Intracellular pH and CAIX inhibition in SCCNij202.

<p>S4 does not change CHOP mRNA (A) and MMP9 mRNA (B). Abbreviations: CHOP, C/EBP homologous protein; MMP9, matrix metallopeptidase 9; NS, not significant; 1× S4, 8 h, one i.p. injection S4, harvest after 8 hours; 1× S4, 24 h, one i.p. injection S4, harvest after 24 hours; 3× S4, one i.p. injection S4 a day for 3 days, harvest 8 hours after the last injection; 5× S4, one i.p. injection S4 a day for 5 days, harvest 8 hours after the last injection.</p

Encapsulation of anti-carbonic anhydrase IX antibody in hydrogel microspheres for tumor targeting

<p>Encapsulation is a well-established method of biomaterial protection, controlled release, and efficient delivery. Here we evaluated encapsulation of monoclonal antibody M75 directed to tumor biomarker carbonic anhydrase IX (CA IX) into alginate microbeads (SA-beads) or microcapsules made of sodium alginate, cellulose sulfate, and poly(methylene-co-guanidine) (PMCG). M75 antibody release was quantified using ELISA and its binding properties were assessed by immunodetection methods. SA-beads showed rapid M75 antibody release in the first hour, followed by steady release during the whole experiment of 7 days. In contrast, the M75 release from PMCG capsules was gradual, reaching the maximum concentration on the 7th day. The release was more efficient at pH 6.8 compared to pH 7.4. The released antibody could recognize CA IX, and target the CA IX-positive cells in 3D spheroids. In conclusion, SA-beads and PMCG microcapsules can be considered as promising antibody reservoirs for targeting of cancer cells.</p

No cytotoxic effects of CAIX inhibition in SCCNij202.

<p>S4 does not affect proliferation within CAIX positive areas (A), apoptosis within CAIX positive areas (B) or the amount of necrosis (C). Abbreviations: BrdUrd, bromodeoxyuridine; CAIX, carbonic anhydrase IX; LI, labeling index; NS, not significant, 1× S4, 8 h, one i.p. injection S4, harvest after 8 hours; 1× S4, 24 h, one i.p. injection S4, harvest after 24 hours; 3× S4, one i.p. injection S4 a day for 3 days, harvest 8 hours after the last injection; 5× S4, one i.p. injection S4 a day for 5 days, harvest 8 hours after the last injection.</p

CAIX ectodomain shedding after treatment with S4.

<p>S4 decreases CAIX ectodomain shedding in the serum, especially 8 hours after the first injection. Abbreviations: CAIX, carbonic anhydrase IX; 1× S4, 8 h, one i.p. injection S4, harvest after 8 hours; 1× S4, 24 h, one i.p. injection S4, harvest after 24 hours; 3× S4, one i.p. injection S4 a day for 3 days, harvest 8 hours after the last injection; 5× S4, one i.p. injection S4 a day for 5 days, harvest 8 hours after the last injection.</p

Characterization of Carbonic Anhydrase IX Interactome Reveals Proteins Assisting Its Nuclear Localization in Hypoxic Cells

Carbonic anhydrase IX (CA IX) is a transmembrane protein affecting pH regulation, cell migration/invasion, and survival in hypoxic tumors. Although the pathways related to CA IX have begun to emerge, molecular partners mediating its functions remain largely unknown. Here we characterize the CA IX interactome in hypoxic HEK-293 cells. Most of the identified CA IX-binding partners contain the HEAT/ARM repeat domain and belong to the nuclear transport machinery. We show that the interaction with two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs via the C-terminal region of CA IX and increases with protein phosphorylation. We also demonstrate that nuclear CA IX is enriched in hypoxic cells and is present in renal cell carcinoma tissues. These data place CA IX among the cell-surface signal transducers undergoing nuclear translocation. Accordingly, CA IX interactome involves also CAND1, which participates in both gene transcription and assembly of SCF ubiquitin ligase complexes. It is noteworthy that down-regulation of CAND1 leads to decreased CA IX protein levels apparently via affecting its stability. Our findings provide the first evidence that CA IX interacts with proteins involved in nuclear/cytoplasmic transport, gene transcription, and protein stability, and suggest the existence of nuclear CA IX protein subpopulations with a potential intracellular function, distinct from the crucial CA IX role at the cell surface

Characterization of Carbonic Anhydrase IX Interactome Reveals Proteins Assisting Its Nuclear Localization in Hypoxic Cells

Carbonic anhydrase IX (CA IX) is a transmembrane protein affecting pH regulation, cell migration/invasion, and survival in hypoxic tumors. Although the pathways related to CA IX have begun to emerge, molecular partners mediating its functions remain largely unknown. Here we characterize the CA IX interactome in hypoxic HEK-293 cells. Most of the identified CA IX-binding partners contain the HEAT/ARM repeat domain and belong to the nuclear transport machinery. We show that the interaction with two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs via the C-terminal region of CA IX and increases with protein phosphorylation. We also demonstrate that nuclear CA IX is enriched in hypoxic cells and is present in renal cell carcinoma tissues. These data place CA IX among the cell-surface signal transducers undergoing nuclear translocation. Accordingly, CA IX interactome involves also CAND1, which participates in both gene transcription and assembly of SCF ubiquitin ligase complexes. It is noteworthy that down-regulation of CAND1 leads to decreased CA IX protein levels apparently via affecting its stability. Our findings provide the first evidence that CA IX interacts with proteins involved in nuclear/cytoplasmic transport, gene transcription, and protein stability, and suggest the existence of nuclear CA IX protein subpopulations with a potential intracellular function, distinct from the crucial CA IX role at the cell surface

Characterization of Carbonic Anhydrase IX Interactome Reveals Proteins Assisting Its Nuclear Localization in Hypoxic Cells

Carbonic anhydrase IX (CA IX) is a transmembrane protein affecting pH regulation, cell migration/invasion, and survival in hypoxic tumors. Although the pathways related to CA IX have begun to emerge, molecular partners mediating its functions remain largely unknown. Here we characterize the CA IX interactome in hypoxic HEK-293 cells. Most of the identified CA IX-binding partners contain the HEAT/ARM repeat domain and belong to the nuclear transport machinery. We show that the interaction with two of these proteins, namely XPO1 exportin and TNPO1 importin, occurs via the C-terminal region of CA IX and increases with protein phosphorylation. We also demonstrate that nuclear CA IX is enriched in hypoxic cells and is present in renal cell carcinoma tissues. These data place CA IX among the cell-surface signal transducers undergoing nuclear translocation. Accordingly, CA IX interactome involves also CAND1, which participates in both gene transcription and assembly of SCF ubiquitin ligase complexes. It is noteworthy that down-regulation of CAND1 leads to decreased CA IX protein levels apparently via affecting its stability. Our findings provide the first evidence that CA IX interacts with proteins involved in nuclear/cytoplasmic transport, gene transcription, and protein stability, and suggest the existence of nuclear CA IX protein subpopulations with a potential intracellular function, distinct from the crucial CA IX role at the cell surface