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The small molecule inhibitor QLT0267 Radiosensitizes squamous cell carcinoma cells of the head and neck.

Eke I, Leonhardt F, Storch K, Hehlgans S, Cordes N - PLoS ONE (2009)

Bottom Line: QLT0267 significantly reduced basal cell survival and enhanced the radiosensitivity of FaDu and UTSCC45 cells in a time- and concentration-dependent manner.QLT0267 exerted differential, cell culture model-dependent effects with regard to radiogenic rDSB and accumulation of cells in the G2 cell cycle phase.A knockdown of ILK revealed no change in clonogenic survival of the tested cell lines as compared to controls.

View Article: PubMed Central - PubMed

Affiliation: OncoRay - Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden, Germany.

ABSTRACT

Background: The constant increase of cancer cell resistance to radio- and chemotherapy hampers improvement of patient survival and requires novel targeting approaches. Integrin-Linked Kinase (ILK) has been postulated as potent druggable cancer target. On the basis of our previous findings clearly showing that ILK transduces antisurvival signals in cells exposed to ionizing radiation, this study evaluated the impact of the small molecule inhibitor QLT0267, reported as putative ILK inhibitor, on the cellular radiation survival response of human head and neck squamous cell carcinoma cells (hHNSCC).

Methodology/principal findings: Parental FaDu cells and FaDu cells stably transfected with a constitutively active ILK mutant (FaDu-IH) or empty vectors, UTSCC45 cells, ILK(floxed/floxed(fl/fl)) and ILK(-/-) mouse fibroblasts were used. Cells grew either two-dimensionally (2D) on or three-dimensionally (3D) in laminin-rich extracellular matrix. Cells were treated with QLT0267 alone or in combination with irradiation (X-rays, 0-6 Gy single dose). ILK knockdown was achieved by small interfering RNA transfection. ILK kinase activity, clonogenic survival, number of residual DNA double strand breaks (rDSB; gammaH2AX/53BP1 foci assay), cell cycle distribution, protein expression and phosphorylation (e.g. Akt, p44/42 mitogen-activated protein kinase (MAPK)) were measured. Data on ILK kinase activity and phosphorylation of Akt and p44/42 MAPK revealed a broad inhibitory spectrum of QLT0267 without specificity for ILK. QLT0267 significantly reduced basal cell survival and enhanced the radiosensitivity of FaDu and UTSCC45 cells in a time- and concentration-dependent manner. QLT0267 exerted differential, cell culture model-dependent effects with regard to radiogenic rDSB and accumulation of cells in the G2 cell cycle phase. Relative to corresponding controls, FaDu-IH and ILK(fl/fl) fibroblasts showed enhanced radiosensitivity, which failed to be antagonized by QLT0267. A knockdown of ILK revealed no change in clonogenic survival of the tested cell lines as compared to controls.

Conclusions/significance: Our data clearly show that the small molecule inhibitor QLT0267 has potent cytotoxic and radiosensitizing capability in hHNSCC cells. However, QLT0267 is not specific for ILK. Further in vitro and in vivo studies are necessary to clarify the potential of QLT0267 as a targeted therapeutic in the clinic.

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QLT0267 significantly reduces basal cell survival and sensitizes hHNSCC cells to ionizing radiation.(A) For 2D or 3D clonogenic assays, single cells were plated onto lrECM or inserted into lrECM and exposed to increasing concentrations of QLT0267 (0–20 µM) for 1 h or 24 h. Colonies were counted microscopically after 8–11 days. Results are means±s.d. (n = 3). Student's t-test compared clonogenic survival of QLT0267-treated cells under 2D vs. 3D growth conditions. *P<0.05. (B) Photographs show colonies 11 days (FaDu) or 8 days (UTSCC45) after treatment with DMSO or 10 µM QLT0267. (C) Subsequent to a 1-h or a 24-h exposure with DMSO or QLT0267, 2D and 3D cell cultures were irradiated with 2 Gy X-rays. Results represent means±s.d. (n = 3). Student's t-test compared QLT0267-treated/irradiated vs. DMSO-treated/irradiated cells. *P<0.05; **P<0.01
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pone-0006434-g002: QLT0267 significantly reduces basal cell survival and sensitizes hHNSCC cells to ionizing radiation.(A) For 2D or 3D clonogenic assays, single cells were plated onto lrECM or inserted into lrECM and exposed to increasing concentrations of QLT0267 (0–20 µM) for 1 h or 24 h. Colonies were counted microscopically after 8–11 days. Results are means±s.d. (n = 3). Student's t-test compared clonogenic survival of QLT0267-treated cells under 2D vs. 3D growth conditions. *P<0.05. (B) Photographs show colonies 11 days (FaDu) or 8 days (UTSCC45) after treatment with DMSO or 10 µM QLT0267. (C) Subsequent to a 1-h or a 24-h exposure with DMSO or QLT0267, 2D and 3D cell cultures were irradiated with 2 Gy X-rays. Results represent means±s.d. (n = 3). Student's t-test compared QLT0267-treated/irradiated vs. DMSO-treated/irradiated cells. *P<0.05; **P<0.01

