Journal: Scientific Reports

Article Title: Pharmacological inhibition of hypoxia induced acidosis employing a CAIX inhibitor sensitizes gemcitabine resistant PDAC cells

doi: 10.1038/s41598-025-93388-5

Figure Lengend Snippet: Expression of HIF-1α and CAIX in MIA PaCa-2 and PANC-1 post MZM treatment. ( A ) Basal levels of mRNA transcript for HIF-1α and CAIX in MIA PaCa-2 and PANC-1 cells at 3, 18 and 24 h under hypoxia and normoxia. ( B ) Basal expression of HIF-1α and CAIX protein in MIA PaCa-2 and PANC-1 cells at 24 and 48 h. ( C , D ) Expression of HIF-1α and CAIX in MIA PaCa-2 cells treated with MZM at increasing concentrations (0.1–3 mM) for 24 and 48 h under hypoxia and normoxia. ( E , F ) Expression of HIF-1α and CAIX in PANC-1 cells treated with MZM at increasing concentrations (0.1–3 mM) for 24 and 48 h under hypoxia and normoxia.

Article Snippet: The membranes were incubated with various primary antibodies against different proteins viz, HIF-1α (#610658, BD Biosciences, England, UK), CAIX (#5649, Cell signaling, MA, USA), Ki-67 (#ab15580, Abcam, MA, USA), VEGF (#50661, Cell Signaling, MA, USA), Cyclin D1 (#2922, Cell Signaling, MA, USA) and GAPDH (#MA5-15738, Invitrogen, Canada) overnight followed by 3 washes with PBST.

Techniques: Expressing

Journal: Scientific Reports

Article Title: Pharmacological inhibition of hypoxia induced acidosis employing a CAIX inhibitor sensitizes gemcitabine resistant PDAC cells

doi: 10.1038/s41598-025-93388-5

Figure Lengend Snippet: Inhibition of HIF-1α and CAIX in PDAC cells. ( A–D ) Under hypoxia , mRNA transcripts levels of HIF-1α and CAIX in MIA PaCa-2 ( A and B) and PANC-1 ( C and D) post treatment with MZM alone or in combination with GEM. The relative expression of genes was calculated with the 2 –∆∆Ct method, using β-actin as housekeeping gene for normalization. Statistical differences between the groups were determined by one-way ANOVA and post hoc multiple variances using Tukey test. The statistically significant difference is represented as * p < 0.05, ** p < 0.01 and *** p < 0.001 for control v/s specific group. ( E–H ) Western blot showing HIF-1α and CAIX protein expression levels in MIA PaCa-2 ( E and F) and PANC-1 ( G and H) post treatment with MZM alone or in combination with GEM (400 nM) at 24 and 48 h under normoxia (21% O 2 ) , and hypoxia condition (1% O 2 ) . Numbers on top indicate fold difference in expression which was determined after densitometry analysis using Image J software. β-actin was used as loading control.

Article Snippet: The membranes were incubated with various primary antibodies against different proteins viz, HIF-1α (#610658, BD Biosciences, England, UK), CAIX (#5649, Cell signaling, MA, USA), Ki-67 (#ab15580, Abcam, MA, USA), VEGF (#50661, Cell Signaling, MA, USA), Cyclin D1 (#2922, Cell Signaling, MA, USA) and GAPDH (#MA5-15738, Invitrogen, Canada) overnight followed by 3 washes with PBST.

Techniques: Inhibition, Expressing, Control, Western Blot, Software

Journal: Scientific Reports

Article Title: Pharmacological inhibition of hypoxia induced acidosis employing a CAIX inhibitor sensitizes gemcitabine resistant PDAC cells

