Limits...
Carbonic anhydrase XII is a new therapeutic target to overcome chemoresistance in cancer cells.

Kopecka J, Campia I, Jacobs A, Frei AP, Ghigo D, Wollscheid B, Riganti C - Oncotarget (2015)

Bottom Line: Multidrug resistance (MDR) in cancer cells is a challenging phenomenon often associated with P-glycoprotein (Pgp) surface expression.CAXII and Pgp physically interacted at the cell surface.CAXII silencing or pharmacological inhibition with acetazolamide decreased the ATPase activity of Pgp by altering the optimal pH at which Pgp operated and promoted chemosensitization to Pgp substrates in MDR cells.We propose CAXII as a new secondary marker of the MDR phenotype that influences Pgp activity directly and can be used as a pharmacological target for MDR research and potential treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Torino, 10126 Torino, Italy.

ABSTRACT
Multidrug resistance (MDR) in cancer cells is a challenging phenomenon often associated with P-glycoprotein (Pgp) surface expression. Finding new ways to bypass Pgp-mediated MDR still remains a daunting challenge towards the successful treatment of malignant neoplasms such as colorectal cancer.We applied the Cell Surface Capture technology to chemosensitive and chemoresistant human colon cancer to explore the cell surface proteome of Pgp-expressing cells in a discovery-driven fashion. Comparative quantitative analysis of identified cell surface glycoproteins revealed carbonic anhydrase type XII (CAXII) to be up-regulated on the surface of chemoresistant cells, similarly to Pgp. In cellular models showing an acquired MDR phenotype due to the selective pressure of chemotherapy, the progressive increase of the transcription factor hypoxia-inducible factor-1 alpha was paralleled by the simultaneous up-regulation of Pgp and CAXII. CAXII and Pgp physically interacted at the cell surface. CAXII silencing or pharmacological inhibition with acetazolamide decreased the ATPase activity of Pgp by altering the optimal pH at which Pgp operated and promoted chemosensitization to Pgp substrates in MDR cells.We propose CAXII as a new secondary marker of the MDR phenotype that influences Pgp activity directly and can be used as a pharmacological target for MDR research and potential treatment.

Show MeSH

Related in: MedlinePlus

Depletion of CAXII does not affect proliferation and survival of chemoresistant cellsHT29/dx cells were cultured for 48 h with fresh medium (CTRL), treated with a non targeting scrambled siRNA (scr) or with a CAXII-targeting specific siRNA pool (siCAXII). HT29 cells were included as control. (A) The expression of CAXII was measured in whole cell lysates by Western blotting. The β-tubulin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (B) Confocal microscope analysis of cells stained for the proliferation marker Ki67. The samples were analyzed by laser scanning confocal microscopy for Ki67 protein signal (green fluorescence) or for PI (red fluorescence), used to visualize nuclei. Magnification: 60 × objective; 10 × ocular lens. Bar = 20 μm. (C) The percentage of cells positive to annexin V-FITC, as index of early apoptosis, and to PI, as index of late apoptosis, was measured by flow cytometry. Percentages indicate annexin V-positive cells (lower right quadrant), PI-positive cells (upper left quadrant), annexin V/PI-positive cells (upper right quadrant). The figures are representative of three experiments with similar results. (D) Western blot analysis of the autophagy markers beclin, ATG12 and LC3B. The β-tubulin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (E) Cells were fixed and stained for β-galactosidase activity, then examined by fluorescence microscopy. Magnification: 20 × objective; 10 × ocular lens. Bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4466649&req=5

Figure 5: Depletion of CAXII does not affect proliferation and survival of chemoresistant cellsHT29/dx cells were cultured for 48 h with fresh medium (CTRL), treated with a non targeting scrambled siRNA (scr) or with a CAXII-targeting specific siRNA pool (siCAXII). HT29 cells were included as control. (A) The expression of CAXII was measured in whole cell lysates by Western blotting. The β-tubulin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (B) Confocal microscope analysis of cells stained for the proliferation marker Ki67. The samples were analyzed by laser scanning confocal microscopy for Ki67 protein signal (green fluorescence) or for PI (red fluorescence), used to visualize nuclei. Magnification: 60 × objective; 10 × ocular lens. Bar = 20 μm. (C) The percentage of cells positive to annexin V-FITC, as index of early apoptosis, and to PI, as index of late apoptosis, was measured by flow cytometry. Percentages indicate annexin V-positive cells (lower right quadrant), PI-positive cells (upper left quadrant), annexin V/PI-positive cells (upper right quadrant). The figures are representative of three experiments with similar results. (D) Western blot analysis of the autophagy markers beclin, ATG12 and LC3B. The β-tubulin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (E) Cells were fixed and stained for β-galactosidase activity, then examined by fluorescence microscopy. Magnification: 20 × objective; 10 × ocular lens. Bar = 100 μm.

Mentions: To investigate the functional role of CAXII in chemoresistant cells, we produced a HT29/dx subclone silenced for CAXII (Figure 5A). HT29 and HT29/dx cells did not show any appreciable difference in terms of: cell proliferation, as revealed by the proportion of Ki67-positive cells (Figure 5B); spontaneous apoptotic cell death, as indicated by the percentage of annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI)-positive cells (Figure 5C); autophagy, as indicated by the expression level of classical autophagic markers such as beclin, ATG12 and LC3B (Figure 5D). Interestingly, untreated HT29/dx cells appeared more senescent than parental HT29 cells, as suggested by higher staining with β-galactosidase (Figure 5E). Despite the documented role of CAXII as a pro-oncogenic factor [22], enzyme silencing did not alter any of these parameters in chemoresistant cells (Figure 5B–5E).


