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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

Combined silencing of ca9 and ca12 compromises in vivo LS174Tr cell proliferation when combined with ionizing radiation.(A,B). Anti-tumor activity of silencing ca9 or ca12 individually or combined is increased in conjunction with ionizing radiation in LS174Tr xenograft tumors. At 4 days before injection of LS-shca9/ctl(A) or LS-shca9/ca12- cells (B), cells were incubated with or without Tet (+/-Tet) to silence ca9. In vivo xenograft assays were performed by s.c injection of viable and individual tumor cells (1 × 106) into the flanks of athymic nude mice. To maintain ca9 knock-down mice received DOX in the drinking water 4 days before cell injection. Fifteen days after cell injection, when the tumor size reached 4–5 mm, a single dose (8 Gy) of irradiation (IR) was delivered to the tumors only with “contact X-rays.” Xenograft growth was determined by measuring the tumor volume. (C) Xenograft tumor growth of control tumors LS-shev/ctl irradiated (IR) or not, in the presence (+DOX) or in the absence (-DOX) of doxycycline in the drinking water. Five mice were used per condition.
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Figure 4: Combined silencing of ca9 and ca12 compromises in vivo LS174Tr cell proliferation when combined with ionizing radiation.(A,B). Anti-tumor activity of silencing ca9 or ca12 individually or combined is increased in conjunction with ionizing radiation in LS174Tr xenograft tumors. At 4 days before injection of LS-shca9/ctl(A) or LS-shca9/ca12- cells (B), cells were incubated with or without Tet (+/-Tet) to silence ca9. In vivo xenograft assays were performed by s.c injection of viable and individual tumor cells (1 × 106) into the flanks of athymic nude mice. To maintain ca9 knock-down mice received DOX in the drinking water 4 days before cell injection. Fifteen days after cell injection, when the tumor size reached 4–5 mm, a single dose (8 Gy) of irradiation (IR) was delivered to the tumors only with “contact X-rays.” Xenograft growth was determined by measuring the tumor volume. (C) Xenograft tumor growth of control tumors LS-shev/ctl irradiated (IR) or not, in the presence (+DOX) or in the absence (-DOX) of doxycycline in the drinking water. Five mice were used per condition.

Mentions: Using contact radiotherapy (Gérard et al., 2011), we specifically targeted the established tumor mass grown on the back of nude mice. Irradiation of control tumors (LS-shca9/ctl -DOX + IR) stopped tumor progression for 5 days after irradiation before proliferating again at a high rate, which was similar to that of non-irradiated control tumors (LS-shca9/ctl -DOX; Figure 4A). Tumor progression was delayed with ca-9-silencing as observed previously while irradiation of ca9-silenced tumors (LS-shca9/ctl +DOX +IR) showed more pronounced arrest in tumor progression (25 days after irradiation to reach 600 mm3), which may reflect cell death within the tumor (Figure 4A). Irradiation of ca12-silenced tumors (LS-shca9/ca12- -DOX +IR) reduced the growth rate compared to non-irradiated tumors (LS-shca9/ca12- -DOX) to the same extent observed for irradiation of control tumors suggesting that ca12 alone is not able to confer tumor radioresistance (Figure 4B). However, irradiation of double silenced tumors (LS-shca9/ca12- +DOX +IR) showed a substantial reduction in the progression of the tumor (33 days after irradiation to reach 600 mm3). No interaction between DOX and irradiation was observed as shown with control LS-shev/ctl -/+DOX tumors (Figure 4C). Calculations of the tumor growth delay for time required to reach 300 and 600 cm3, respectively were 17.6/19.7 days (LS174shCA9 -Dox, -IR), 25.7 days/29.1 days (LS174shCA9 -Dox, +IR), 25.7 days/29 days (LS174shCA9 +Dox, -IR) and 36 days/46.3 days (LS174shCA9 +Dox, +IR). Growth delay times for CA9/CA12 double-silencing were 33.9 days/39.9 days (LS174shCA9/CA12 +Dox, -IR) and 36.1 days/45.9 days (LS174shCA9/CA12 +Dox, + IR). We further calculated the specific tumor growth delay (STGD) with the following formula: STGD = (DT experimental - DT control)/DT control (DT, doubling time). Irradiation alone and CA9 silencing alone resulted in similar STGD values of 0.61 and 0.57, respectively. Combined irradiation and silencing of CA9 increased the STGD to 3.9 compared to control. Meanwhile silencing of CA12 resulted in a STGD value of 1.85 while double CA9 and CA12 silencing with irradiation had a similar STGD value to CA9 silencing of 3.67.


