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Knock-down of hypoxia-induced carbonic anhydrases IX and XII radiosensitizes tumor cells by increasing intracellular acidosis.

Doyen J, Parks SK, Marcié S, Pouysségur J, Chiche J - Front Oncol (2013)

Bottom Line: We found that diminishing the pH(i)-regulating capacity of fibroblasts through inhibition of Na(+)/H(+) exchanger 1 sensitize cells to radiation-induced cell death.Thirdly, we demonstrate that irradiation of LS174Tr spheroids, silenced for either ca9 or both ca9/ca12, showed a respective 50 and 75% increase in cell death as a result of a decrease in cell number in the radioresistant S phase and a disruption of CA-mediated pH(i) regulation.Finally, LS174Tr tumor progression was strongly decreased when ca9/ca12 silencing was combined with irradiation in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute for Research on Cancer and Aging of Nice, CNRS UMR 7284, University of Nice Sophia-Antipolis, Nice, France ; Department of Radiation Oncology, Centre Antoine-Lacassagne , Nice, France.

ABSTRACT
The relationship between acidosis within the tumor microenvironment and radioresistance of hypoxic tumor cells remains unclear. Previously we reported that hypoxia-induced carbonic anhydrases (CA) IX and CAXII constitute a robust intracellular pH (pH(i))-regulating system that confers a survival advantage on hypoxic human colon carcinoma LS174Tr cells in acidic microenvironments. Here we investigate the role of acidosis, CAIX and CAXII knock-down in combination with ionizing radiation. Fibroblasts cells (-/+ CAIX) and LS174Tr cells (inducible knock-down for ca9/ca12) were analyzed for cell cycle phase distribution and survival after irradiation in extracellular pH(o) manipulations and hypoxia (1% O(2)) exposure. Radiotherapy was used to target ca9/ca12-silenced LS174Tr tumors grown in nude mice. We found that diminishing the pH(i)-regulating capacity of fibroblasts through inhibition of Na(+)/H(+) exchanger 1 sensitize cells to radiation-induced cell death. Secondly, the pH(i)-regulating function of CAIX plays a key protective role in irradiated fibroblasts in an acidic environment as accompanied by a reduced number of cells in the radiosensitive phases of the cell cycle. Thirdly, we demonstrate that irradiation of LS174Tr spheroids, silenced for either ca9 or both ca9/ca12, showed a respective 50 and 75% increase in cell death as a result of a decrease in cell number in the radioresistant S phase and a disruption of CA-mediated pH(i) regulation. Finally, LS174Tr tumor progression was strongly decreased when ca9/ca12 silencing was combined with irradiation in vivo. These findings highlight the combinatory use of radiotherapy with targeting of the pH(i)-regulating CAs as an anti-cancer strategy.

No MeSH data available.


