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Positron emission tomographic monitoring of dual phosphatidylinositol-3-kinase and mTOR inhibition in anaplastic large cell lymphoma.

Graf N, Li Z, Herrmann K, Weh D, Aichler M, Slawska J, Walch A, Peschel C, Schwaiger M, Buck AK, Dechow T, Keller U - Onco Targets Ther (2014)

Bottom Line: The biological effects of BGT226 were determined in vitro in the NPM-ALK positive cell lines SU-DHL-1 and Karpas299 by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, propidium iodide staining, and biochemical analysis of PI3K and mTOR downstream signaling.Lymphomas were removed for immunohistochemical analysis of proliferation and apoptosis to correlate PET findings with in vivo treatment effects.Imaging results correlated with a marked decrease in the proliferation marker Ki67, and a slight increase in the apoptotic marker, cleaved caspase 3, as revealed by immunostaining of explanted lymphoma tissue.

View Article: PubMed Central - PubMed

Affiliation: III Medical Department, Technische Universität München, Munich, Germany.

ABSTRACT

Background: Dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibition offers an attractive therapeutic strategy in anaplastic large cell lymphoma depending on oncogenic nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) signaling. We tested the efficacy of a novel dual PI3K/mTOR inhibitor, NVP-BGT226 (BGT226), in two anaplastic large cell lymphoma cell lines in vitro and in vivo and performed an early response evaluation with positron emission tomography (PET) imaging using the standard tracer, 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) and the thymidine analog, 3'-deoxy-3'-[(18)F] fluorothymidine (FLT).

Methods: The biological effects of BGT226 were determined in vitro in the NPM-ALK positive cell lines SU-DHL-1 and Karpas299 by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, propidium iodide staining, and biochemical analysis of PI3K and mTOR downstream signaling. FDG-PET and FLT-PET were performed in immunodeficient mice bearing either SU-DHL-1 or Karpas299 xenografts at baseline and 7 days after initiation of treatment with BGT226. Lymphomas were removed for immunohistochemical analysis of proliferation and apoptosis to correlate PET findings with in vivo treatment effects.

Results: SU-DHL-1 cells showed sensitivity to BGT226 in vitro, with cell cycle arrest in G0/G1 phase and an IC50 in the low nanomolar range, in contrast with Karpas299 cells, which were mainly resistant to BGT226. In vivo, both FDG-PET and FLT-PET discriminated sensitive from resistant lymphoma, as indicated by a significant reduction of tumor-to-background ratios on day 7 in treated SU-DHL-1 lymphoma-bearing animals compared with the control group, but not in animals with Karpas299 xenografts. Imaging results correlated with a marked decrease in the proliferation marker Ki67, and a slight increase in the apoptotic marker, cleaved caspase 3, as revealed by immunostaining of explanted lymphoma tissue.

Conclusion: Dual PI3K/mTOR inhibition using BGT226 is effective in ALK-positive anaplastic large cell lymphoma and can be monitored with both FDG-PET and FLT-PET early on in the course of therapy.

No MeSH data available.


Related in: MedlinePlus

Karpas299 is mainly resistant to phosphatidylinositol-3-kinase/mammalian target of rapamycin inhibition in vitro.Notes: (A) Karpas299 cells were cultured with BGT226 in the presence of increasing concentrations as indicated for 24 hours (left panel) and 48 hours (right panel). Cell viability was assessed by MTT assay. (B) Cell cycle distribution assessed by propidium iodide flow cytometry at 24 hours (left panel) and 48 hours (right panel). The bars represent the mean ± standard deviation of three individual experiments. (C) Assessment of protein levels and target inhibition after 6 hours of incubation with BGT226 by immunoblotting using the indicated antibodies.Abbreviation: MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
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f2-ott-7-789: Karpas299 is mainly resistant to phosphatidylinositol-3-kinase/mammalian target of rapamycin inhibition in vitro.Notes: (A) Karpas299 cells were cultured with BGT226 in the presence of increasing concentrations as indicated for 24 hours (left panel) and 48 hours (right panel). Cell viability was assessed by MTT assay. (B) Cell cycle distribution assessed by propidium iodide flow cytometry at 24 hours (left panel) and 48 hours (right panel). The bars represent the mean ± standard deviation of three individual experiments. (C) Assessment of protein levels and target inhibition after 6 hours of incubation with BGT226 by immunoblotting using the indicated antibodies.Abbreviation: MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.

Mentions: In contrast with SU-DHL-1 cells, Karpas299 cells did not respond to BGT226 in the low nanomolar range. In fact, about 10-fold higher concentrations of BGT226 as compared with SU-DHL-1 cells were necessary to observe inhibitory effects. MTT assay revealed an IC50 of about 100 nM that was independent of incubation time (Figure 2A). Accordingly, a relevant decrease of the S phase fraction and an increase of the G0/G1 fraction was only seen at the same high dose level using propidium iodide cell cycle analysis, whereas the subG1 fraction indicating apoptosis was unaffected at any dose level or time point tested (Figure 2B). Consistent with viability and cell cycle assays, Western blot analysis revealed virtually unchanged Akt phosphorylation and incomplete inhibition of p70S6 kinase phosphorylation at 100 nM (Figure 2C). Thus, Karpas299 is an NPM-ALK+ cell line with low sensitivity to BGT226, and is suitable as a low/nonresponder cell line.


Positron emission tomographic monitoring of dual phosphatidylinositol-3-kinase and mTOR inhibition in anaplastic large cell lymphoma.

