Journal: International Journal of Molecular Sciences

Article Title: Characterisation of 3D Bioprinted Human Breast Cancer Model for In Vitro Drug and Metabolic Targeting

doi: 10.3390/ijms23137444

Figure Lengend Snippet: Sensitivity and metabolic enzyme activity differences of monolayer cultures, 3D-cultured spheroids (cells growing in hanging drops or ultra-low attachment plates) in vitro and the in vivo xenograft model. ( a ) In vitro proliferation results (sulforhodamine B (SRB) and Alamar blue (AB) tests, and cell counting) of ZR75.1 monolayer cell culture after 72-h treatments. Dashed lines show average viability % calculated from the three tests used. All data represent mean ± SD. Results are given in control %. ( b ) The registered tumour weights and the calculated final tumour volumes of the ZR75.1 xenografts after 21-day treatments. ( c ) Alamar blue (AB) assay and cell counting were used to detect the in vitro cell growth of ZR75.1 spheroids cultured in ULA plates (3D) after 72 h treatments. All data represent mean ± SD. Results are given in control %. (Co—control; R—rapamycin, 50 ng/mL or Rapamune 3 mg/kg; doxy—doxycycline, 10 µM or 5 mg/kg; doxo—doxorubicin, 50 ng/mL or 2 mg/kg). All data represent mean ± SD. Synergistic treatment interaction is labelled with S (based on combination index calculation). * p < 0.05. ( d ) Maintaining condition (monolayer—2D; HD spheroid—3D; xenograft—Xeno) affects protein expression pattern of ZR75.1 cells and xenograft tumour. Wes TM Simple technique was used to detect mTOR activity (p-mTOR, p-(Ser473)-Akt, p-S6, p-4EBP1) and metabolic enzyme (FASN—fatty acid synthase, CPT1A—carnitine palmitoyltransferase 1A, PKM2—pyruvate kinase M2, LDHB—lactate dehydrogenase B, COX4—cytochrome c oxidase subunit 4) expressions (left panel). Densitometric analysis was performed to present the normalized protein expression differences (right panel) (β-actin was used as loading control). Red frame highlights the similarities of the studied enzyme expressions of 2D and spheroid cultures and underlines the differences in protein expression pattern obtained using ZR75.1 in vivo model. * p < 0.05.

Article Snippet: Pyruvate Kinase Isoenzyme M2 , PKM2 , Cell Signaling , #4053 , 1:50 , - , glycolysis.

Techniques: Activity Assay, Cell Culture, In Vitro, In Vivo, Cell Counting, Control, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Characterisation of 3D Bioprinted Human Breast Cancer Model for In Vitro Drug and Metabolic Targeting

doi: 10.3390/ijms23137444

Figure Lengend Snippet: Tissue heterogeneity of several metabolic markers. Primary antibody panel/list of the primary antibodies used for immunohistochemistry and Wes TM analyses.

Article Snippet: Pyruvate Kinase Isoenzyme M2 , PKM2 , Cell Signaling , #4053 , 1:50 , - , glycolysis.

Techniques: Immunohistochemistry, Control, Phospho-proteomics, Activity Assay

Journal: Carcinogenesis

Article Title: Reversal of the Warburg phenomenon in chemoprevention of prostate cancer by sulforaphane