doi: 10.1038/s41598-025-93388-5

Figure Lengend Snippet: ( A ) Inhibition of carbonic anhydrase IX as a surrogate marker of hypoxia, acidosis, and glycolytic metabolism by MZM in PC. Left side of figure indicates hypoxia related upregulation of CAIX and the related effects of this spike. The right of the figure indicates the inhibitory potential of MZM against all the markers studied and therefore all the pathways affected. CFA, colony forming assay, WHA , wound healing assay; PDX, patient derived xenograft, GEM, Gemcitabine. ( B ) Proposed cellular mechanism of action for MZM in PDAC cells. The illustration depicts proposed mechanism of MZM against carbonic anhydrase IX (CAIX) as a potential target in KRAS mutated pancreatic cancer. Under hypoxia, activated CAIX alters pH of intracellular milieu after intake of bicarbonate ions, generation of proton pool, increased uptake of glucose and accumulation of lactate. The altered pH favours stimulation of oncogenic signals by pro survival pathways which cater PC cells to proliferate, survive, invade, and metastasize under acidic environment. Pharmacological inhibition of CAIX by MZM modulates pericellular and intracellular activity owing to hypoxia, acidosis and glycolytic flux and further reduces tumor growth in GEM resistant PC cells. Abbreviations: MZM, Methazolamide; GEM, Gemcitabine, PDAC, Pancreatic ductal adenocarcinoma, NHE1, Sodium/proton exchanger 1; GLUT, Glucose transporter; MCT4, Monocarboxylate transporter 4; PTEN, Phosphatase and tensin homolog; NBC, Sodium bicarbonate cotransporter; IGF, Insulin growth factor; HGF, Hepatocyte growth factor; EGF, Epithelial growth factor; VEGF, Vascular endothelial growth factor; VEGFR, Vascular endothelial growth factor receptor; ATPase, Adenosine triphosphatase; HRE, Hypoxia response element; Hif-1α, Hypoxia inducible factor; MDR1, Multidrug resistance protein 1; RTK, Receptor tyrosine kinase; PHD1/2, Hypoxia-inducible factor prolyl hydroxylase 1/2; VHL, Von Hippel-Lindau Syndrome; LDHA, Lactate dehydrogenase A. Green upside arrow indicates upregulation under hypoxia. Red downside arrow indicates downregulation of effect mediated by MZM. pHe, extracellular pH; pHi, intracellular pH; red star indicates activated KRAS pathway.

Article Snippet: The membranes were incubated with various primary antibodies against different proteins viz, HIF-1α (#610658, BD Biosciences, England, UK), CAIX (#5649, Cell signaling, MA, USA), Ki-67 (#ab15580, Abcam, MA, USA), VEGF (#50661, Cell Signaling, MA, USA), Cyclin D1 (#2922, Cell Signaling, MA, USA) and GAPDH (#MA5-15738, Invitrogen, Canada) overnight followed by 3 washes with PBST.

Techniques: Inhibition, Marker, Wound Healing Assay, Derivative Assay, Activity Assay

Journal: The Prostate

Article Title: The Diagnostic Value of Plasma Small Extracellular Vesicle‐Derived CAIX Protein in Prostate Cancer and Clinically Significant Prostate Cancer: A Study on Predictive Models

doi: 10.1002/pros.24879

Figure Lengend Snippet: Characterization of sEVs isolated from human PCa plasma samples. (A) NTA shows the modal sizes of sEV particles in the plasma of patients with negative and positive biopsies (B) Representative TEM images show sEVs (white arrows) isolated from biopsy‐negative (Left image) and biopsy‐positive (Right image) patients, with visible lipid bilayers and cup‐shaped structures, scale bar‐200 nm. (C) Representative Western blots demonstrating the expression of CD9, calnexin, syntenin‐1, TSG101, and CAIX in sEV preparations isolated from plasma samples of patients with negative and positive biopsies, compared to whole cell lysates from DU145 prostate cancer cells. (D) Comparison of protein expression (ApoA1, LDHB, HBG1) in hemolyzed plasma, non‐hemolyzed plasma, and sEVs isolated from non‐hemolyzed plasma samples from PCa patients ( n = 3 each). (E) The hemolyzed and non‐hemolyzed plasma samples exhibited distinct color differences upon visual inspection(n = 3). Abbreviations: ApoA1, Apolipoprotein A1; CAIX, carbonic anhydrase IX; HBG1, Hemoglobin subunit gamma 1; LDHB, Lactate Dehydrogenase B; NTA, nanoparticle tracking analysis; PCa, prostate cancer; sEV, small extracellular vesicle; TEM, transmission electron microscopy; WCL, whole cell lysate. [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Subsequently, slides were sealed with goat serum at RT for 1 h, followed by overnight incubation at 4°C with rabbit polyclonal antibodies against CAIX (Proteintech,11071‐1‐AP) diluted 1:1000.

