dilazep  (MedChemExpress)

Journal: bioRxiv

Article Title: Macropinocytosis enables metabolic recycling of extracellular DNA in cancer cells

doi: 10.1101/2025.10.22.684010

Figure Lengend Snippet: Macropinocytosis of exDNA overcomes cancer cell dependence on glutamine-derived nitrogen. a , Overview of cellular metabolic fates of glutamine. b , Chemical structures of nucleic acids. The glutamine amide-derived nitrogen is highlighted in red. c , MDA-MB-231 cells were treated for 6 days with 0.1 μM CB-839 or glutamine deprivation, with or without 1 mM α-ketoglutarate (α-KG) or pyruvate (Pyr). Cell counts were normalized to the RPMI control and are presented as mean ± SEM, n = 3. d , MDA-MB-231 cells for 7 days in glutamine-free medium supplemented with either 3% or 5% BSA, with 1 mM Pyr plus 200 µM inosine monophosphate (IMP) or with 2 mM glutamine (Gln). Cell density was quantified by crystal violet staining and normalized to the glutamine-free control. Statistical significance was assessed by one-way ANOVA against the glutamine-free control. Data are shown as mean ± SEM from three independent replicates. e , MDA-MB-231 cells were treated for 10 days under glutamine-free conditions supplemented with 0.2 mM each of the following: non-essential amino acids (NEAA: glycine, alanine, asparagine, aspartate, glutamate, proline, serine), N-acetyl-D-glucosamine (GlcNAc), D-glucosamine 6-phosphate (Glc-P), nicotinamide adenine dinucleotide (NAD + ), ammonium (NH₄⁺), or mixed dNTPs. Treatments were performed with or without 1 mM Pyr or α-KG. Cell densities were normalized to the glutamine-free control and are presented as mean ± SEM, n = 3. f , MDA-MB-231 cells were treated with 200 µM urate, IMP, or UMP ± 1 mM α-KG or Pyr for a week. Cell numbers, determined by counting, were normalized to the glutamine-free control. Statistical comparisons between groups and the glutamine-free control were performed using one-way ANOVA. Data are presented as mean ± SEM from 3 independent replicates. g , Relative cell numbers of MDA-MB-231, JIMT-1, MIA PaCa-2, or SUIT-2 after 1-week culture with various supplements: 1 mM α-KG, 200 µM urate or IMP, and 2.5 µM exDNA. Data were normalized to the glutamine-free control and represented as mean ± SEM, n = 3, one-way ANOVA with Dunnett’s multiple comparisons test. h , MDA-MB-231 and JIMT-1 breast cancer cells were cultured under glutamine-free conditions for ∼1-week, with or without 25 nM cytD. Treatments included supplementation with 1 mM α-KG or Pyr and 2.5 µM exDNA. Cell numbers were quantified using the CyQUANT assay, normalized to glutamine-free controls, and presented as fold change relative to control (mean ± SEM, n = 6). Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test. i , Cell numbers were measured using the CyQUANT assay after 12 days culture in glutamine-deprived medium supplemented with 1 mM α-KG and 2.5 µM exDNA, with or without co-treatment using nucleoside transporter inhibitors (NBTI, DPL, or dilazep) at 0.01 µM (+), 0.1 µM (++), or 1 µM (+++).

Article Snippet: Chemicals used in this study were as follows: Inosine 5’-monophosphate, IMP (TCI I0036); Uridine 5’-monophosphate, UMP (Sigma U6375); Deoxynucleotide mix, dNTPs (Sigma D7295); Adenosine 5’-triphosphate, ATP (Sigma A6419); Bovine serum albumin, BSA (Sigma A7030); Sodium pyruvate, Pyr (Sigma P2256); Dimethyl alpha-ketoglutarate, α-KG (Sigma 349631); Sodium urate (Sigma U2875); Ammonium chloride (Sigma A9434); N-acetyl-D-glucosamine, GlcNAc (Sigma 1079); D-glucosamine 6-phosphate, Glc-P (Sigma G5509); NAD + (Sigma NAD100-RO); Acetic acid (Sigma A6283); Nucleoside transporter inhibitors: S-(4-Nitrobenzyl)-6-thioinosine, NBTI (Sigma N2255), dipyridamole, DPL (Sigma D9766), and dilazep dihydrochloride (MCE HY-100957).

Techniques: Derivative Assay, Control, Staining, Cell Culture, CyQUANT Assay