doi: 10.1093/carcin/bgz155

Figure Lengend Snippet: SFN treatment decreased ECAR and protein levels of HKII, PKM2 and LDHA in prostate cancer cells. Pharmacologic profiling of ECAR in LNCaP cells through real-time measurements using the Seahorse flux analyzer following 6- (A) or 9-h (B) treatment with DMSO or SFN (5 or 10 µM). After measurement of basal ECAR, the cells were treated with metabolic inhibitors, including 1 µM oligomycin (O), 300 nM carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (F), 1 mM 2-deoxy glucose (2-DG) or 1 µM rotenone (R) at the indicated times. Basal ECAR was calculated using the difference between the mean of time points prior to injection of O (#1 to #4). Oligomycin-sensitive rate was calculated using the difference between the mean of time points prior to injection of F (#5 to #7). Quantitation of basal ECAR (C) and oligomycin-sensitive ECAR (D) in LNCaP cells following 9-h treatment with DMSO or SFN. Quantitative data for the 6-h time point are not shown as the difference was not significant. The experiment was repeated three times in triplicate, and combined data are expressed as mean ± SEM. Significantly different (*P < 0.05) compared with control by one-way ANOVA followed by Dunnett’s test. (E) Confocal images (×63 oil objective magnification) for HKII, PKM2 and LDHA (green fluorescence) in LNCaP and 22Rv1 cells following 24-h treatment with DMSO or 10 µM SFN. Nucleus was stained with DRAQ5 (blue fluorescence). (F) Quantitation of corrected total cell fluorescence (CTCF) using ImageJ software in LNCaP and 22Rv1 cells following treatment with DMSO or SFN for the specified time points. *Significant (P < 0.05) compared with DMSO-treated control by Student’s t-test. Results were consistent in two independent experiments.

Article Snippet: Antibodies against phosphorylated s473 Akt, hexokinase II (HKII; for western blotting), pyruvate kinase M2 (PKM2) and LDHA (for western blotting) were purchased from Cell Signaling Technology (Danvers, MA); an antibody against β-actin was from Sigma–Aldrich (St. Louis, MO); a rabbit monoclonal anti-LDHA antibody used for immunofluorescence microscopy and immunohistochemistry was from Novus Biologicals (Centennial, CO); anti-glucose transporter (GLUT) 1 and anti-GLUT4 antibodies were purchased from Proteintech Group (Rosemont, IL), whereas anti-monocarboxylate transporter 1 (MCT1) antibody was purchased from Santa Cruz Biotechnology (Dallas, TX); and a rabbit polyclonal anti-HKII antibody used for immunofluorescence microscopy and immunohistochemistry was purchased from Abcam (Cambridge, MA).

Techniques: Injection, Quantitation Assay, Fluorescence, Staining, Software

Journal: Carcinogenesis

Article Title: Reversal of the Warburg phenomenon in chemoprevention of prostate cancer by sulforaphane

doi: 10.1093/carcin/bgz155

Figure Lengend Snippet: SFN treatment inhibited expression of glycolysis-related enzyme proteins in prostate cancer cells in vitro and in vivo. (A) Immunoblotting for HKII, PKM2 and LDHA proteins using lysates from LNCaP and 22Rv1 cells after 24-h treatment with DMSO or the indicated doses of SFN. Numbers above bands are fold changes in protein expression relative to respective DMSO-treated controls. (B) Immunohistochemical images for HKII, PKM2 and LDHA protein expression in representative prostate adenocarcinoma sections of TRAMP mice (×40 objective magnification, scale bar = 50 µm). (C) H-score for HKII, PKM2 and LDHA protein expression in TRAMP tumor section. The results shown are mean ± SD (n = 6). Statistical analysis was performed by Student’s t-test (*P < 0.05).

Article Snippet: Antibodies against phosphorylated s473 Akt, hexokinase II (HKII; for western blotting), pyruvate kinase M2 (PKM2) and LDHA (for western blotting) were purchased from Cell Signaling Technology (Danvers, MA); an antibody against β-actin was from Sigma–Aldrich (St. Louis, MO); a rabbit monoclonal anti-LDHA antibody used for immunofluorescence microscopy and immunohistochemistry was from Novus Biologicals (Centennial, CO); anti-glucose transporter (GLUT) 1 and anti-GLUT4 antibodies were purchased from Proteintech Group (Rosemont, IL), whereas anti-monocarboxylate transporter 1 (MCT1) antibody was purchased from Santa Cruz Biotechnology (Dallas, TX); and a rabbit polyclonal anti-HKII antibody used for immunofluorescence microscopy and immunohistochemistry was purchased from Abcam (Cambridge, MA).