Techniques: Isolation, Clinical Proteomics, Western Blot, Expressing, Comparison, Transmission Assay, Electron Microscopy

Journal: The Prostate

Article Title: The Diagnostic Value of Plasma Small Extracellular Vesicle‐Derived CAIX Protein in Prostate Cancer and Clinically Significant Prostate Cancer: A Study on Predictive Models

doi: 10.1002/pros.24879

Figure Lengend Snippet: sEV protein CAIX can be used to predict diagnosis in the initial PCa biopsies. (A, B) In the training cohort, sEV protein CAIX is significantly upregulated in both PCa ( P = 1.8e −15 ) and csPCa patients ( P = 1.3e −13 ). (C, D) The validation cohort confirms the sEV CAIX findings. (E, F) In the training cohort, tPSA is upregulated in both PCa ( P = 1.5e −18 ) and csPCa ( P = 1.1e −08 ). (G, H). In the validation cohort, tPSA upregulation is observed in PCa ( p = 0.0021) and csPCa ( P = 8.6e −05 ). (I, J) The ROC analysis shows the diagnostic power of the sEV protein CAIX score in PCa (AUC: 0.873) and the Model in csPCa (AUC: 0.909) in the training cohort. Comparison of ROC analysis shows that the sEV protein CAIX expression (assay) had a better diagnostic performance than blood tPSA, f/tPSA, and PSAD test in PCa. The Model (CAIX + PSAD) had a better diagnostic performance than sEV protein CAIX, tPSA, f/tPSA and PSAD tests in csPCa alone. (K, L) Similar results were obtained in the validation cohort. Abbreviations: AUC, area under the curve; CAIX, carbonic anhydrase IX; csPCa, clinically significant PCa; f/t PSA, free prostate‐specific antigen/total prostate‐specific antigen; nsPCa, nonsignificant PCa; PCa, prostate cancer; PSAD, prostate‐specific antigen density; ROC, receiver operating characteristic; tPSA, total prostate‐specific antigen. [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Subsequently, slides were sealed with goat serum at RT for 1 h, followed by overnight incubation at 4°C with rabbit polyclonal antibodies against CAIX (Proteintech,11071‐1‐AP) diluted 1:1000.

Techniques: Biomarker Discovery, Diagnostic Assay, Comparison, Expressing

Journal: The Prostate

Article Title: The Diagnostic Value of Plasma Small Extracellular Vesicle‐Derived CAIX Protein in Prostate Cancer and Clinically Significant Prostate Cancer: A Study on Predictive Models

doi: 10.1002/pros.24879

Figure Lengend Snippet: Performance of the sEV protein CAIX expression with PCa‐associated clinical features to predict biopsy results by logistic regression.

Article Snippet: Subsequently, slides were sealed with goat serum at RT for 1 h, followed by overnight incubation at 4°C with rabbit polyclonal antibodies against CAIX (Proteintech,11071‐1‐AP) diluted 1:1000.

Techniques: Expressing, Biomarker Discovery

Journal: The Prostate

Article Title: The Diagnostic Value of Plasma Small Extracellular Vesicle‐Derived CAIX Protein in Prostate Cancer and Clinically Significant Prostate Cancer: A Study on Predictive Models

doi: 10.1002/pros.24879

Figure Lengend Snippet: Performance of sEV protein CAIX expression with PCa‐associated clinical features in predicting csPCa in patients undergoing prostate biopsy using logistic regression.

Article Snippet: Subsequently, slides were sealed with goat serum at RT for 1 h, followed by overnight incubation at 4°C with rabbit polyclonal antibodies against CAIX (Proteintech,11071‐1‐AP) diluted 1:1000.

Techniques: Expressing, Biomarker Discovery

Journal: The Prostate

Article Title: The Diagnostic Value of Plasma Small Extracellular Vesicle‐Derived CAIX Protein in Prostate Cancer and Clinically Significant Prostate Cancer: A Study on Predictive Models

doi: 10.1002/pros.24879

Figure Lengend Snippet: The clinical utility of the sEV protein CAIX test assay. (A, B) DCA shows that the CAIX assay demonstrates the highest net benefit across 0.2–0.8 threshold probabilities for PCa in the training and validation cohort. The horizontal black lines parallel to the x‐axis represent no patient undergoing a biopsy (Treat None). The gray line indicates that all the patients will have PCa (Treat All). (C) The waterfall plot of the sEV protein CAIX test assay scores is related to the prostate biopsy results ( n = 230). Each bar represents an individual. Red indicates the ISUP grade ≥ two tumors (Gleason score ≥ 7); dusty blue indicates the ISUP grade of one tumor (Gleason score = 6); green indicates the negative biopsies. Two black horizontal lines represent the cutoff points of 0.454 at the sensitivity of 90% and 0.376 at the sensitivity of 95%. Abbreviations: AUC, area under the curve; CAIX, carbonic anhydrase IX; f/t PSA, free prostate‐specific antigen/total prostate‐specific antigen; ISUP, International Society of Urological Pathology; PCa, prostate cancer; PSAD, prostate‐specific antigen density; tPSA, total PSA. [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Subsequently, slides were sealed with goat serum at RT for 1 h, followed by overnight incubation at 4°C with rabbit polyclonal antibodies against CAIX (Proteintech,11071‐1‐AP) diluted 1:1000.

