Journal: International Journal of Molecular Sciences

Article Title: Synergistic Efficacy of Gedatolisib and Darolutamide in Prostate Cancer to Overcome Resistance to Androgen-Targeted Therapy

doi: 10.3390/ijms262411810

Figure Lengend Snippet: Effects of the gedatolisib/darolutamide combination on metabolic functions. ( A ) Scheme showing selected key steps of glycolysis and fatty acid synthesis. ( B , C ) Analysis of glucose uptake and lactate production in PC cell lines treated with the indicated drugs for 4 or 24 h, respectively. The values are relative to DMSO-treated cells (set as 1) and are normalized to cell number (assessed by measuring DNA content using CyQuant assay). Data represent mean +/− SD (see for number of replicates). a = p < 0.05 for single drugs vs. DMSO, b = p < 0.05 for gedatolisib + darolutamide vs. darolutamide, c = p < 0.05 for gedatolisib + darolutamide vs. gedatolisib by two-way Anova, Fisher test. ( D ) qPCR analysis showing relative HK2 and LDHB mRNA levels in PC cell lines treated with the indicated drugs for 24 h. ( E ) Flow cytometry analysis of lipid metabolism by BODIPY 493/503 staining in PC cell lines treated with the indicated drugs for 24 h. Data represent mean +/− SD ( n = 2 biological replicates). a = p < 0.05 for single drugs vs. DMSO, b = p < 0.05 for gedatolisib + darolutamide vs. darolutamide, c = p < 0.05 for gedatolisib + darolutamide vs. gedatolisib by two-way Anova, Fisher test. ( F ) qPCR analysis showing relative FASN mRNA levels in PC cell lines treated with the indicated drugs for 24 h. FASN = Fatty acid synthase; HK = Hexokinase; LDH = Lactate dehydrogenase; TCA = tricarboxylic acid cycle; RU = relative units. In the heatmaps, white indicates no relative change, blue indicates reduction, and red indicates increase in mRNA levels. See for values.

Article Snippet: The Taqman probes used were AR exon 4–5, Hs00171172_m1; AR exon 1–2, Hs00907242_m1; TMPRSS2 , Hs01122322_m1; KLK3 , Hs02576345_m1; NDFIP1 , Hs00228968_m1; FASN , Hs01005622_m1; LDHB , Hs00929956_m1; HK2 , Hs00892681_m1; E2F , Hs00153451_m1; CDC6 , Hs00154374_m1.

Techniques: CyQUANT Assay, Flow Cytometry, Staining

Journal: Scientific reports

Article Title: LDHB silencing enhances the effects of radiotherapy by impairing nucleotide metabolism and promoting persistent DNA damage.

doi: 10.1038/s41598-025-95633-3

Figure Lengend Snippet: Fig.1. LDHB expression is linked to the activation of DNA damage response and its silencing increases DNA damage accumulation. A-B, correlation analysis of LDHB expression with cell cycle and DNA repair score was performed 3 using CancerSEA. C-E, the protein expression of LDHB and γH2AX was analyzed by flow cytometry 72 h after 4 transfection with siCTRL or siLDHB (n = 3–4), and significance was evaluated using paired T-test. F, the cell cycle 5 distribution was analyzed 72 h after transfection with siCTRL or siLDHB by flow cytometry using DAPI staining. G, 6 the γH2AX expression were analysis by flow cytometry at different cell cycle (n = 3–4). data were means ± SD, unpaired 7 Welch’s t-test. H, LDHB expression in A549, H358, PF139-siCTRL and siLDHB cells was analyzed by Western blot after 8 72 h of transfection. 9

Article Snippet: For stable knockdown, the lentiviruses were produced from the LDHB Human shRNA Plasmid Kit (Origene TL311768) according to The RNAi Consortium (TRC) Broad Institute protocol.