Related in: MedlinePlus

Silencing of the hypoxia-induced pHi-regulating enzymes ca9 and ca12 induced in vitro cell death of LS174Tr cells when combined with ionizing radiation.(A)Inset: Expression of CAIX and CAXII in colon carcinoma Tetracycline (Tet)-inducible LS-shca9/ctl -Tet cells either silenced for ca9 (LS-shca9/ctl +Tet) or for ca12 (LS-shca9/ca12- -Tet) or both ca9 and ca12 (LS-shca9/ca12- +Tet) in hypoxia 1% O2 (Hx) for 48 h. Hsp90 was used as a loading control. The cell cycle phase distribution was determined by FACScan analysis of LS-shca9/ctl -/+Tet and LS-shca9/ca12- -/+Tet cells exposed hypoxia of 1% O2 (Hx) for 24 h in a -containing medium. (B) Immunoblotting of p21, β1 integrin, and Hsp90 (loading control) in LS-shca9/ctl and LS-shca9/ca12- cells pre-incubated for 4 days in the presence (+Tet) or absence (-Tet) of Tet to silence ca9, before exposure to hypoxia of 1% O2 (Hx) for 48 h (H). (C,D). Tet-inducible LS174Tr cells silenced for ca9 or ca12 or both ca9 and ca12 were cultured as spheroids in a CO2 atmosphere and HEPES-buffered -free medium (pHo 7.7 in the absence (-Tet) or presence (+Tet) of Tet for 8 days before they were irradiated (8 Gy) or not (0 Gy). After irradiation, spheroids were transferred to polyhema-coated 96-well plates containing fresh medium for 5 days. Spheroids were then subjected to Accutase dissociation and individualized live cells (C) and dead cells (D) were counted using trypan blue. The spheroid proliferation index was calculated as the ratio of the number of living cells counted at day 13 to the number of cells at day 0. Data represent the average of three independent experiments. (E) The clonogenic capacity of LS174Tr-shca9/ctl -/+Tet and LS174TR-shca9/ca12- -/+Tet cells exposed to hypoxia (1% O2) for 48 h in a regular medium, was measured 10 days after irradiation (0,1, 2, 4, 6, and 8 Gy). Dishes were stained with Giemsa (Fluka). The colonies were counted with Image J software.
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Figure 3: Silencing of the hypoxia-induced pHi-regulating enzymes ca9 and ca12 induced in vitro cell death of LS174Tr cells when combined with ionizing radiation.(A)Inset: Expression of CAIX and CAXII in colon carcinoma Tetracycline (Tet)-inducible LS-shca9/ctl -Tet cells either silenced for ca9 (LS-shca9/ctl +Tet) or for ca12 (LS-shca9/ca12- -Tet) or both ca9 and ca12 (LS-shca9/ca12- +Tet) in hypoxia 1% O2 (Hx) for 48 h. Hsp90 was used as a loading control. The cell cycle phase distribution was determined by FACScan analysis of LS-shca9/ctl -/+Tet and LS-shca9/ca12- -/+Tet cells exposed hypoxia of 1% O2 (Hx) for 24 h in a -containing medium. (B) Immunoblotting of p21, β1 integrin, and Hsp90 (loading control) in LS-shca9/ctl and LS-shca9/ca12- cells pre-incubated for 4 days in the presence (+Tet) or absence (-Tet) of Tet to silence ca9, before exposure to hypoxia of 1% O2 (Hx) for 48 h (H). (C,D). Tet-inducible LS174Tr cells silenced for ca9 or ca12 or both ca9 and ca12 were cultured as spheroids in a CO2 atmosphere and HEPES-buffered -free medium (pHo 7.7 in the absence (-Tet) or presence (+Tet) of Tet for 8 days before they were irradiated (8 Gy) or not (0 Gy). After irradiation, spheroids were transferred to polyhema-coated 96-well plates containing fresh medium for 5 days. Spheroids were then subjected to Accutase dissociation and individualized live cells (C) and dead cells (D) were counted using trypan blue. The spheroid proliferation index was calculated as the ratio of the number of living cells counted at day 13 to the number of cells at day 0. Data represent the average of three independent experiments. (E) The clonogenic capacity of LS174Tr-shca9/ctl -/+Tet and LS174TR-shca9/ca12- -/+Tet cells exposed to hypoxia (1% O2) for 48 h in a regular medium, was measured 10 days after irradiation (0,1, 2, 4, 6, and 8 Gy). Dishes were stained with Giemsa (Fluka). The colonies were counted with Image J software.

Mentions: LS174Tr cells cultured in hypoxia before exposure to an increasing dose of ionizing radiation demonstrated a higher cloning efficiency than normoxic cells along with equal distribution of cell cycle phases before irradiation (data not shown). This established the classical radioresistance of LS174Tr cells as observed in other hypoxic cells and validated this model for our study. In a regular -containing medium a higher number of cells in the radiosensitive G1/G2/M phases was observed when ca9 or both ca9 and ca12 were silenced (Figure 3A). Protein expression levels of CAIX and CAXII in the Tet-inducible silencing of ca9 in control LS174Tr cells (LS-shca9/ctl) or ca12 silenced cells (LS-shca9/ca12-) were confirmed for efficient knock-down (Figure 3A, inset). In the same conditions, ca9 or both ca9/ca12 silencing was accompanied by an increase in p21, E-cadherin, and β1 integrin expression, which were associated with a reduced cell proliferation (Figure 3B). To mimic both the tumor hypoxic and proton gradient observed in vivo, we grew LS174 cells in three dimensions. Spheroids were grown in nominally bicarbonate free media to enhance the pH gradients that develop during spheroid growth. Irradiation of ca9-silenced spheroids (LS-shca9/ctl +Tet, 8 Gy) revealed a cumulative decrease in the proliferation index (Figure 3C) and a twofold increase in cell death from 27.5% (0 Gy) to 51.7% (8 Gy) when compared to non-irradiated ca9-silenced spheroids (Figure 3D). While ca12 silencing alone did not alter the proliferation rate of non-irradiated spheroids, irradiation of ca12-silenced cells (LS-shca9/ca12- -Tet 8 Gy) reduced the proliferation index (Figure 3C) and increased cell death from 27.5% (0 Gy) to 37.6% (8 Gy; Figure 3D). Irradiation of double silenced cells (LS-shca9/ca12- +Tet 8 Gy) strongly compromised proliferation and viability (75.5% cell death; Figures 3C,D). Clonogenic test confirmed that double silenced cells (LS-shca9/ca12- +Tet) exposed to hypoxia were less capable to recover from irradiation compared to control cells (LS-shca9/ctl -Tet) or single ca9 or ca12-silenced cells (LS-shca9/ctl +Tet and LS-shca9/ca12- -Tet; Figure 3E).


