A lipid-free and insulin-supplemented medium supports De Novo fatty acid synthesis gene activation in melanoma cells

Abstract

While investigating the role played by de novo lipid (DNL) biosynthesis in cancer cells, we sought a medium condition that would support cell proliferation without providing any serum lipids. Here we report that a defined serum free cell culture medium condition containing insulin, transferrin and selenium (ITS) supports controlled study of transcriptional regulation of de novo fatty acid (DNFA) production and de novo cholesterol synthesis (DNCS) in melanoma cell lines. This lipid-free ITS medium is able to support continuous proliferation of several melanoma cell lines that utilize DNL to support their lipid requirements. We show that the ITS medium stimulates gene transcription in support of both DNFA and DNCS, specifically mediated by SREBP1/2 in melanoma cells. We further found that the ITS medium promoted SREBP1 nuclear localization and occupancy on DNFA gene promoters. Our data show clear utility of this serum and lipid-free medium for melanoma cancer cell culture and lipid-related areas of investigation.

SREBP1-dependent de novo fatty acid synthesis gene expression is elevated in malignant melanoma and represents a cellular survival trait

“…To understand the dynamics of DNFA gene expression, we performed a time-course study in A375 cells cultured under SREBP1-activating conditions (1% ITS medium) 55 . FASN and SCD displayed increased expression at consecutive time points (Supplementary Fig.…”

The amount of membrane cholesterol required for robust cell adhesion and proliferation in serum-free condition

“…Cell lines using in this research were obtained from the Cell Resource Center for Biomedical Research (Institute of Development, Aging and Cancer, Tohoku University, Japan) and maintained in 10% Fetal Bovine Serum (Gibco, 10270)-contained DMEM medium (nacalai tesque, 08458–45) which is supplemented with 1x Penicillin-Streptomycin Mixed Solution (nacalai tesque, 26253–84), and passaged using TrypLE (Gibco, 12604013) every 4–5 days. Briefly for in RPMI-G [ 11 ], 2x10 4 cells were seeded into one well of a 4-well plate without pre-coating in RPMI1640 medium (nacalai tesque, 3026485) supplemented with 1x ITS-G: Insulin-Transferrin-Selenium (Gibco, 41400045), 1x L-Glutamine (nacalai tesque, 16948–04), 1x Penicillin-Streptomycin Mixed Solution. In DA-X condition (shown as ITS-X/ DMEM/ BSA (FN) in Fig 1A ), 2x10 4 cells were seeded into one well of a 4-well plate pre-coated with Fibronectin in DMEM medium (High Glucose) (nacalai tesque, 08458–45) supplemented with 1x ITS-X: Insulin-Transferrin-Selenium-Ethanolamine (Gibco, 51500056), 5 mg/mL BSA: Bovine Serum Albumin (Sigma, A3059), 1x L-Glutamine, 1x Penicillin-Streptomycin Mixed Solution.…”

Serum-Free Medium for Recombinant Protein Expression in Chinese Hamster Ovary Cells

“…At the current stage of CHO cell production, the biosynthesis of recombinant proteins is controlled by some cellular processes that are dependent on lipid metabolism ( Wu and Näär, 2019 ; Budge et al, 2020 ). The distribution of key media components during glycolysis, the tricarboxylic acid cycle, lactate production, and biosynthetic pathways switch dramatically between exponential growth and stationary (production) phases ( Dean and Reddy, 2013 ).…”