Graf N, Li Z, Herrmann K, Weh D, Aichler M, Slawska J, Walch A, Peschel C, Schwaiger M, Buck AK, Dechow T, Keller U - Onco Targets Ther (2014)

Karpas299 is mainly resistant to phosphatidylinositol-3-kinase/mammalian target of rapamycin inhibition in vitro.Notes: (A) Karpas299 cells were cultured with BGT226 in the presence of increasing concentrations as indicated for 24 hours (left panel) and 48 hours (right panel). Cell viability was assessed by MTT assay. (B) Cell cycle distribution assessed by propidium iodide flow cytometry at 24 hours (left panel) and 48 hours (right panel). The bars represent the mean ± standard deviation of three individual experiments. (C) Assessment of protein levels and target inhibition after 6 hours of incubation with BGT226 by immunoblotting using the indicated antibodies.Abbreviation: MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043809&req=5

f2-ott-7-789: Karpas299 is mainly resistant to phosphatidylinositol-3-kinase/mammalian target of rapamycin inhibition in vitro.Notes: (A) Karpas299 cells were cultured with BGT226 in the presence of increasing concentrations as indicated for 24 hours (left panel) and 48 hours (right panel). Cell viability was assessed by MTT assay. (B) Cell cycle distribution assessed by propidium iodide flow cytometry at 24 hours (left panel) and 48 hours (right panel). The bars represent the mean ± standard deviation of three individual experiments. (C) Assessment of protein levels and target inhibition after 6 hours of incubation with BGT226 by immunoblotting using the indicated antibodies.Abbreviation: MTT, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
Mentions: In contrast with SU-DHL-1 cells, Karpas299 cells did not respond to BGT226 in the low nanomolar range. In fact, about 10-fold higher concentrations of BGT226 as compared with SU-DHL-1 cells were necessary to observe inhibitory effects. MTT assay revealed an IC50 of about 100 nM that was independent of incubation time (Figure 2A). Accordingly, a relevant decrease of the S phase fraction and an increase of the G0/G1 fraction was only seen at the same high dose level using propidium iodide cell cycle analysis, whereas the subG1 fraction indicating apoptosis was unaffected at any dose level or time point tested (Figure 2B). Consistent with viability and cell cycle assays, Western blot analysis revealed virtually unchanged Akt phosphorylation and incomplete inhibition of p70S6 kinase phosphorylation at 100 nM (Figure 2C). Thus, Karpas299 is an NPM-ALK+ cell line with low sensitivity to BGT226, and is suitable as a low/nonresponder cell line.

Bottom Line: The biological effects of BGT226 were determined in vitro in the NPM-ALK positive cell lines SU-DHL-1 and Karpas299 by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, propidium iodide staining, and biochemical analysis of PI3K and mTOR downstream signaling.Lymphomas were removed for immunohistochemical analysis of proliferation and apoptosis to correlate PET findings with in vivo treatment effects.Imaging results correlated with a marked decrease in the proliferation marker Ki67, and a slight increase in the apoptotic marker, cleaved caspase 3, as revealed by immunostaining of explanted lymphoma tissue.

View Article: PubMed Central - PubMed

Affiliation: III Medical Department, Technische Universität München, Munich, Germany.

ABSTRACT

Background: Dual phosphatidylinositol-3-kinase (PI3K)/mammalian target of rapamycin (mTOR) inhibition offers an attractive therapeutic strategy in anaplastic large cell lymphoma depending on oncogenic nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) signaling. We tested the efficacy of a novel dual PI3K/mTOR inhibitor, NVP-BGT226 (BGT226), in two anaplastic large cell lymphoma cell lines in vitro and in vivo and performed an early response evaluation with positron emission tomography (PET) imaging using the standard tracer, 2-deoxy-2-[(18)F]fluoro-D-glucose (FDG) and the thymidine analog, 3'-deoxy-3'-[(18)F] fluorothymidine (FLT).

Methods: The biological effects of BGT226 were determined in vitro in the NPM-ALK positive cell lines SU-DHL-1 and Karpas299 by 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, propidium iodide staining, and biochemical analysis of PI3K and mTOR downstream signaling. FDG-PET and FLT-PET were performed in immunodeficient mice bearing either SU-DHL-1 or Karpas299 xenografts at baseline and 7 days after initiation of treatment with BGT226. Lymphomas were removed for immunohistochemical analysis of proliferation and apoptosis to correlate PET findings with in vivo treatment effects.

Results: SU-DHL-1 cells showed sensitivity to BGT226 in vitro, with cell cycle arrest in G0/G1 phase and an IC50 in the low nanomolar range, in contrast with Karpas299 cells, which were mainly resistant to BGT226. In vivo, both FDG-PET and FLT-PET discriminated sensitive from resistant lymphoma, as indicated by a significant reduction of tumor-to-background ratios on day 7 in treated SU-DHL-1 lymphoma-bearing animals compared with the control group, but not in animals with Karpas299 xenografts. Imaging results correlated with a marked decrease in the proliferation marker Ki67, and a slight increase in the apoptotic marker, cleaved caspase 3, as revealed by immunostaining of explanted lymphoma tissue.

Conclusion: Dual PI3K/mTOR inhibition using BGT226 is effective in ALK-positive anaplastic large cell lymphoma and can be monitored with both FDG-PET and FLT-PET early on in the course of therapy.

No MeSH data available.


Related in: MedlinePlus