Techniques: Expressing, Activation Assay, Flow Cytometry, Transfection, Staining, Western Blot

Journal: Scientific reports

Article Title: LDHB silencing enhances the effects of radiotherapy by impairing nucleotide metabolism and promoting persistent DNA damage.

doi: 10.1038/s41598-025-95633-3

Figure Lengend Snippet: Fig.2. Long-term LDHB silencing renders cells sensitive to radiotherapy. A, the expression of LDHB and γH2AX in A549shCTRL and shLDHB cells was analyzed by flow cytometry (n = 3–6), and data were means ± SD, unpaired Welch’s 12 t-test or unpaired two-tailed t-test. B-I, Cell cycle, and DNA damage were analyzed with DAPI and γH2AX by flow 13 cytometry at the indicated time points after 4Gy irradiation (n = 3–4), data were means ± SD, unpaired Welch’s t-test. 14.

Article Snippet: For stable knockdown, the lentiviruses were produced from the LDHB Human shRNA Plasmid Kit (Origene TL311768) according to The RNAi Consortium (TRC) Broad Institute protocol.

Techniques: Expressing, Flow Cytometry, Two Tailed Test, Cytometry, Irradiation

Journal: Scientific reports

Article Title: LDHB silencing enhances the effects of radiotherapy by impairing nucleotide metabolism and promoting persistent DNA damage.

doi: 10.1038/s41598-025-95633-3

Figure Lengend Snippet: Fig.3. Long-term LDHB silencing combined with radiotherapy results in genomic instability. A-B, cells were stained with γH2AX and LDHB and imaged with confocal microscopy after treatment with 4Gy at different time points (n = 3), the 17 MFI was normalized to untreated shCTRL cells. data were means ± SD, unpaired Welch’s t-test. C, the expression of 18 PARP, p-Chk2, P53, P21, and LDHB was analyzed by western blot after 24 h of 4Gy irradiation. D-E, β-galactosidase 19 staining on shCTRL and shLDHB cells 5 days after irradiation with 4Gy (n = 3), data were means ± SD, unpaired Welch’s 20 t-test. 21.

Article Snippet: For stable knockdown, the lentiviruses were produced from the LDHB Human shRNA Plasmid Kit (Origene TL311768) according to The RNAi Consortium (TRC) Broad Institute protocol.

Techniques: Staining, Confocal Microscopy, Expressing, Western Blot, Irradiation

Journal: Scientific reports

Article Title: LDHB silencing enhances the effects of radiotherapy by impairing nucleotide metabolism and promoting persistent DNA damage.

doi: 10.1038/s41598-025-95633-3

Figure Lengend Snippet: Fig.4. LDHB silencing depletes nucleotide metabolism in lung cancer tumors. A-C, Enrichment analysis of decreased metabolites in shLDHB tumors compared to shCTRL tumors. D-E, LDHB and γH2AX levels were analyzed in 23 A549 siCTRL and siLDHB cells after one hour of irradiation with or without supplementary with nucleotide precursors (100 24 μM hypoxanthine, 100 μM adenine, 400 μM uridine) (n = 3), data were means ± SD, unpaired Welch’s t-test. 25.

Article Snippet: For stable knockdown, the lentiviruses were produced from the LDHB Human shRNA Plasmid Kit (Origene TL311768) according to The RNAi Consortium (TRC) Broad Institute protocol.

Techniques: Irradiation

Journal: Scientific reports

Article Title: LDHB silencing enhances the effects of radiotherapy by impairing nucleotide metabolism and promoting persistent DNA damage.

doi: 10.1038/s41598-025-95633-3

Figure Lengend Snippet: Fig.5. LDHB silencing depletes nucleotide metabolism in lung cancer tumors. A, schematic representation of local irradiation of xenograft tumors in mice. B-E, IHC analysis of LDHB, γH2AX and p21 expression in shCTRL and shLDHB 28 tumors 10 days after irradiation with 10Gy or control treatment (n = 6), data were means ± SD, two-tailed t-test. 29.

Article Snippet: For stable knockdown, the lentiviruses were produced from the LDHB Human shRNA Plasmid Kit (Origene TL311768) according to The RNAi Consortium (TRC) Broad Institute protocol.

Techniques: Irradiation, Expressing, Control, Two Tailed Test

Journal: Molecules

Article Title: Flipping the Target: Evaluating Natural LDHA Inhibitors for Selective LDHB Modulation

doi: 10.3390/molecules30142923

Figure Lengend Snippet: The top-scoring natural compounds identified through molecular docking as potential allosteric inhibitors of LDHB. Docking was conducted using the crystal structure of LDHB (PDB ID: 7DBJ). The chemical structures and corresponding docking scores (in kcal/mol) of the 16 compounds that achieved scores equal to or better than that of AXKO-0046 (−7.6 kcal/mol), a known uncompetitive inhibitor of LDHB, are illustrated.

