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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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Related in: MedlinePlus

QLT0267 differentially modifies phosphorylation of various protein kinases cell culture model-dependently.(A) Following a 24-h treatment with DMSO (D) or indicated concentrations of QLT0267, 2D or 3D cultured cells were lysed as described under Materials and Methods. Total protein lysates were subjected to SDS-PAGE and Western blotting with specific antibodies. β-Actin served as loading control. (B) Protein phosphorylation was analyzed by densitometry and normalized to total protein expression. Results represent means±s.d. (n = 3). Student's t-test compared QLT0267- vs. DMSO-treated cells. *P<0.05; **P<0.01.
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pone-0006434-g005: QLT0267 differentially modifies phosphorylation of various protein kinases cell culture model-dependently.(A) Following a 24-h treatment with DMSO (D) or indicated concentrations of QLT0267, 2D or 3D cultured cells were lysed as described under Materials and Methods. Total protein lysates were subjected to SDS-PAGE and Western blotting with specific antibodies. β-Actin served as loading control. (B) Protein phosphorylation was analyzed by densitometry and normalized to total protein expression. Results represent means±s.d. (n = 3). Student's t-test compared QLT0267- vs. DMSO-treated cells. *P<0.05; **P<0.01.

Mentions: To examine the downstream effects of QLT0267 on important prosurvival signaling pathways, the phosphorylation and protein expression of ILK, FAK, p44/42 MAPK and the putative ILK downstream targets Akt and Glycogen Synthase Kinase 3β (GSK3β) were explored. Overall, the QLT0267-related modulation of examined protein kinases showed a great similarity between both cell lines (Figure 5A and B). In 2D, Akt Serine(S)473 and focal adhesion kinase (FAK) Tyrosine(Y)397 phosphorylation were strongly reduced by QLT0267 while both GSK3β S9 and p44/42 MAPK phosphorylation were moderately or greatly induced, respectively (Figure 5A and B). Although in part significant, the QLT0267-related changes in phosphorylation of examined protein kinases were lower under 3D conditions than under 2D conditions (Figure 5A and B). Expression of total proteins remained stable upon QLT0267 or DMSO treatment.


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 differentially modifies phosphorylation of various protein kinases cell culture model-dependently.(A) Following a 24-h treatment with DMSO (D) or indicated concentrations of QLT0267, 2D or 3D cultured cells were lysed as described under Materials and Methods. Total protein lysates were subjected to SDS-PAGE and Western blotting with specific antibodies. β-Actin served as loading control. (B) Protein phosphorylation was analyzed by densitometry and normalized to total protein expression. Results represent means±s.d. (n = 3). Student's t-test compared QLT0267- vs. DMSO-treated cells. *P<0.05; **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0006434-g005: QLT0267 differentially modifies phosphorylation of various protein kinases cell culture model-dependently.(A) Following a 24-h treatment with DMSO (D) or indicated concentrations of QLT0267, 2D or 3D cultured cells were lysed as described under Materials and Methods. Total protein lysates were subjected to SDS-PAGE and Western blotting with specific antibodies. β-Actin served as loading control. (B) Protein phosphorylation was analyzed by densitometry and normalized to total protein expression. Results represent means±s.d. (n = 3). Student's t-test compared QLT0267- vs. DMSO-treated cells. *P<0.05; **P<0.01.
Mentions: To examine the downstream effects of QLT0267 on important prosurvival signaling pathways, the phosphorylation and protein expression of ILK, FAK, p44/42 MAPK and the putative ILK downstream targets Akt and Glycogen Synthase Kinase 3β (GSK3β) were explored. Overall, the QLT0267-related modulation of examined protein kinases showed a great similarity between both cell lines (Figure 5A and B). In 2D, Akt Serine(S)473 and focal adhesion kinase (FAK) Tyrosine(Y)397 phosphorylation were strongly reduced by QLT0267 while both GSK3β S9 and p44/42 MAPK phosphorylation were moderately or greatly induced, respectively (Figure 5A and B). Although in part significant, the QLT0267-related changes in phosphorylation of examined protein kinases were lower under 3D conditions than under 2D conditions (Figure 5A and B). Expression of total proteins remained stable upon QLT0267 or DMSO treatment.

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