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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 treatment increases the number of radiation-induced DSBs without affecting apoptosis.(A) After treatment with 10 µM QLT0267 for 24 h, 2D or 3D lrECM grown cells remained unirradiated or received a single dose of 2 Gy. After 24 h, cells were isolated, fixed and co-stained against 53BP1 and γH2AX. Double stained foci from 150 cells were microscopically counted per experiment. Number of foci of irradiated cells was normalized to number of foci of unirradiated cells. Results represent means±s.d. (n = 3). Student's t-test compared QLT0267- vs. DMSO-treated or QLT0267/2Gy- vs. DMSO/2Gy-treated cells. *P<0.05, **P<0.01. Photographs illustrate immunofluorescence staining of 53BP1 (green) and γH2AX (red) of 3D grown cell cultures. Nuclei were stained with DAPI (blue). (B) In parallel, cells were treated as indicated, fixed and stained with DAPI to microscopically determine cells with typically apoptotic nuclear morphology. Results are means±s.d. (n = 3).
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pone-0006434-g003: QLT0267 treatment increases the number of radiation-induced DSBs without affecting apoptosis.(A) After treatment with 10 µM QLT0267 for 24 h, 2D or 3D lrECM grown cells remained unirradiated or received a single dose of 2 Gy. After 24 h, cells were isolated, fixed and co-stained against 53BP1 and γH2AX. Double stained foci from 150 cells were microscopically counted per experiment. Number of foci of irradiated cells was normalized to number of foci of unirradiated cells. Results represent means±s.d. (n = 3). Student's t-test compared QLT0267- vs. DMSO-treated or QLT0267/2Gy- vs. DMSO/2Gy-treated cells. *P<0.05, **P<0.01. Photographs illustrate immunofluorescence staining of 53BP1 (green) and γH2AX (red) of 3D grown cell cultures. Nuclei were stained with DAPI (blue). (B) In parallel, cells were treated as indicated, fixed and stained with DAPI to microscopically determine cells with typically apoptotic nuclear morphology. Results are means±s.d. (n = 3).

Mentions: DNA double strand breaks (DSB) are considered to be the most severe, life-threatening DNA lesions caused by ionizing radiation [37]. With regard to the cytotoxic and radiosensitizing effects of QLT0267, we measured the number of residual DSB ( = unrepaired DSB at 24 h after treatment; rDSB) induced by QLT0267 alone or in combination with 2-Gy irradiation using the γH2AX/53BP1 foci assay. Analysis of QLT0267 exposure only revealed a significant (P<0.01) elevation of rDSB in 2D grown FaDu cells (Figure 3A). In combination with irradiation, QLT0267-treated 3D FaDu and UTSCC45 cell cultures showed significantly (P<0.01) more rDSB 24 h after 2 Gy as compared to DMSO (Figure 3A).


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 treatment increases the number of radiation-induced DSBs without affecting apoptosis.(A) After treatment with 10 µM QLT0267 for 24 h, 2D or 3D lrECM grown cells remained unirradiated or received a single dose of 2 Gy. After 24 h, cells were isolated, fixed and co-stained against 53BP1 and γH2AX. Double stained foci from 150 cells were microscopically counted per experiment. Number of foci of irradiated cells was normalized to number of foci of unirradiated cells. Results represent means±s.d. (n = 3). Student's t-test compared QLT0267- vs. DMSO-treated or QLT0267/2Gy- vs. DMSO/2Gy-treated cells. *P<0.05, **P<0.01. Photographs illustrate immunofluorescence staining of 53BP1 (green) and γH2AX (red) of 3D grown cell cultures. Nuclei were stained with DAPI (blue). (B) In parallel, cells were treated as indicated, fixed and stained with DAPI to microscopically determine cells with typically apoptotic nuclear morphology. Results are means±s.d. (n = 3).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2713401&req=5

pone-0006434-g003: QLT0267 treatment increases the number of radiation-induced DSBs without affecting apoptosis.(A) After treatment with 10 µM QLT0267 for 24 h, 2D or 3D lrECM grown cells remained unirradiated or received a single dose of 2 Gy. After 24 h, cells were isolated, fixed and co-stained against 53BP1 and γH2AX. Double stained foci from 150 cells were microscopically counted per experiment. Number of foci of irradiated cells was normalized to number of foci of unirradiated cells. Results represent means±s.d. (n = 3). Student's t-test compared QLT0267- vs. DMSO-treated or QLT0267/2Gy- vs. DMSO/2Gy-treated cells. *P<0.05, **P<0.01. Photographs illustrate immunofluorescence staining of 53BP1 (green) and γH2AX (red) of 3D grown cell cultures. Nuclei were stained with DAPI (blue). (B) In parallel, cells were treated as indicated, fixed and stained with DAPI to microscopically determine cells with typically apoptotic nuclear morphology. Results are means±s.d. (n = 3).
Mentions: DNA double strand breaks (DSB) are considered to be the most severe, life-threatening DNA lesions caused by ionizing radiation [37]. With regard to the cytotoxic and radiosensitizing effects of QLT0267, we measured the number of residual DSB ( = unrepaired DSB at 24 h after treatment; rDSB) induced by QLT0267 alone or in combination with 2-Gy irradiation using the γH2AX/53BP1 foci assay. Analysis of QLT0267 exposure only revealed a significant (P<0.01) elevation of rDSB in 2D grown FaDu cells (Figure 3A). In combination with irradiation, QLT0267-treated 3D FaDu and UTSCC45 cell cultures showed significantly (P<0.01) more rDSB 24 h after 2 Gy as compared to DMSO (Figure 3A).

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