Article Snippet: Recombinant human LDHB (Catalog #: 9205-HB) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques:

Journal: Molecules

Article Title: Flipping the Target: Evaluating Natural LDHA Inhibitors for Selective LDHB Modulation

doi: 10.3390/molecules30142923

Figure Lengend Snippet: The evaluation of the top candidate compounds for LDHB inhibition. The inhibitory activity of the selected compounds was assessed using a 96-well endpoint colorimetric assay, as described in the . ( A ) The inhibition of LDHB activity by the top four candidates identified from virtual screening, compared to the known LDHB inhibitor AXKO-0046. The horizontal bars indicate the mean values from three independent experiments. The dashed line indicates 50% inhibition. Dose–response curves show the inhibitory effect of AXKO-0046 ( B ), luteolin ( C ), and quercetin ( D ) on LDHB activity. The percentage of LDHB inhibition was determined by comparing the enzyme activity in the presence of each compound to that of a control without any of the inhibitors. IC 50 values were calculated using nonlinear regression analysis. The molecular structures of the compounds are shown in each panel.

Article Snippet: Recombinant human LDHB (Catalog #: 9205-HB) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Inhibition, Activity Assay, Colorimetric Assay, Control

Journal: Molecules

Article Title: Flipping the Target: Evaluating Natural LDHA Inhibitors for Selective LDHB Modulation

doi: 10.3390/molecules30142923

Figure Lengend Snippet: A kinetic analysis of LDHB inhibition by luteolin and quercetin. The inhibitory effects of luteolin ( A , B ) and quercetin ( C , D ) on LDHB activity were assessed using varying concentrations of sodium lactate ( A , C ) and NAD + ( B , D ). Lineweaver–Burk plots were constructed using linear regression analysis from the kinetic data (see ), with each curve corresponding to a different inhibitor concentration (0–100 μM).

Article Snippet: Recombinant human LDHB (Catalog #: 9205-HB) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Inhibition, Activity Assay, Construct, Concentration Assay

Journal: Molecules

Article Title: Flipping the Target: Evaluating Natural LDHA Inhibitors for Selective LDHB Modulation

doi: 10.3390/molecules30142923

Figure Lengend Snippet: The predicted binding of AXKO-0046, luteolin, and quercetin on LDHB. Structural superposition shows LDHB in complex with NADH (blue sticks) bound at the active sites of each monomer (chain A: orange; chain C: cyan). AXKO-0046 (purple), luteolin (red), and quercetin (green) are all localized at the allosteric site formed at the dimer interface, distal to the catalytic site. This binding mode is consistent with their experimentally determined inhibition behavior, supporting an uncompetitive mechanism of action.

Article Snippet: Recombinant human LDHB (Catalog #: 9205-HB) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Binding Assay, Inhibition

Journal: Molecules

Article Title: Flipping the Target: Evaluating Natural LDHA Inhibitors for Selective LDHB Modulation

doi: 10.3390/molecules30142923

Figure Lengend Snippet: The molecular interactions of AXKO-0046 ( A , B ), luteolin ( C , D ), and quercetin ( E , F ) with the allosteric site of LDHB located in the interphase of chains A and C (in 7DBJ), illustrated as 2D interaction diagrams (left panels) and 3D hydrophobic surface maps (right panels). In the 2D diagrams, the amino acid residues from LDHB chains A and C involved in the interactions are labeled and highlighted with colored circles. These circles correspond to different interaction types, as indicated in the interaction key located in the bottom left panel of the figure. In the 3D hydrophobic surface maps, key hydrophobic (brown) and hydrophilic (blue) regions of the binding pocket are shown, along with hydrogen bonds and other non-covalent interactions. The color gradient reflects surface hydrophobicity, as explained in the key shown in the bottom right panel of the figure. The docking studies were carried out using PyRx v1.1 (AutoDock Vina). The 2D and 3D images were obtained using BIOVIA Discovery Studio.

