untreated cells; PLC–enzyme units normalised to protein content and expressed relative to untreated cells).

Fig 4

Proportion of [³H-methyl]choline accumulated in the lipid fraction by untreated (control) and metformin-treated MDA-MB-468 breast cancer cells incubated for 4h with the PtdCho-PLC specific inhibitor D609 expressed as a percentage of total cell uptake (A).

Fig 5

Proportion of [3H-methyl]choline accumulated in the lipid fraction by untreated (control) and metformin-treated MDA-MB-468 breast cancer cells incubated for 4h with the ACC inhibitor TOFA (A) or incubated for 4h with the lipase inhibitor agliastatin (B).

Fig 6. Incorporation of [¹⁴C] radioactivity into lipid and glycerol by control and metformin-treated cells incubated with [¹⁴C-U]glucose for 2h after saponification of cell lipid fraction (units [14C]incorporation/mg protein (x1000)).

Fig 7. Cell cycle distribution in MDA-MB-468 cells treated with metformin for 24h (white), 48h (speckle) and 72h (squares).

Fig 8. Alterations in phosphatidylcholine metabolism and conversion of glucose to fatty acid and glycerol associated with metformin treatment.

(Abbreviations: PtdCho, phosphatidylcholine; PCho phosphocholine; CDP-choline, cytidine tri-phosphate–choline; CK, choline kinase; CCT, CTP:phosphocholine cytidylyltransferase; DAG diacylglycerol; PLC phospholipase C; TCA, tricarboxylic acid; Gly-3-P, glyceraldehyde-3-phosphate.