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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

NHE-1 protects cells against irradiation-induced cell death in an acidic environment.(A) Cell cycle distribution of CCL39 cells treated (+) or not (-) with 100 μM of the NHE-1 inhibitor (HOE#694) in normoxia in a /CO2-free environment at pHo 7.5 or 7.0 for 24 h. (B,C) CCL39 cells (1 × 104) were plated in 60 mm dishes. Once attached cells were incubated in a -free medium adjusted to pHo 7.5 or 7.0, and treated in the presence (+) or absence (-) of 100 μM of NHE-1 inhibitor (HOE#694) for 24 h in a CO2-free atmosphere. Dishes were then irradiated (0, 2, 4, 6, 8, and 10 Gy) and returned to a 5% CO2 incubator with fresh -containing medium for 4 days. Cell death was determined by the trypan blue exclusion assay.
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Figure 1: NHE-1 protects cells against irradiation-induced cell death in an acidic environment.(A) Cell cycle distribution of CCL39 cells treated (+) or not (-) with 100 μM of the NHE-1 inhibitor (HOE#694) in normoxia in a /CO2-free environment at pHo 7.5 or 7.0 for 24 h. (B,C) CCL39 cells (1 × 104) were plated in 60 mm dishes. Once attached cells were incubated in a -free medium adjusted to pHo 7.5 or 7.0, and treated in the presence (+) or absence (-) of 100 μM of NHE-1 inhibitor (HOE#694) for 24 h in a CO2-free atmosphere. Dishes were then irradiated (0, 2, 4, 6, 8, and 10 Gy) and returned to a 5% CO2 incubator with fresh -containing medium for 4 days. Cell death was determined by the trypan blue exclusion assay.

Mentions: The contribution of intracellular acidosis to cell radiosensitivity was studied on fibroblasts growing in pHo 7.5 or a more acidic pHo 7.0 medium, in the presence or in the absence of NHE-1 inhibitor. We choose to work at pHo of 7.0 as it is low enough to reduce the pHi compared to the pHi obtained at pHo of 7.5 but is not low enough to prevent an observation of radiosensitization in acidic conditions due to a reduction in cell viability caused by acidosis alone. The impact of inhibiting NHE-1 on pHi regulation in these cells has been well established with NHE-1 inhibition causing a significant reduction in pHi in a pHo of 7.0 (Pouysségur et al., 1984). Prior to irradiation, we determined the effect of NHE-1 inhibition on cell cycle phase distribution. Selective inhibition of NHE-1 using HOE#694 (100 μM; see Masereel et al., 2003 for a review of NHE inhibitors and HOE#694 effectiveness) at the more acidic pHo 7.0 condition reduced the percentage of CCL39 cells in the most radioresistant S phase (34% decrease of cells in the radioresistant S phase in the presence of HOE#694 compared to non-treated cells) while it had no effect in a more neutral pHo 7.5 medium (Figure 1A). Consistent with this finding, irradiation of NHE-1-inhibited fibroblasts in a pHo 7.0 medium led to an increase in cell death (57% for 10 Gy) compared to either untreated cells (35% for 10 Gy; Figure 1B) or cells exposed to a pHo 7.5 medium treated or not with HOE#694 (32% for 10 Gy; Figure 1C).


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)

NHE-1 protects cells against irradiation-induced cell death in an acidic environment.(A) Cell cycle distribution of CCL39 cells treated (+) or not (-) with 100 μM of the NHE-1 inhibitor (HOE#694) in normoxia in a /CO2-free environment at pHo 7.5 or 7.0 for 24 h. (B,C) CCL39 cells (1 × 104) were plated in 60 mm dishes. Once attached cells were incubated in a -free medium adjusted to pHo 7.5 or 7.0, and treated in the presence (+) or absence (-) of 100 μM of NHE-1 inhibitor (HOE#694) for 24 h in a CO2-free atmosphere. Dishes were then irradiated (0, 2, 4, 6, 8, and 10 Gy) and returned to a 5% CO2 incubator with fresh -containing medium for 4 days. Cell death was determined by the trypan blue exclusion assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539669&req=5

Figure 1: NHE-1 protects cells against irradiation-induced cell death in an acidic environment.(A) Cell cycle distribution of CCL39 cells treated (+) or not (-) with 100 μM of the NHE-1 inhibitor (HOE#694) in normoxia in a /CO2-free environment at pHo 7.5 or 7.0 for 24 h. (B,C) CCL39 cells (1 × 104) were plated in 60 mm dishes. Once attached cells were incubated in a -free medium adjusted to pHo 7.5 or 7.0, and treated in the presence (+) or absence (-) of 100 μM of NHE-1 inhibitor (HOE#694) for 24 h in a CO2-free atmosphere. Dishes were then irradiated (0, 2, 4, 6, 8, and 10 Gy) and returned to a 5% CO2 incubator with fresh -containing medium for 4 days. Cell death was determined by the trypan blue exclusion assay.
Mentions: The contribution of intracellular acidosis to cell radiosensitivity was studied on fibroblasts growing in pHo 7.5 or a more acidic pHo 7.0 medium, in the presence or in the absence of NHE-1 inhibitor. We choose to work at pHo of 7.0 as it is low enough to reduce the pHi compared to the pHi obtained at pHo of 7.5 but is not low enough to prevent an observation of radiosensitization in acidic conditions due to a reduction in cell viability caused by acidosis alone. The impact of inhibiting NHE-1 on pHi regulation in these cells has been well established with NHE-1 inhibition causing a significant reduction in pHi in a pHo of 7.0 (Pouysségur et al., 1984). Prior to irradiation, we determined the effect of NHE-1 inhibition on cell cycle phase distribution. Selective inhibition of NHE-1 using HOE#694 (100 μM; see Masereel et al., 2003 for a review of NHE inhibitors and HOE#694 effectiveness) at the more acidic pHo 7.0 condition reduced the percentage of CCL39 cells in the most radioresistant S phase (34% decrease of cells in the radioresistant S phase in the presence of HOE#694 compared to non-treated cells) while it had no effect in a more neutral pHo 7.5 medium (Figure 1A). Consistent with this finding, irradiation of NHE-1-inhibited fibroblasts in a pHo 7.0 medium led to an increase in cell death (57% for 10 Gy) compared to either untreated cells (35% for 10 Gy; Figure 1B) or cells exposed to a pHo 7.5 medium treated or not with HOE#694 (32% for 10 Gy; Figure 1C).

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