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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

CAIX protects cells against irradiation-induced cell death in an acidic environment.(A) Cell cycle distribution of NHE-1-disrupted fibroblasts PS120 cells expressing (pca9) or not (pev) CAIX, in normoxia in a /CO2-free environment at pHo 7.0 or 7.5 for 24 h. (B–D) PS120-pev and PS120-pca9 cells (1 × 104) were plated in 60 mm dishes. Once attached cells were incubated in 30 mM HEPES-buffered -free medium adjusted to pHo 7.0 in the absence (B) or in the presence of 10 mM (D) or to pHo 7.5 (C) for 24 h in a CO2-free atmosphere. Dishes were then irradiated (0, 2, 4, 6, 8, and 10 Gy) and returned to a CO2-containing incubator with fresh regular -containing (44 mM) medium for 4 days. Cell death was determined by the trypan blue exclusion assay. Data represent the average of three independent experiments. (E) The clonogenic capacity of PS120-pev and PS120-pca9 cells exposed to a medium adjusted to pHo 7.0 or 7.5 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 ImageJ software according to the following parameters: particles size = 0.15–5 mm2 and circularity = 0.1–1.
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Figure 2: CAIX protects cells against irradiation-induced cell death in an acidic environment.(A) Cell cycle distribution of NHE-1-disrupted fibroblasts PS120 cells expressing (pca9) or not (pev) CAIX, in normoxia in a /CO2-free environment at pHo 7.0 or 7.5 for 24 h. (B–D) PS120-pev and PS120-pca9 cells (1 × 104) were plated in 60 mm dishes. Once attached cells were incubated in 30 mM HEPES-buffered -free medium adjusted to pHo 7.0 in the absence (B) or in the presence of 10 mM (D) or to pHo 7.5 (C) for 24 h in a CO2-free atmosphere. Dishes were then irradiated (0, 2, 4, 6, 8, and 10 Gy) and returned to a CO2-containing incubator with fresh regular -containing (44 mM) medium for 4 days. Cell death was determined by the trypan blue exclusion assay. Data represent the average of three independent experiments. (E) The clonogenic capacity of PS120-pev and PS120-pca9 cells exposed to a medium adjusted to pHo 7.0 or 7.5 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 ImageJ software according to the following parameters: particles size = 0.15–5 mm2 and circularity = 0.1–1.

Mentions: We have previously demonstrated that expression of catalytically active CAIX in NHE-1-deficient CCL39 fibroblasts (PS120 cells) maintains a higher pHi compared to control PS120 cells lacking CAIX, when cells were exposed to a nominally bicarbonate free acidic medium (Chiche et al., 2010a). Here we showed that in the condition where CAIX is required for pHi regulation (pHo 7.0 compared to pHo 7.5 medium), expression of CAIX in PS120 cells (PS120-pca9) maintains the distribution of the cell cycle phases, while in the absence of CAIX, PS120-pev cells demonstrate a 35% reduction in the most radioresistant S phase (Figure 2A). Consequently, control PS120-pev cells growing at pHo 7.0 were shown to be more radiosensitive than PS120-pca9 cells, with 70% cell death following irradiation of 10 Gy for PS120-pev cells compared to 37% for PS120-pca9 cells (Figure 2B). Of note, PS120-pca9 cells irradiated with 10 Gy at pHo 7.0 exhibited similar cell death rates to that at pHo 7.5 while PS120-pev cells had much higher cell death at low pHo (Figures 2B,C). Thus, active CAIX protects cells against ionizing irradiation at low pH. To definitively validate that the pHi-regulating functions of CAIX are indeed involved in cellular radioprotection, PS120-pev and PS120-pca9 cells were exposed to a pHo 7.0 medium containing 10 mM . This addition has been shown previously to maintain pHi in acidic pHo environments (Chiche et al., 2010a). Irradiation of PS120-pev cells in the presence of reduced the percentage of cell death to that obtained for PS120-pca9 cells in a pHo 7.0 medium (Figure 2D). Cloning efficiency experiments also confirm the capacity of irradiated cells to survive and recover following irradiation. From 4 to 8 Gy single doses of ionizing radiation of PS120-pev cells exposed to a pHo 7.0 medium drastically reduced the cloning efficiency, compared to that observed in a pHo 7.5 medium (Figure 2E, left panel). In contrast, PS120-pca9 cells exposed to a pHo 7.0 medium were capable to recover after irradiation, to the same extent that we observed in a pHo 7.5 (Figure 2E, right panel). Taken together these results suggest that the pHi-regulating properties of NHE-1 and CAIX protect cells against irradiation.