Article Snippet: Recombinant human LDHB (Catalog #: 9205-HB) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Labeling, Binding Assay

Journal: Molecules

Article Title: Flipping the Target: Evaluating Natural LDHA Inhibitors for Selective LDHB Modulation

doi: 10.3390/molecules30142923

Figure Lengend Snippet: ( A ) The pairwise sequence alignment of human LDHA and LDHB. Identical amino acids are marked with an asterisk (*), conserved amino acids with a colon (:), and semi-conserved amino acids with a period (.). The residues are color-coded based on their chemical properties: acidic (red), basic (blue), polar (cyan), hydrophobic (green), and others (magenta). The alignment shows 75.1% identity (251 out of 334 residues) and 88.6% similarity (296 out of 334 residues), with notable substitutions near the active site and cofactor-binding regions. ( B ) Structural superposition of human LDHA (PDB ID: 4zvv; chain A, gold) bound to NADH (red) and LDHB (PDB ID: 1t2f; chain A, purple) bound to NAD + (cyan), generated using UCSF ChimeraX v 1.9. Despite the overall structural conservation, differences are evident in the substrate-binding pockets and NADH/NAD + interaction sites, highlighted in the two-dimensional interaction diagrams below the structure. Critical residues participating in hydrogen bonding and hydrophobic interactions are depicted, and ligand interaction types are labeled according to their interaction characteristics. These localized variations may influence isoform-specific ligand binding and inhibitory potency.

Article Snippet: Recombinant human LDHB (Catalog #: 9205-HB) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Sequencing, Binding Assay, Generated, Labeling, Ligand Binding Assay

Journal: Molecules

Article Title: Flipping the Target: Evaluating Natural LDHA Inhibitors for Selective LDHB Modulation

doi: 10.3390/molecules30142923

Figure Lengend Snippet: The key structural and functional differences between human LDHA and LDHB relevant to isoform-specific inhibitor design. Despite their high structural similarity, LDHA and LDHB differ in tissue distribution, substrate and cofactor preference, net charge, and inhibition mechanisms. LDHA is predominant in glycolytic tissues and catalyzes the conversion of pyruvate to lactate using NADH, while LDHB, expressed in oxidative tissues, favors the reverse reaction using NAD + . LDHB also shows substrate inhibition at high pyruvate levels. Importantly, LDHA is typically inhibited competitively at the active site, whereas LDHB is inhibited allosterically via uncompetitive mechanisms, as shown for luteolin and quercetin in this study. These differences highlight the need for isoform-specific assay conditions and targeted inhibitor development.

Article Snippet: Recombinant human LDHB (Catalog #: 9205-HB) was purchased from R&D Systems (Minneapolis, MN, USA).

Techniques: Functional Assay, Inhibition

Journal: Nature Communications

Article Title: Ubiquinol-mediated suppression of mitochondria-associated ferroptosis is a targetable function of lactate dehydrogenase B in cancer

doi: 10.1038/s41467-025-57906-3

Figure Lengend Snippet: Measurement of mitochondrial lipid peroxidation in A549, MSTO-211H, HT1080 and PANC-1 cells after 72 h of transfection with siRNA (siCTRL) or with LDHB-targeted siRNAs (siLDHB) ( a , b ), n = 6, n = 5, n = 3 independent replicates for A549, MSTO-211H, HT1080, and PANC-1 respectively. Mitochondrial lipid peroxidation ( c , d scale bar, 20 µm), 4-HNE, and TOM20 colocalization ( e , f scale bar, 20 µm) were examined by immunofluorescence in A549 siRNAs cells after 72 h of transfection, n = 3 independent replicates. Transmission electron microscopy images of A549 siRNAs cells after 72 h of transfection ( g ). Images of the clonogenic assays of A549, PANC-1 siRNAs cells at day 10 after treatment with DMSO or 2 µM ferrostatin-1 (FER1) or 20 µM mitoTEMPO ( h ). Analysis of colony numbers of A549, PANC-1 siRNAs cells at day 10 after treatment with DMSO or 2 µM ferrostatin-1 (FER1) or 20 µM mitoTEMPO, n = 9 independent replicates for DMSO and FER1 treated groups, n = 3 independent replicates for mitoTEMPO treated groups for A549 cell line. n = 3 independent replicates for PANC-1 cell line ( i ). Data were presented as mean ± SD. Unpaired, two-tailed t -test. ns no significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Samples were stained with appropriate antibodies against LDHB (1:5000, R&D Systems MAB9205-100) and 4-HNE (1:500, Abcam ab46545).