Mentions: As QLT0267 modulated ILK kinase activity, we next analyzed the effect of QLT0267 on basal clonogenic survival and radiation clonogenic survival of FaDu and UTSCC45 hHNSCC cells under 2D and 3D cell culture conditions. On the basis of our previous studies [20], [25]–[27], we expected a radioprotective effect by QLT0267. A 1-h QLT0267 exposure showed no significant effect on both basal and radiation cell survival (Figure 2A-C, Table 1). Surprisingly, a 24-h QLT0267 treatment exerted significant (P<0.05) cytotoxicity (Fig. 2A and B) and radiosensitization of both FaDu and UTSCC45 cells to the clinical relevant radiation dose per fraction of 2 Gy relative to DMSO controls (Figure 2C, Table 1). Except in 3D FaDu cell cultures showing a 2.6-fold enhancement of the cellular radiosensitivity under QLT0267 exposure, 2D FaDu and 2D and 3D UTSCC45 cells were radiosensitized by QLT0267 by a factor of ∼1.5 (Table 1). Conclusively, neither the cytotoxic nor the radiosensitizing effects mediated by QLT0267 could be correlated with the changes in ILK kinase activity displayed in figure 1. Further, QLT0267 failed to show a radioprotective effect, which suggests a low or absent specificity for ILK.


The small molecule inhibitor QLT0267 Radiosensitizes squamous cell carcinoma cells of the head and neck.

Eke I, Leonhardt F, Storch K, Hehlgans S, Cordes N - PLoS ONE (2009)

QLT0267 significantly reduces basal cell survival and sensitizes hHNSCC cells to ionizing radiation.(A) For 2D or 3D clonogenic assays, single cells were plated onto lrECM or inserted into lrECM and exposed to increasing concentrations of QLT0267 (0–20 µM) for 1 h or 24 h. Colonies were counted microscopically after 8–11 days. Results are means±s.d. (n = 3). Student's t-test compared clonogenic survival of QLT0267-treated cells under 2D vs. 3D growth conditions. *P<0.05. (B) Photographs show colonies 11 days (FaDu) or 8 days (UTSCC45) after treatment with DMSO or 10 µM QLT0267. (C) Subsequent to a 1-h or a 24-h exposure with DMSO or QLT0267, 2D and 3D cell cultures were irradiated with 2 Gy X-rays. Results represent means±s.d. (n = 3). Student's t-test compared QLT0267-treated/irradiated vs. DMSO-treated/irradiated cells. *P<0.05; **P<0.01
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pone-0006434-g002: QLT0267 significantly reduces basal cell survival and sensitizes hHNSCC cells to ionizing radiation.(A) For 2D or 3D clonogenic assays, single cells were plated onto lrECM or inserted into lrECM and exposed to increasing concentrations of QLT0267 (0–20 µM) for 1 h or 24 h. Colonies were counted microscopically after 8–11 days. Results are means±s.d. (n = 3). Student's t-test compared clonogenic survival of QLT0267-treated cells under 2D vs. 3D growth conditions. *P<0.05. (B) Photographs show colonies 11 days (FaDu) or 8 days (UTSCC45) after treatment with DMSO or 10 µM QLT0267. (C) Subsequent to a 1-h or a 24-h exposure with DMSO or QLT0267, 2D and 3D cell cultures were irradiated with 2 Gy X-rays. Results represent means±s.d. (n = 3). Student's t-test compared QLT0267-treated/irradiated vs. DMSO-treated/irradiated cells. *P<0.05; **P<0.01
Mentions: As QLT0267 modulated ILK kinase activity, we next analyzed the effect of QLT0267 on basal clonogenic survival and radiation clonogenic survival of FaDu and UTSCC45 hHNSCC cells under 2D and 3D cell culture conditions. On the basis of our previous studies [20], [25]–[27], we expected a radioprotective effect by QLT0267. A 1-h QLT0267 exposure showed no significant effect on both basal and radiation cell survival (Figure 2A-C, Table 1). Surprisingly, a 24-h QLT0267 treatment exerted significant (P<0.05) cytotoxicity (Fig. 2A and B) and radiosensitization of both FaDu and UTSCC45 cells to the clinical relevant radiation dose per fraction of 2 Gy relative to DMSO controls (Figure 2C, Table 1). Except in 3D FaDu cell cultures showing a 2.6-fold enhancement of the cellular radiosensitivity under QLT0267 exposure, 2D FaDu and 2D and 3D UTSCC45 cells were radiosensitized by QLT0267 by a factor of ∼1.5 (Table 1). Conclusively, neither the cytotoxic nor the radiosensitizing effects mediated by QLT0267 could be correlated with the changes in ILK kinase activity displayed in figure 1. Further, QLT0267 failed to show a radioprotective effect, which suggests a low or absent specificity for ILK.

