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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.

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Expression of CAXII in chemosensitive and chemoresistant human cancer cellsHuman chemosensitive colon cancer HT29 cells and their chemoresistant counterpart HT29/dx cells, human chemosensitive lung cancer A549 cells and their chemoresistant counterpart A549/dx cells, human chemosensitive osteosarcoma U2-OS cells and the chemoresistant clones U2-OS/dx 30, U2-OS/dx 100, U2-OS/dx 580 were subjected to the following assays. (A) Confocal microscope analysis of HT29 and HT29/dx cells stained for CAXII. The samples were analyzed by laser scanning confocal microscope for green fluorescence signal (CAXII) or by Nomarski differential interference contrast (DIC) optics. Magnification: 60 × objective; 10 × ocular lens. Bar = 20 μm. (B) Western blot analysis of biotinylated plasma membrane associated CAXII, Pgp and MRP1 in HT29 and HT29/dx cells. The pan-cadherin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (C) Whole cell lysates were analyzed by Western blotting for the expression of CAXII, Pgp and MRP1. The β-tubulin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (D) Linear regression analysis between CAXII and Pgp expression. The mean band density of CAXII and Pgp (panel C), expressed as arbitrary units, was calculated by ImageJ software (http://www.rsb.info.nih.gov/ij/). r coefficient was calculated using Fig. P software (Fig. P Software Inc., Hamilton, Canada).
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Figure 2: Expression of CAXII in chemosensitive and chemoresistant human cancer cellsHuman chemosensitive colon cancer HT29 cells and their chemoresistant counterpart HT29/dx cells, human chemosensitive lung cancer A549 cells and their chemoresistant counterpart A549/dx cells, human chemosensitive osteosarcoma U2-OS cells and the chemoresistant clones U2-OS/dx 30, U2-OS/dx 100, U2-OS/dx 580 were subjected to the following assays. (A) Confocal microscope analysis of HT29 and HT29/dx cells stained for CAXII. The samples were analyzed by laser scanning confocal microscope for green fluorescence signal (CAXII) or by Nomarski differential interference contrast (DIC) optics. Magnification: 60 × objective; 10 × ocular lens. Bar = 20 μm. (B) Western blot analysis of biotinylated plasma membrane associated CAXII, Pgp and MRP1 in HT29 and HT29/dx cells. The pan-cadherin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (C) Whole cell lysates were analyzed by Western blotting for the expression of CAXII, Pgp and MRP1. The β-tubulin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (D) Linear regression analysis between CAXII and Pgp expression. The mean band density of CAXII and Pgp (panel C), expressed as arbitrary units, was calculated by ImageJ software (http://www.rsb.info.nih.gov/ij/). r coefficient was calculated using Fig. P software (Fig. P Software Inc., Hamilton, Canada).

Mentions: Among the ABC transporters detected on the surface of chemoresistant cells, ABCC1/MRP1 was more abundant on HT29/dx cells, and ABCC3/MRP3, ABCA1, ABCA2 showed lower expression levels. One of the proteins with highest relative expression in chemoresistant cells was CAXII, which was 16-fold more expressed in HT29/dx cells than in parental HT29 cells (Figure 1B and Supplemental Table 1). CAXII topology and identified glycopeptides are shown in Supplemental Figure 1. These data were confirmed by confocal microscopy analysis (Figure 2A) and by immunoblot analysis of biotinylated extracts from HT29 and HT29/dx cells (Figure 2B). Biotinylation assays confirmed that Pgp and MRP1 were more abundant on the surface of HT29/dx compared to HT29 cells (Figure 2B). The ratio of N-glycosylated to deglycosylated Pgp in plasma membrane extracts was about 1 in HT29/dx cells, whereas the ratio of N-glycosylated to deglycosylated MRP1 was higher than 1 (Supplemental Figure 2A and 2B).


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)

Expression of CAXII in chemosensitive and chemoresistant human cancer cellsHuman chemosensitive colon cancer HT29 cells and their chemoresistant counterpart HT29/dx cells, human chemosensitive lung cancer A549 cells and their chemoresistant counterpart A549/dx cells, human chemosensitive osteosarcoma U2-OS cells and the chemoresistant clones U2-OS/dx 30, U2-OS/dx 100, U2-OS/dx 580 were subjected to the following assays. (A) Confocal microscope analysis of HT29 and HT29/dx cells stained for CAXII. The samples were analyzed by laser scanning confocal microscope for green fluorescence signal (CAXII) or by Nomarski differential interference contrast (DIC) optics. Magnification: 60 × objective; 10 × ocular lens. Bar = 20 μm. (B) Western blot analysis of biotinylated plasma membrane associated CAXII, Pgp and MRP1 in HT29 and HT29/dx cells. The pan-cadherin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (C) Whole cell lysates were analyzed by Western blotting for the expression of CAXII, Pgp and MRP1. The β-tubulin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (D) Linear regression analysis between CAXII and Pgp expression. The mean band density of CAXII and Pgp (panel C), expressed as arbitrary units, was calculated by ImageJ software (http://www.rsb.info.nih.gov/ij/). r coefficient was calculated using Fig. P software (Fig. P Software Inc., Hamilton, Canada).
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Figure 2: Expression of CAXII in chemosensitive and chemoresistant human cancer cellsHuman chemosensitive colon cancer HT29 cells and their chemoresistant counterpart HT29/dx cells, human chemosensitive lung cancer A549 cells and their chemoresistant counterpart A549/dx cells, human chemosensitive osteosarcoma U2-OS cells and the chemoresistant clones U2-OS/dx 30, U2-OS/dx 100, U2-OS/dx 580 were subjected to the following assays. (A) Confocal microscope analysis of HT29 and HT29/dx cells stained for CAXII. The samples were analyzed by laser scanning confocal microscope for green fluorescence signal (CAXII) or by Nomarski differential interference contrast (DIC) optics. Magnification: 60 × objective; 10 × ocular lens. Bar = 20 μm. (B) Western blot analysis of biotinylated plasma membrane associated CAXII, Pgp and MRP1 in HT29 and HT29/dx cells. The pan-cadherin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (C) Whole cell lysates were analyzed by Western blotting for the expression of CAXII, Pgp and MRP1. The β-tubulin expression was used as a control of equal protein loading. The figure is representative of three experiments with similar results. (D) Linear regression analysis between CAXII and Pgp expression. The mean band density of CAXII and Pgp (panel C), expressed as arbitrary units, was calculated by ImageJ software (http://www.rsb.info.nih.gov/ij/). r coefficient was calculated using Fig. P software (Fig. P Software Inc., Hamilton, Canada).
Mentions: Among the ABC transporters detected on the surface of chemoresistant cells, ABCC1/MRP1 was more abundant on HT29/dx cells, and ABCC3/MRP3, ABCA1, ABCA2 showed lower expression levels. One of the proteins with highest relative expression in chemoresistant cells was CAXII, which was 16-fold more expressed in HT29/dx cells than in parental HT29 cells (Figure 1B and Supplemental Table 1). CAXII topology and identified glycopeptides are shown in Supplemental Figure 1. These data were confirmed by confocal microscopy analysis (Figure 2A) and by immunoblot analysis of biotinylated extracts from HT29 and HT29/dx cells (Figure 2B). Biotinylation assays confirmed that Pgp and MRP1 were more abundant on the surface of HT29/dx compared to HT29 cells (Figure 2B). The ratio of N-glycosylated to deglycosylated Pgp in plasma membrane extracts was about 1 in HT29/dx cells, whereas the ratio of N-glycosylated to deglycosylated MRP1 was higher than 1 (Supplemental Figure 2A and 2B).

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