Techniques: Transfection, Immunofluorescence, Transmission Assay, Electron Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Ubiquinol-mediated suppression of mitochondria-associated ferroptosis is a targetable function of lactate dehydrogenase B in cancer

doi: 10.1038/s41467-025-57906-3

Figure Lengend Snippet: Immunoblot analysis of LDHB, GPX4, DHODH, LDHA, SLC7A11, and FSP1 in HT1080, MSTO-211H, A549, PANC-1 siRNAs cells, n = 3–6 independent repeats, and the statistical analysis was shown in Supplementary Fig. ( a ). Colocalization analysis by immunofluorescence in HT1080 siRNAs cells after 72 h of transfection ( b , scale bar, 20 µm). Images of microscopic analysis ( c , 20x objective) and assessment of mitochondrial lipid peroxidation by flow cytometry in HT1080 sgNTC and sgGPX4 cells after 72 h of transfection with siRNAs, n = 3 independent replicates ( d , e ). Clonogenic assays of parental HT1080 (HT1080-P) and HT1080 cells treated with 4 µM RSL3 after ten cycles (HT1080-R) ( f ). Immunoblot analysis of LDHB, GPX4, DHODH, and LDHA in HT1080 parental and RSL3 resistant cells. The experiment was repeated three times, yielding consistent results ( g ). Cell viability of HT1080-P and HT1080-R cells after transfection with siRNAs treated with DMSO or RSL3 alone or in combination with 5 µM FER1 for 48 h, following pretreatment with vehicle, 5 µM FER1 for 24 h, n = 3 independent replicates ( h ). Immunoblot analysis of LDHB, GPX4, LDHA in HT1080 control and LDHB overexpression cells ( i ). Cell viability assays of HT1080 cells and HT1080 LDHB ORF cells treated with RSL3 with or without 5 µM FER1 for 48 h, following pretreatment with vehicle, 5 µM FER1 for 24 h. n = 3 independent replicates ( j ). Clonogenic assay of HT1080 siRNAs cells treated with DMSO or RSL3 alone or in combination with 2 mM glutathione reduced ethyl ester (GSH-mee) for 48 h after pretreatment with vehicle, 2 mM GSH-mee for 24 h ( k ). Tumor volume and weight of A549 sgNTC shCTRL ( n = 12), sgNTC shLDHB ( n = 11), sgGPX4 shCTRL ( n = 12), sgGPX4 shLDHB ( n = 12), sgGPX4 shLDHB treated with liproxstatin-1 ( n = 12) xenograft tumors from different mice ( l , m ), tumor volume and weight of HT1080 sgNTC shCTRL ( n = 10), sgNTC shLDHB ( n = 10), sgGPX4 shCTRL ( n = 10), sgGPX4 shLDHB ( n = 12), sgGPX4 shLDHB treated with liproxstatin-1 ( n = 12) xenograft tumors from different mice ( o , p ), 4-HNE expression of A549 and HT1080 sgNTC shCTRL, sgNTC shLDHB, sgGPX4 shCTRL, and sgGPX4 shLDHB xenograft tumors treated with or without liproxstatin-1, n = 10 or n = 12 different regions from different tumors from different mice respectively ( n , q ). Data were presented as mean ± SEM ( l , o ) or mean ± SD ( e , h , j , m , n , p , q ). Two-way ANOVA ( h , j , l , o ), Unpaired, two-tailed t -test ( e , m , n , p , q ). ns no significant difference, * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Source data are provided as a Source Data file.

Article Snippet: Samples were stained with appropriate antibodies against LDHB (1:5000, R&D Systems MAB9205-100) and 4-HNE (1:500, Abcam ab46545).

Techniques: Western Blot, Immunofluorescence, Transfection, Flow Cytometry, Control, Over Expression, Clonogenic Assay, Expressing, Two Tailed Test