Bottom Line: QLT0267 significantly reduced basal cell survival and enhanced the radiosensitivity of FaDu and UTSCC45 cells in a time- and concentration-dependent manner.QLT0267 exerted differential, cell culture model-dependent effects with regard to radiogenic rDSB and accumulation of cells in the G2 cell cycle phase.A knockdown of ILK revealed no change in clonogenic survival of the tested cell lines as compared to controls.

View Article: PubMed Central - PubMed

Affiliation: OncoRay - Center for Radiation Research in Oncology, Medical Faculty Carl Gustav Carus, Dresden University of Technology, Dresden, Germany.

ABSTRACT

Background: The constant increase of cancer cell resistance to radio- and chemotherapy hampers improvement of patient survival and requires novel targeting approaches. Integrin-Linked Kinase (ILK) has been postulated as potent druggable cancer target. On the basis of our previous findings clearly showing that ILK transduces antisurvival signals in cells exposed to ionizing radiation, this study evaluated the impact of the small molecule inhibitor QLT0267, reported as putative ILK inhibitor, on the cellular radiation survival response of human head and neck squamous cell carcinoma cells (hHNSCC).

Methodology/principal findings: Parental FaDu cells and FaDu cells stably transfected with a constitutively active ILK mutant (FaDu-IH) or empty vectors, UTSCC45 cells, ILK(floxed/floxed(fl/fl)) and ILK(-/-) mouse fibroblasts were used. Cells grew either two-dimensionally (2D) on or three-dimensionally (3D) in laminin-rich extracellular matrix. Cells were treated with QLT0267 alone or in combination with irradiation (X-rays, 0-6 Gy single dose). ILK knockdown was achieved by small interfering RNA transfection. ILK kinase activity, clonogenic survival, number of residual DNA double strand breaks (rDSB; gammaH2AX/53BP1 foci assay), cell cycle distribution, protein expression and phosphorylation (e.g. Akt, p44/42 mitogen-activated protein kinase (MAPK)) were measured. Data on ILK kinase activity and phosphorylation of Akt and p44/42 MAPK revealed a broad inhibitory spectrum of QLT0267 without specificity for ILK. QLT0267 significantly reduced basal cell survival and enhanced the radiosensitivity of FaDu and UTSCC45 cells in a time- and concentration-dependent manner. QLT0267 exerted differential, cell culture model-dependent effects with regard to radiogenic rDSB and accumulation of cells in the G2 cell cycle phase. Relative to corresponding controls, FaDu-IH and ILK(fl/fl) fibroblasts showed enhanced radiosensitivity, which failed to be antagonized by QLT0267. A knockdown of ILK revealed no change in clonogenic survival of the tested cell lines as compared to controls.

Conclusions/significance: Our data clearly show that the small molecule inhibitor QLT0267 has potent cytotoxic and radiosensitizing capability in hHNSCC cells. However, QLT0267 is not specific for ILK. Further in vitro and in vivo studies are necessary to clarify the potential of QLT0267 as a targeted therapeutic in the clinic.

Show MeSH
Related in: MedlinePlus