Carbonic anhydrase XII is a new therapeutic target to overcome chemoresistance in cancer cells.

Kopecka J, Campia I, Jacobs A, Frei AP, Ghigo D, Wollscheid B, Riganti C - Oncotarget (2015)

Depletion of CAXII does not affect proliferation and survival of chemoresistant cellsHT29/dx cells were cultured for 48 h with fresh medium (CTRL), treated with a non targeting scrambled siRNA (scr) or with a CAXII-targeting specific siRNA pool (siCAXII). HT29 cells were included as control. (A) The expression of CAXII was measured in whole cell lysates by Western blotting. The β-tubulin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (B) Confocal microscope analysis of cells stained for the proliferation marker Ki67. The samples were analyzed by laser scanning confocal microscopy for Ki67 protein signal (green fluorescence) or for PI (red fluorescence), used to visualize nuclei. Magnification: 60 × objective; 10 × ocular lens. Bar = 20 μm. (C) The percentage of cells positive to annexin V-FITC, as index of early apoptosis, and to PI, as index of late apoptosis, was measured by flow cytometry. Percentages indicate annexin V-positive cells (lower right quadrant), PI-positive cells (upper left quadrant), annexin V/PI-positive cells (upper right quadrant). The figures are representative of three experiments with similar results. (D) Western blot analysis of the autophagy markers beclin, ATG12 and LC3B. The β-tubulin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (E) Cells were fixed and stained for β-galactosidase activity, then examined by fluorescence microscopy. Magnification: 20 × objective; 10 × ocular lens. Bar = 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4466649&req=5

Figure 5: Depletion of CAXII does not affect proliferation and survival of chemoresistant cellsHT29/dx cells were cultured for 48 h with fresh medium (CTRL), treated with a non targeting scrambled siRNA (scr) or with a CAXII-targeting specific siRNA pool (siCAXII). HT29 cells were included as control. (A) The expression of CAXII was measured in whole cell lysates by Western blotting. The β-tubulin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (B) Confocal microscope analysis of cells stained for the proliferation marker Ki67. The samples were analyzed by laser scanning confocal microscopy for Ki67 protein signal (green fluorescence) or for PI (red fluorescence), used to visualize nuclei. Magnification: 60 × objective; 10 × ocular lens. Bar = 20 μm. (C) The percentage of cells positive to annexin V-FITC, as index of early apoptosis, and to PI, as index of late apoptosis, was measured by flow cytometry. Percentages indicate annexin V-positive cells (lower right quadrant), PI-positive cells (upper left quadrant), annexin V/PI-positive cells (upper right quadrant). The figures are representative of three experiments with similar results. (D) Western blot analysis of the autophagy markers beclin, ATG12 and LC3B. The β-tubulin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (E) Cells were fixed and stained for β-galactosidase activity, then examined by fluorescence microscopy. Magnification: 20 × objective; 10 × ocular lens. Bar = 100 μm.
Mentions: To investigate the functional role of CAXII in chemoresistant cells, we produced a HT29/dx subclone silenced for CAXII (Figure 5A). HT29 and HT29/dx cells did not show any appreciable difference in terms of: cell proliferation, as revealed by the proportion of Ki67-positive cells (Figure 5B); spontaneous apoptotic cell death, as indicated by the percentage of annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI)-positive cells (Figure 5C); autophagy, as indicated by the expression level of classical autophagic markers such as beclin, ATG12 and LC3B (Figure 5D). Interestingly, untreated HT29/dx cells appeared more senescent than parental HT29 cells, as suggested by higher staining with β-galactosidase (Figure 5E). Despite the documented role of CAXII as a pro-oncogenic factor [22], enzyme silencing did not alter any of these parameters in chemoresistant cells (Figure 5B–5E).

Bottom Line: Multidrug resistance (MDR) in cancer cells is a challenging phenomenon often associated with P-glycoprotein (Pgp) surface expression.CAXII and Pgp physically interacted at the cell surface.CAXII silencing or pharmacological inhibition with acetazolamide decreased the ATPase activity of Pgp by altering the optimal pH at which Pgp operated and promoted chemosensitization to Pgp substrates in MDR cells.We propose CAXII as a new secondary marker of the MDR phenotype that influences Pgp activity directly and can be used as a pharmacological target for MDR research and potential treatment.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, University of Torino, 10126 Torino, Italy.

ABSTRACT
Multidrug resistance (MDR) in cancer cells is a challenging phenomenon often associated with P-glycoprotein (Pgp) surface expression. Finding new ways to bypass Pgp-mediated MDR still remains a daunting challenge towards the successful treatment of malignant neoplasms such as colorectal cancer.We applied the Cell Surface Capture technology to chemosensitive and chemoresistant human colon cancer to explore the cell surface proteome of Pgp-expressing cells in a discovery-driven fashion. Comparative quantitative analysis of identified cell surface glycoproteins revealed carbonic anhydrase type XII (CAXII) to be up-regulated on the surface of chemoresistant cells, similarly to Pgp. In cellular models showing an acquired MDR phenotype due to the selective pressure of chemotherapy, the progressive increase of the transcription factor hypoxia-inducible factor-1 alpha was paralleled by the simultaneous up-regulation of Pgp and CAXII. CAXII and Pgp physically interacted at the cell surface. CAXII silencing or pharmacological inhibition with acetazolamide decreased the ATPase activity of Pgp by altering the optimal pH at which Pgp operated and promoted chemosensitization to Pgp substrates in MDR cells.We propose CAXII as a new secondary marker of the MDR phenotype that influences Pgp activity directly and can be used as a pharmacological target for MDR research and potential treatment.

Show MeSH
Related in: MedlinePlus