Knock-down of hypoxia-induced carbonic anhydrases IX and XII radiosensitizes tumor cells by increasing intracellular acidosis.

Doyen J, Parks SK, Marcié S, Pouysségur J, Chiche J - Front Oncol (2013)

Silencing of the hypoxia-induced pHi-regulating enzymes ca9 and ca12 induced in vitro cell death of LS174Tr cells when combined with ionizing radiation.(A)Inset: Expression of CAIX and CAXII in colon carcinoma Tetracycline (Tet)-inducible LS-shca9/ctl -Tet cells either silenced for ca9 (LS-shca9/ctl +Tet) or for ca12 (LS-shca9/ca12- -Tet) or both ca9 and ca12 (LS-shca9/ca12- +Tet) in hypoxia 1% O2 (Hx) for 48 h. Hsp90 was used as a loading control. The cell cycle phase distribution was determined by FACScan analysis of LS-shca9/ctl -/+Tet and LS-shca9/ca12- -/+Tet cells exposed hypoxia of 1% O2 (Hx) for 24 h in a -containing medium. (B) Immunoblotting of p21, β1 integrin, and Hsp90 (loading control) in LS-shca9/ctl and LS-shca9/ca12- cells pre-incubated for 4 days in the presence (+Tet) or absence (-Tet) of Tet to silence ca9, before exposure to hypoxia of 1% O2 (Hx) for 48 h (H). (C,D). Tet-inducible LS174Tr cells silenced for ca9 or ca12 or both ca9 and ca12 were cultured as spheroids in a CO2 atmosphere and HEPES-buffered -free medium (pHo 7.7 in the absence (-Tet) or presence (+Tet) of Tet for 8 days before they were irradiated (8 Gy) or not (0 Gy). After irradiation, spheroids were transferred to polyhema-coated 96-well plates containing fresh medium for 5 days. Spheroids were then subjected to Accutase dissociation and individualized live cells (C) and dead cells (D) were counted using trypan blue. The spheroid proliferation index was calculated as the ratio of the number of living cells counted at day 13 to the number of cells at day 0. Data represent the average of three independent experiments. (E) The clonogenic capacity of LS174Tr-shca9/ctl -/+Tet and LS174TR-shca9/ca12- -/+Tet cells exposed to hypoxia (1% O2) for 48 h in a regular medium, was measured 10 days after irradiation (0,1, 2, 4, 6, and 8 Gy). Dishes were stained with Giemsa (Fluka). The colonies were counted with Image J software.
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Figure 3: Silencing of the hypoxia-induced pHi-regulating enzymes ca9 and ca12 induced in vitro cell death of LS174Tr cells when combined with ionizing radiation.(A)Inset: Expression of CAIX and CAXII in colon carcinoma Tetracycline (Tet)-inducible LS-shca9/ctl -Tet cells either silenced for ca9 (LS-shca9/ctl +Tet) or for ca12 (LS-shca9/ca12- -Tet) or both ca9 and ca12 (LS-shca9/ca12- +Tet) in hypoxia 1% O2 (Hx) for 48 h. Hsp90 was used as a loading control. The cell cycle phase distribution was determined by FACScan analysis of LS-shca9/ctl -/+Tet and LS-shca9/ca12- -/+Tet cells exposed hypoxia of 1% O2 (Hx) for 24 h in a -containing medium. (B) Immunoblotting of p21, β1 integrin, and Hsp90 (loading control) in LS-shca9/ctl and LS-shca9/ca12- cells pre-incubated for 4 days in the presence (+Tet) or absence (-Tet) of Tet to silence ca9, before exposure to hypoxia of 1% O2 (Hx) for 48 h (H). (C,D). Tet-inducible LS174Tr cells silenced for ca9 or ca12 or both ca9 and ca12 were cultured as spheroids in a CO2 atmosphere and HEPES-buffered -free medium (pHo 7.7 in the absence (-Tet) or presence (+Tet) of Tet for 8 days before they were irradiated (8 Gy) or not (0 Gy). After irradiation, spheroids were transferred to polyhema-coated 96-well plates containing fresh medium for 5 days. Spheroids were then subjected to Accutase dissociation and individualized live cells (C) and dead cells (D) were counted using trypan blue. The spheroid proliferation index was calculated as the ratio of the number of living cells counted at day 13 to the number of cells at day 0. Data represent the average of three independent experiments. (E) The clonogenic capacity of LS174Tr-shca9/ctl -/+Tet and LS174TR-shca9/ca12- -/+Tet cells exposed to hypoxia (1% O2) for 48 h in a regular medium, was measured 10 days after irradiation (0,1, 2, 4, 6, and 8 Gy). Dishes were stained with Giemsa (Fluka). The colonies were counted with Image J software.
Mentions: LS174Tr cells cultured in hypoxia before exposure to an increasing dose of ionizing radiation demonstrated a higher cloning efficiency than normoxic cells along with equal distribution of cell cycle phases before irradiation (data not shown). This established the classical radioresistance of LS174Tr cells as observed in other hypoxic cells and validated this model for our study. In a regular -containing medium a higher number of cells in the radiosensitive G1/G2/M phases was observed when ca9 or both ca9 and ca12 were silenced (Figure 3A). Protein expression levels of CAIX and CAXII in the Tet-inducible silencing of ca9 in control LS174Tr cells (LS-shca9/ctl) or ca12 silenced cells (LS-shca9/ca12-) were confirmed for efficient knock-down (Figure 3A, inset). In the same conditions, ca9 or both ca9/ca12 silencing was accompanied by an increase in p21, E-cadherin, and β1 integrin expression, which were associated with a reduced cell proliferation (Figure 3B). To mimic both the tumor hypoxic and proton gradient observed in vivo, we grew LS174 cells in three dimensions. Spheroids were grown in nominally bicarbonate free media to enhance the pH gradients that develop during spheroid growth. Irradiation of ca9-silenced spheroids (LS-shca9/ctl +Tet, 8 Gy) revealed a cumulative decrease in the proliferation index (Figure 3C) and a twofold increase in cell death from 27.5% (0 Gy) to 51.7% (8 Gy) when compared to non-irradiated ca9-silenced spheroids (Figure 3D). While ca12 silencing alone did not alter the proliferation rate of non-irradiated spheroids, irradiation of ca12-silenced cells (LS-shca9/ca12- -Tet 8 Gy) reduced the proliferation index (Figure 3C) and increased cell death from 27.5% (0 Gy) to 37.6% (8 Gy; Figure 3D). Irradiation of double silenced cells (LS-shca9/ca12- +Tet 8 Gy) strongly compromised proliferation and viability (75.5% cell death; Figures 3C,D). Clonogenic test confirmed that double silenced cells (LS-shca9/ca12- +Tet) exposed to hypoxia were less capable to recover from irradiation compared to control cells (LS-shca9/ctl -Tet) or single ca9 or ca12-silenced cells (LS-shca9/ctl +Tet and LS-shca9/ca12- -Tet; Figure 3E).