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)

CAIX protects cells against irradiation-induced cell death in an acidic environment.(A) Cell cycle distribution of NHE-1-disrupted fibroblasts PS120 cells expressing (pca9) or not (pev) CAIX, in normoxia in a /CO2-free environment at pHo 7.0 or 7.5 for 24 h. (B–D) PS120-pev and PS120-pca9 cells (1 × 104) were plated in 60 mm dishes. Once attached cells were incubated in 30 mM HEPES-buffered -free medium adjusted to pHo 7.0 in the absence (B) or in the presence of 10 mM (D) or to pHo 7.5 (C) for 24 h in a CO2-free atmosphere. Dishes were then irradiated (0, 2, 4, 6, 8, and 10 Gy) and returned to a CO2-containing incubator with fresh regular -containing (44 mM) medium for 4 days. Cell death was determined by the trypan blue exclusion assay. Data represent the average of three independent experiments. (E) The clonogenic capacity of PS120-pev and PS120-pca9 cells exposed to a medium adjusted to pHo 7.0 or 7.5 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 ImageJ software according to the following parameters: particles size = 0.15–5 mm2 and circularity = 0.1–1.
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Figure 2: CAIX protects cells against irradiation-induced cell death in an acidic environment.(A) Cell cycle distribution of NHE-1-disrupted fibroblasts PS120 cells expressing (pca9) or not (pev) CAIX, in normoxia in a /CO2-free environment at pHo 7.0 or 7.5 for 24 h. (B–D) PS120-pev and PS120-pca9 cells (1 × 104) were plated in 60 mm dishes. Once attached cells were incubated in 30 mM HEPES-buffered -free medium adjusted to pHo 7.0 in the absence (B) or in the presence of 10 mM (D) or to pHo 7.5 (C) for 24 h in a CO2-free atmosphere. Dishes were then irradiated (0, 2, 4, 6, 8, and 10 Gy) and returned to a CO2-containing incubator with fresh regular -containing (44 mM) medium for 4 days. Cell death was determined by the trypan blue exclusion assay. Data represent the average of three independent experiments. (E) The clonogenic capacity of PS120-pev and PS120-pca9 cells exposed to a medium adjusted to pHo 7.0 or 7.5 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 ImageJ software according to the following parameters: particles size = 0.15–5 mm2 and circularity = 0.1–1.
Mentions: We have previously demonstrated that expression of catalytically active CAIX in NHE-1-deficient CCL39 fibroblasts (PS120 cells) maintains a higher pHi compared to control PS120 cells lacking CAIX, when cells were exposed to a nominally bicarbonate free acidic medium (Chiche et al., 2010a). Here we showed that in the condition where CAIX is required for pHi regulation (pHo 7.0 compared to pHo 7.5 medium), expression of CAIX in PS120 cells (PS120-pca9) maintains the distribution of the cell cycle phases, while in the absence of CAIX, PS120-pev cells demonstrate a 35% reduction in the most radioresistant S phase (Figure 2A). Consequently, control PS120-pev cells growing at pHo 7.0 were shown to be more radiosensitive than PS120-pca9 cells, with 70% cell death following irradiation of 10 Gy for PS120-pev cells compared to 37% for PS120-pca9 cells (Figure 2B). Of note, PS120-pca9 cells irradiated with 10 Gy at pHo 7.0 exhibited similar cell death rates to that at pHo 7.5 while PS120-pev cells had much higher cell death at low pHo (Figures 2B,C). Thus, active CAIX protects cells against ionizing irradiation at low pH. To definitively validate that the pHi-regulating functions of CAIX are indeed involved in cellular radioprotection, PS120-pev and PS120-pca9 cells were exposed to a pHo 7.0 medium containing 10 mM . This addition has been shown previously to maintain pHi in acidic pHo environments (Chiche et al., 2010a). Irradiation of PS120-pev cells in the presence of reduced the percentage of cell death to that obtained for PS120-pca9 cells in a pHo 7.0 medium (Figure 2D). Cloning efficiency experiments also confirm the capacity of irradiated cells to survive and recover following irradiation. From 4 to 8 Gy single doses of ionizing radiation of PS120-pev cells exposed to a pHo 7.0 medium drastically reduced the cloning efficiency, compared to that observed in a pHo 7.5 medium (Figure 2E, left panel). In contrast, PS120-pca9 cells exposed to a pHo 7.0 medium were capable to recover after irradiation, to the same extent that we observed in a pHo 7.5 (Figure 2E, right panel). Taken together these results suggest that the pHi-regulating properties of NHE-1 and CAIX protect cells against irradiation.

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