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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 modifies ILK kinase activity in cell culture model-dependent manner.(A) 2D or (B) 3D cultured FaDu and UTSCC45 cells were exposed to 1 µM or 10 µM QLT0267 (DMSO served as control) and lysed after 1 h or 24 h. After immunoprecipitation of ILK and incubation with GSK fusion protein, samples were subjected to SDS-PAGE and Western blotting. Phosphorylation of GSK fusion protein with phospho-GSK3α/β S21/9 antibody indicates ILK kinase activity. Fold changes were calculated from densitometry and normalized to ILK and DMSO controls.
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pone-0006434-g001: QLT0267 modifies ILK kinase activity in cell culture model-dependent manner.(A) 2D or (B) 3D cultured FaDu and UTSCC45 cells were exposed to 1 µM or 10 µM QLT0267 (DMSO served as control) and lysed after 1 h or 24 h. After immunoprecipitation of ILK and incubation with GSK fusion protein, samples were subjected to SDS-PAGE and Western blotting. Phosphorylation of GSK fusion protein with phospho-GSK3α/β S21/9 antibody indicates ILK kinase activity. Fold changes were calculated from densitometry and normalized to ILK and DMSO controls.

Mentions: To assure efficient ILK kinase inhibition by QLT0267, we performed a kinase assay on immunoprecipitates from total cell lysates of 2D and 3D lrECM cell cultures treated with different QLT0267 concentrations over 1 h or 24 h. The data showed a strongly reduced ILK kinase activity in both 2D grown FaDu and UTSCC45 hHNSCC cell lines exposed to 1 µM QLT0267 as compared to an equivalent volume of DMSO (Figure 1A). No further ILK kinase inhibition was achieved when the dose was increased to 10 µM indicating that the maximal inhibitory effect of QLT0267 was already obtained at a concentration of 1 µM. In 3D, however, the inhibitory efficiency of QLT0267 on ILK was reduced in FaDu in comparison to 2D FaDu cell cultures, whereas in 3D UTSCC45 cells QLT0267 even exerted an inverse effect on ILK kinase activity relative to 2D conditions (Figure 1B).


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 modifies ILK kinase activity in cell culture model-dependent manner.(A) 2D or (B) 3D cultured FaDu and UTSCC45 cells were exposed to 1 µM or 10 µM QLT0267 (DMSO served as control) and lysed after 1 h or 24 h. After immunoprecipitation of ILK and incubation with GSK fusion protein, samples were subjected to SDS-PAGE and Western blotting. Phosphorylation of GSK fusion protein with phospho-GSK3α/β S21/9 antibody indicates ILK kinase activity. Fold changes were calculated from densitometry and normalized to ILK and DMSO controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2713401&req=5

pone-0006434-g001: QLT0267 modifies ILK kinase activity in cell culture model-dependent manner.(A) 2D or (B) 3D cultured FaDu and UTSCC45 cells were exposed to 1 µM or 10 µM QLT0267 (DMSO served as control) and lysed after 1 h or 24 h. After immunoprecipitation of ILK and incubation with GSK fusion protein, samples were subjected to SDS-PAGE and Western blotting. Phosphorylation of GSK fusion protein with phospho-GSK3α/β S21/9 antibody indicates ILK kinase activity. Fold changes were calculated from densitometry and normalized to ILK and DMSO controls.
Mentions: To assure efficient ILK kinase inhibition by QLT0267, we performed a kinase assay on immunoprecipitates from total cell lysates of 2D and 3D lrECM cell cultures treated with different QLT0267 concentrations over 1 h or 24 h. The data showed a strongly reduced ILK kinase activity in both 2D grown FaDu and UTSCC45 hHNSCC cell lines exposed to 1 µM QLT0267 as compared to an equivalent volume of DMSO (Figure 1A). No further ILK kinase inhibition was achieved when the dose was increased to 10 µM indicating that the maximal inhibitory effect of QLT0267 was already obtained at a concentration of 1 µM. In 3D, however, the inhibitory efficiency of QLT0267 on ILK was reduced in FaDu in comparison to 2D FaDu cell cultures, whereas in 3D UTSCC45 cells QLT0267 even exerted an inverse effect on ILK kinase activity relative to 2D conditions (Figure 1B).

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