Bottom Line: We found that diminishing the pH(i)-regulating capacity of fibroblasts through inhibition of Na(+)/H(+) exchanger 1 sensitize cells to radiation-induced cell death.Thirdly, we demonstrate that irradiation of LS174Tr spheroids, silenced for either ca9 or both ca9/ca12, showed a respective 50 and 75% increase in cell death as a result of a decrease in cell number in the radioresistant S phase and a disruption of CA-mediated pH(i) regulation.Finally, LS174Tr tumor progression was strongly decreased when ca9/ca12 silencing was combined with irradiation in vivo.

View Article: PubMed Central - PubMed

Affiliation: Institute for Research on Cancer and Aging of Nice, CNRS UMR 7284, University of Nice Sophia-Antipolis, Nice, France ; Department of Radiation Oncology, Centre Antoine-Lacassagne , Nice, France.

ABSTRACT
The relationship between acidosis within the tumor microenvironment and radioresistance of hypoxic tumor cells remains unclear. Previously we reported that hypoxia-induced carbonic anhydrases (CA) IX and CAXII constitute a robust intracellular pH (pH(i))-regulating system that confers a survival advantage on hypoxic human colon carcinoma LS174Tr cells in acidic microenvironments. Here we investigate the role of acidosis, CAIX and CAXII knock-down in combination with ionizing radiation. Fibroblasts cells (-/+ CAIX) and LS174Tr cells (inducible knock-down for ca9/ca12) were analyzed for cell cycle phase distribution and survival after irradiation in extracellular pH(o) manipulations and hypoxia (1% O(2)) exposure. Radiotherapy was used to target ca9/ca12-silenced LS174Tr tumors grown in nude mice. We found that diminishing the pH(i)-regulating capacity of fibroblasts through inhibition of Na(+)/H(+) exchanger 1 sensitize cells to radiation-induced cell death. Secondly, the pH(i)-regulating function of CAIX plays a key protective role in irradiated fibroblasts in an acidic environment as accompanied by a reduced number of cells in the radiosensitive phases of the cell cycle. Thirdly, we demonstrate that irradiation of LS174Tr spheroids, silenced for either ca9 or both ca9/ca12, showed a respective 50 and 75% increase in cell death as a result of a decrease in cell number in the radioresistant S phase and a disruption of CA-mediated pH(i) regulation. Finally, LS174Tr tumor progression was strongly decreased when ca9/ca12 silencing was combined with irradiation in vivo. These findings highlight the combinatory use of radiotherapy with targeting of the pH(i)-regulating CAs as an anti-cancer strategy.

No MeSH data available.


Related in: MedlinePlus