Techniques: Biomarker Discovery

Journal: The Prostate

Article Title: The Diagnostic Value of Plasma Small Extracellular Vesicle‐Derived CAIX Protein in Prostate Cancer and Clinically Significant Prostate Cancer: A Study on Predictive Models

doi: 10.1002/pros.24879

Figure Lengend Snippet: Multivariate stepwise logistic regression analysis for predicting csPCa in the training group.

Article Snippet: Subsequently, slides were sealed with goat serum at RT for 1 h, followed by overnight incubation at 4°C with rabbit polyclonal antibodies against CAIX (Proteintech,11071‐1‐AP) diluted 1:1000.

Techniques: Biomarker Discovery

Journal: The Prostate

Article Title: The Diagnostic Value of Plasma Small Extracellular Vesicle‐Derived CAIX Protein in Prostate Cancer and Clinically Significant Prostate Cancer: A Study on Predictive Models

doi: 10.1002/pros.24879

Figure Lengend Snippet: Nomograms can predict the probability of csPCa. A Nomogram was established based on sEV protein CAIX test assay and PSAD. (A) Nomogram for diagnosis of csPCa. Higher total points indicate a higher prevalence for csPCa. (B) Nomogram‐predicted probability of csPCa in the training cohort. (C) DCA of the sEV protein CAIX test, tPSA, f/t PSA, PSAD and prediction models for csPCa. DCA shows that the model presents the highest net benefit across 0.2 to 0.8 threshold probabilities for csPCa in the training cohort. The horizontal black lines parallel to the x‐axis represent no patient undergoing a biopsy (Treat None). The gray line indicates that all the patients will have csPCa (Treat All). Abbreviations: CAIX, carbonic anhydrase IX; CsPCa, clinically significant PCa; DCA, decision curve analysis; Predicted Pr, predicted probability; PSAD, PSA density; sEV, small extracellular vehicle; tPSA, total PSA; f/tPSA, free prostate‐specific antigen/total prostate‐specific antigen. [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Subsequently, slides were sealed with goat serum at RT for 1 h, followed by overnight incubation at 4°C with rabbit polyclonal antibodies against CAIX (Proteintech,11071‐1‐AP) diluted 1:1000.

Techniques: Biomarker Discovery

Journal: The Prostate

Article Title: The Diagnostic Value of Plasma Small Extracellular Vesicle‐Derived CAIX Protein in Prostate Cancer and Clinically Significant Prostate Cancer: A Study on Predictive Models

doi: 10.1002/pros.24879

Figure Lengend Snippet: Clinical association of sEV protein CAIX biomarker identified with human PCa tissue. The expression of CAIX protein in adjacent normal tissue and PCa tissue was examined using a TMA including PCa tissue ( n = 94) and normal prostate control tissue ( n = 47). (A) Typical images show a high expression of CAIX in PCa tissue compared to normal prostate tissue, with the predominant localization observed in the cytoplasm. Brown color indicates positive staining while blue color indicates nuclei. Magnification x 20, scale bar = 100 um. (B) Quantitative analysis indicates distinct expression of CAIX (*** p < 0.001) between PCa tissue and adjacent normal prostate tissue. (C) Comparison of CAIX protein IHC staining scores between cancerous and adjacent noncancerous tissues in the same PCa patients ( n = 44, paired two‐tailed t ‐test, p < 0.001). Abbreviations: CAIX, carbonic anhydrase IX; IHC, immunohistochemistry; PCa, prostate cancer. [Color figure can be viewed at wileyonlinelibrary.com ]

Article Snippet: Subsequently, slides were sealed with goat serum at RT for 1 h, followed by overnight incubation at 4°C with rabbit polyclonal antibodies against CAIX (Proteintech,11071‐1‐AP) diluted 1:1000.

Techniques: Biomarker Discovery, Expressing, Control, Staining, Comparison, Immunohistochemistry, Two Tailed Test