Techniques: Expressing, In Vitro, In Vivo, Western Blot, Immunohistochemical staining

Journal: Carcinogenesis

Article Title: Reversal of the Warburg phenomenon in chemoprevention of prostate cancer by sulforaphane

doi: 10.1093/carcin/bgz155

Figure Lengend Snippet: SFN treatment downregulated expression of glycolysis-related proteins in PIN lesions of Hi-Myc mice. Immunohistochemical images for HKII, PKM2 and LDHA protein expression in representative prostate sections of control and SFN-treated Hi-Myc mice (×40 objective magnification, scale bar = 50 µm) are shown in the left panel. Quantitative data for H-score are shown are mean ± SD (n = 5) in the right panel. Statistical analysis was performed by Student’s t-test (*P < 0.05).

Article Snippet: Antibodies against phosphorylated s473 Akt, hexokinase II (HKII; for western blotting), pyruvate kinase M2 (PKM2) and LDHA (for western blotting) were purchased from Cell Signaling Technology (Danvers, MA); an antibody against β-actin was from Sigma–Aldrich (St. Louis, MO); a rabbit monoclonal anti-LDHA antibody used for immunofluorescence microscopy and immunohistochemistry was from Novus Biologicals (Centennial, CO); anti-glucose transporter (GLUT) 1 and anti-GLUT4 antibodies were purchased from Proteintech Group (Rosemont, IL), whereas anti-monocarboxylate transporter 1 (MCT1) antibody was purchased from Santa Cruz Biotechnology (Dallas, TX); and a rabbit polyclonal anti-HKII antibody used for immunofluorescence microscopy and immunohistochemistry was purchased from Abcam (Cambridge, MA).

Techniques: Expressing, Immunohistochemical staining

Journal: Carcinogenesis

Article Title: Reversal of the Warburg phenomenon in chemoprevention of prostate cancer by sulforaphane

doi: 10.1093/carcin/bgz155

Figure Lengend Snippet: Overexpression of Myc, but not constitutively active Akt (caAkt) partially attenuated SFN-mediated decrease in protein levels of HKII, PKM2, and LDHA in 22Rv1 cells. (A) Immunoblotting for Myc, total Akt, and phosphorylated s473 Akt using lysates from 22Rv1 cells stably transfected with empty vector, Myc or caAkt. (B) Immunoblotting for HKII, PKM2 and LDHA using lysates from 22Rv1 cells stably transfected with empty vector (EV) or Myc or caAkt plasmids and treated for 24 h with DMSO or the indicated doses of SFN. (C) Representative images of colonies resulting from 22Rv1 cells after 8 days of treatment with DMSO or the indicated doses of SFN. (D) Quantitation of colony formation. Results shown are mean ± SD (n = 3). Statistically significant (P < 0.05) compared with the *corresponding DMSO-treated control or #between cells transfected with EV and Myc or caAkt by one-way ANOVA followed by Bonferroni’s multiple comparisons test. Comparable results were obtained from replicate experiments. (E) Intracellular lactate levels in 22Rv1 cells stably transfected with EV or Myc or caAkt plasmids and treated for 24 h with DMSO or the indicated doses of SFN. Results shown are mean ± SD (n = 3). Statistically significant (P < 0.05) compared with the *corresponding DMSO-treated control or #between cells transfected with EV and Myc or caAkt by one-way ANOVA followed by Bonferroni’s multiple comparisons test. Each experiment was repeated at least two times.

Article Snippet: Antibodies against phosphorylated s473 Akt, hexokinase II (HKII; for western blotting), pyruvate kinase M2 (PKM2) and LDHA (for western blotting) were purchased from Cell Signaling Technology (Danvers, MA); an antibody against β-actin was from Sigma–Aldrich (St. Louis, MO); a rabbit monoclonal anti-LDHA antibody used for immunofluorescence microscopy and immunohistochemistry was from Novus Biologicals (Centennial, CO); anti-glucose transporter (GLUT) 1 and anti-GLUT4 antibodies were purchased from Proteintech Group (Rosemont, IL), whereas anti-monocarboxylate transporter 1 (MCT1) antibody was purchased from Santa Cruz Biotechnology (Dallas, TX); and a rabbit polyclonal anti-HKII antibody used for immunofluorescence microscopy and immunohistochemistry was purchased from Abcam (Cambridge, MA).

Techniques: Over Expression, Western Blot, Stable Transfection, Transfection, Plasmid Preparation, Quantitation Assay