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)

Combined silencing of ca9 and ca12 compromises in vivo LS174Tr cell proliferation when combined with ionizing radiation.(A,B). Anti-tumor activity of silencing ca9 or ca12 individually or combined is increased in conjunction with ionizing radiation in LS174Tr xenograft tumors. At 4 days before injection of LS-shca9/ctl(A) or LS-shca9/ca12- cells (B), cells were incubated with or without Tet (+/-Tet) to silence ca9. In vivo xenograft assays were performed by s.c injection of viable and individual tumor cells (1 × 106) into the flanks of athymic nude mice. To maintain ca9 knock-down mice received DOX in the drinking water 4 days before cell injection. Fifteen days after cell injection, when the tumor size reached 4–5 mm, a single dose (8 Gy) of irradiation (IR) was delivered to the tumors only with “contact X-rays.” Xenograft growth was determined by measuring the tumor volume. (C) Xenograft tumor growth of control tumors LS-shev/ctl irradiated (IR) or not, in the presence (+DOX) or in the absence (-DOX) of doxycycline in the drinking water. Five mice were used per condition.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Combined silencing of ca9 and ca12 compromises in vivo LS174Tr cell proliferation when combined with ionizing radiation.(A,B). Anti-tumor activity of silencing ca9 or ca12 individually or combined is increased in conjunction with ionizing radiation in LS174Tr xenograft tumors. At 4 days before injection of LS-shca9/ctl(A) or LS-shca9/ca12- cells (B), cells were incubated with or without Tet (+/-Tet) to silence ca9. In vivo xenograft assays were performed by s.c injection of viable and individual tumor cells (1 × 106) into the flanks of athymic nude mice. To maintain ca9 knock-down mice received DOX in the drinking water 4 days before cell injection. Fifteen days after cell injection, when the tumor size reached 4–5 mm, a single dose (8 Gy) of irradiation (IR) was delivered to the tumors only with “contact X-rays.” Xenograft growth was determined by measuring the tumor volume. (C) Xenograft tumor growth of control tumors LS-shev/ctl irradiated (IR) or not, in the presence (+DOX) or in the absence (-DOX) of doxycycline in the drinking water. Five mice were used per condition.
Mentions: Using contact radiotherapy (Gérard et al., 2011), we specifically targeted the established tumor mass grown on the back of nude mice. Irradiation of control tumors (LS-shca9/ctl -DOX + IR) stopped tumor progression for 5 days after irradiation before proliferating again at a high rate, which was similar to that of non-irradiated control tumors (LS-shca9/ctl -DOX; Figure 4A). Tumor progression was delayed with ca-9-silencing as observed previously while irradiation of ca9-silenced tumors (LS-shca9/ctl +DOX +IR) showed more pronounced arrest in tumor progression (25 days after irradiation to reach 600 mm3), which may reflect cell death within the tumor (Figure 4A). Irradiation of ca12-silenced tumors (LS-shca9/ca12- -DOX +IR) reduced the growth rate compared to non-irradiated tumors (LS-shca9/ca12- -DOX) to the same extent observed for irradiation of control tumors suggesting that ca12 alone is not able to confer tumor radioresistance (Figure 4B). However, irradiation of double silenced tumors (LS-shca9/ca12- +DOX +IR) showed a substantial reduction in the progression of the tumor (33 days after irradiation to reach 600 mm3). No interaction between DOX and irradiation was observed as shown with control LS-shev/ctl -/+DOX tumors (Figure 4C). Calculations of the tumor growth delay for time required to reach 300 and 600 cm3, respectively were 17.6/19.7 days (LS174shCA9 -Dox, -IR), 25.7 days/29.1 days (LS174shCA9 -Dox, +IR), 25.7 days/29 days (LS174shCA9 +Dox, -IR) and 36 days/46.3 days (LS174shCA9 +Dox, +IR). Growth delay times for CA9/CA12 double-silencing were 33.9 days/39.9 days (LS174shCA9/CA12 +Dox, -IR) and 36.1 days/45.9 days (LS174shCA9/CA12 +Dox, + IR). We further calculated the specific tumor growth delay (STGD) with the following formula: STGD = (DT experimental - DT control)/DT control (DT, doubling time). Irradiation alone and CA9 silencing alone resulted in similar STGD values of 0.61 and 0.57, respectively. Combined irradiation and silencing of CA9 increased the STGD to 3.9 compared to control. Meanwhile silencing of CA12 resulted in a STGD value of 1.85 while double CA9 and CA12 silencing with irradiation had a similar STGD value to CA9 silencing of 3.67.

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