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Activation of Carbonic Anhydrase IX by Alternatively Spliced Tissue Factor Under Late-Stage Tumor Conditions

View Article: PubMed Central - PubMed

ABSTRACT

Molecules of the coagulation pathway predispose patients to cancer-associated thrombosis and also trigger intracellular signaling pathways that promote cancer progression. The primary transcript of Tissue Factor, the main physiologic trigger of blood clotting, can undergo alternative splicing yielding a secreted variant, termed asTF (alternatively spliced Tissue Factor). asTF is not required for normal hemostasis, but its expression levels positively correlate with advanced tumor stages in several cancers, including pancreatic adenocarcinoma. The asTF-over-expressing pancreatic ductal adenocarcinoma cell line Pt45.P1/asTF+ and its parent cell line Pt45.P1 were tested for growth and mobility under normoxic conditions that model early stage tumors, and in the hypoxic environment of late-stage cancers. asTF over-expression in Pt45.P1 cells conveys increased proliferative ability. According to cell cycle analysis, the major fraction of Pt45.P1/asTF+ cells reside in the dividing G2/M phase of the cell cycle, whereas the parental Pt45.P1 cells are mostly confined to the quiescent G0/G1 phase. asTF over-expression is also associated with significantly higher mobility in cells plated under either normoxia or hypoxia. A hypoxic environment leads to upregulation of Carbonic Anhydrase IX (CAIX), which is more pronounced in Pt45.P1/asTF+ cells. Inhibition of CAIX by the compound U-104 significantly decreases cell growth and mobility of Pt45.P1/asTF+ cells in hypoxia, but not in normoxia. U-104 also reduces the growth of Pt45.P1/asTF+ orthotopic tumors in nude mice. CAIX is a novel downstream mediator of asTF in pancreatic cancer, particularly under hypoxic conditions that model late-stage tumor micro-environment.

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Decreased cell cycle progression under CAIX inhibitionThe effect of 75 μM U-104 on cell cycle progression was assessed by flow cytometry after propidium iodide staining. A) Flow cytometry profiles of PT45.P1 cells transfected with asTF or vector after staining with propidium iodide to analyze the phases of the cell cycle. The plating conditions are indicated above each graph. B,D) Percentage of cells in the G0/G1 phase of the cell cycle for Pt45.P1/asTF+ cells (B) and Pt45.P1 cells (D). C,E) Percentage of cells in the G2/M phase of the cell cycle for Pt45.P1/asTF+ cells (C) and Pt45.P1 cells (E). The error bars are SEM. * indicates significance at p < 0.05.
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Figure 4: Decreased cell cycle progression under CAIX inhibitionThe effect of 75 μM U-104 on cell cycle progression was assessed by flow cytometry after propidium iodide staining. A) Flow cytometry profiles of PT45.P1 cells transfected with asTF or vector after staining with propidium iodide to analyze the phases of the cell cycle. The plating conditions are indicated above each graph. B,D) Percentage of cells in the G0/G1 phase of the cell cycle for Pt45.P1/asTF+ cells (B) and Pt45.P1 cells (D). C,E) Percentage of cells in the G2/M phase of the cell cycle for Pt45.P1/asTF+ cells (C) and Pt45.P1 cells (E). The error bars are SEM. * indicates significance at p < 0.05.

Mentions: When CAIX was inhibited by U-104 in adherent cells under late-stage conditions, the percentage of Pt45.P1/asTF+ cells in the G0/G1 phase significantly increased by 44% as compared to Pt45.P1/asTF+ cells without drug treatment (Figure 4). This inhibition via U-104 also reduced the percentage of cells in the G2/M phase of the cells cycle in the late-stage-like environment, although the p-value only reached 0.07.


Activation of Carbonic Anhydrase IX by Alternatively Spliced Tissue Factor Under Late-Stage Tumor Conditions
Decreased cell cycle progression under CAIX inhibitionThe effect of 75 μM U-104 on cell cycle progression was assessed by flow cytometry after propidium iodide staining. A) Flow cytometry profiles of PT45.P1 cells transfected with asTF or vector after staining with propidium iodide to analyze the phases of the cell cycle. The plating conditions are indicated above each graph. B,D) Percentage of cells in the G0/G1 phase of the cell cycle for Pt45.P1/asTF+ cells (B) and Pt45.P1 cells (D). C,E) Percentage of cells in the G2/M phase of the cell cycle for Pt45.P1/asTF+ cells (C) and Pt45.P1 cells (E). The error bars are SEM. * indicates significance at p < 0.05.
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Related In: Results  -  Collection

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Figure 4: Decreased cell cycle progression under CAIX inhibitionThe effect of 75 μM U-104 on cell cycle progression was assessed by flow cytometry after propidium iodide staining. A) Flow cytometry profiles of PT45.P1 cells transfected with asTF or vector after staining with propidium iodide to analyze the phases of the cell cycle. The plating conditions are indicated above each graph. B,D) Percentage of cells in the G0/G1 phase of the cell cycle for Pt45.P1/asTF+ cells (B) and Pt45.P1 cells (D). C,E) Percentage of cells in the G2/M phase of the cell cycle for Pt45.P1/asTF+ cells (C) and Pt45.P1 cells (E). The error bars are SEM. * indicates significance at p < 0.05.
Mentions: When CAIX was inhibited by U-104 in adherent cells under late-stage conditions, the percentage of Pt45.P1/asTF+ cells in the G0/G1 phase significantly increased by 44% as compared to Pt45.P1/asTF+ cells without drug treatment (Figure 4). This inhibition via U-104 also reduced the percentage of cells in the G2/M phase of the cells cycle in the late-stage-like environment, although the p-value only reached 0.07.

View Article: PubMed Central - PubMed

ABSTRACT

Molecules of the coagulation pathway predispose patients to cancer-associated thrombosis and also trigger intracellular signaling pathways that promote cancer progression. The primary transcript of Tissue Factor, the main physiologic trigger of blood clotting, can undergo alternative splicing yielding a secreted variant, termed asTF (alternatively spliced Tissue Factor). asTF is not required for normal hemostasis, but its expression levels positively correlate with advanced tumor stages in several cancers, including pancreatic adenocarcinoma. The asTF-over-expressing pancreatic ductal adenocarcinoma cell line Pt45.P1/asTF+ and its parent cell line Pt45.P1 were tested for growth and mobility under normoxic conditions that model early stage tumors, and in the hypoxic environment of late-stage cancers. asTF over-expression in Pt45.P1 cells conveys increased proliferative ability. According to cell cycle analysis, the major fraction of Pt45.P1/asTF+ cells reside in the dividing G2/M phase of the cell cycle, whereas the parental Pt45.P1 cells are mostly confined to the quiescent G0/G1 phase. asTF over-expression is also associated with significantly higher mobility in cells plated under either normoxia or hypoxia. A hypoxic environment leads to upregulation of Carbonic Anhydrase IX (CAIX), which is more pronounced in Pt45.P1/asTF+ cells. Inhibition of CAIX by the compound U-104 significantly decreases cell growth and mobility of Pt45.P1/asTF+ cells in hypoxia, but not in normoxia. U-104 also reduces the growth of Pt45.P1/asTF+ orthotopic tumors in nude mice. CAIX is a novel downstream mediator of asTF in pancreatic cancer, particularly under hypoxic conditions that model late-stage tumor micro-environment.

No MeSH data available.


Related in: MedlinePlus