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Preclinical Evaluation of a Potential GSH Ester Based PET/SPECT Imaging Probe DT(GSHMe)₂ to Detect Gamma Glutamyl Transferase Over Expressing Tumors.

Khurana H, Meena VK, Prakash S, Chuttani K, Chadha N, Jaswal A, Dhawan DK, Mishra AK, Hazari PP - PLoS ONE (2015)

Bottom Line: Gamma Glutamyl Transferase (GGT) is an important biomarker in malignant cancers.Preclinical in vitro evaluations on cell lines suggested minimal toxicity of DT(GSHMe)2 at 100 μM concentration.Kinetic analysis revealed transport of 99mTc-DT(GSHMe)2 occurs via a saturable high-affinity carrier with Michaelis constant (Km) of 2.25 μM and maximal transport rate velocity (Vmax) of 0.478 μM/min.

View Article: PubMed Central - PubMed

Affiliation: Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Delhi, India; Department of Biophysics, Biomedical Sciences Block, Panjab University, Chandigarh, India.

ABSTRACT
Gamma Glutamyl Transferase (GGT) is an important biomarker in malignant cancers. The redox processes ensuing from GGT-mediated metabolism of extracellular GSH are implicated in critical aspects of tumor cell biology. Reportedly, Glutathione monoethyl ester (GSHMe) is a substrate of GGT, which has been used for its rapid transport over glutathione. Exploring GGT to be an important target, a homobivalent peptide system, DT(GSHMe)2 was designed to target GGT-over expressing tumors for diagnostic purposes. DT(GSHMe)2 was synthesized, characterized and preclinically evaluated in vitro using toxicity, cell binding assays and time dependent experiments. Stable and defined radiochemistry with 99mTc and 68Ga was optimized for high radiochemical yield. In vivo biodistribution studies were conducted for different time points along with scintigraphic studies of radiolabeled DT(GSHMe)2 on xenografted tumor models. For further validation, in silico docking studies were performed on GGT (hGGT1, P19440). Preclinical in vitro evaluations on cell lines suggested minimal toxicity of DT(GSHMe)2 at 100 μM concentration. Kinetic analysis revealed transport of 99mTc-DT(GSHMe)2 occurs via a saturable high-affinity carrier with Michaelis constant (Km) of 2.25 μM and maximal transport rate velocity (Vmax) of 0.478 μM/min. Quantitative estimation of GGT expression from western blot experiments showed substantial expression with 41.6 ± 7.07 % IDV for tumor. Small animal micro PET (Positron Emission Tomography)/CT(Computed Tomography) coregistered images depicted significantly high uptake of DT(GSHMe)2 at the BMG-1 tumor site. ROI analysis showed high tumor to contra lateral muscle ratio of 9.33 in PET imaging studies. Avid accumulation of radiotracer was observed at tumor versus inflammation site at 2 h post i.v. injection in an Ehrlich Ascites tumor (EAT) mice model, showing evident specificity for tumor. We propose DT(GSHMe)2 to be an excellent candidate for prognostication and tumor imaging using PET/SPECT.

No MeSH data available.


Related in: MedlinePlus

In vitro cytotoxic evaluation of DT(GSHMe)2 A&B) Data is plotted for surviving fraction versus concentration for different intervals.A) Represents MTT and B) represents SRB assay showing similar trend in the surviving fraction. C&D) Cells treated with different concentrations of DT(GSHMe)2 analyzed for cell cycle by flow cytometry. C) Gated population P2, P3, P4, P5 representing cells in G1/G0, S, G2/M and apoptotic cells respectively. B) Bar graph depicting fraction of cells in different phases (P2, P3, P4, P5) of cell cycle.
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pone.0134281.g002: In vitro cytotoxic evaluation of DT(GSHMe)2 A&B) Data is plotted for surviving fraction versus concentration for different intervals.A) Represents MTT and B) represents SRB assay showing similar trend in the surviving fraction. C&D) Cells treated with different concentrations of DT(GSHMe)2 analyzed for cell cycle by flow cytometry. C) Gated population P2, P3, P4, P5 representing cells in G1/G0, S, G2/M and apoptotic cells respectively. B) Bar graph depicting fraction of cells in different phases (P2, P3, P4, P5) of cell cycle.

Mentions: Cell viability was assessed by calculating surviving fraction for various drug concentrations (0.001 μM- 1000 μM) for different time intervals 24, 48 and 72 h. Fig 2A shows time dependent curve which depicts both concentration and time dependent toxicity. The drug was found to be nontoxic at lower concentration, but showed a fall in cell viability from concentration 100 μM and above. Significant cell death about 70 ± 4.38% was observed at 1000 μM at 72 h. IC50 of DT(GSHMe)2 was found to be approximately 500 μM.


Preclinical Evaluation of a Potential GSH Ester Based PET/SPECT Imaging Probe DT(GSHMe)₂ to Detect Gamma Glutamyl Transferase Over Expressing Tumors.

Khurana H, Meena VK, Prakash S, Chuttani K, Chadha N, Jaswal A, Dhawan DK, Mishra AK, Hazari PP - PLoS ONE (2015)

In vitro cytotoxic evaluation of DT(GSHMe)2 A&B) Data is plotted for surviving fraction versus concentration for different intervals.A) Represents MTT and B) represents SRB assay showing similar trend in the surviving fraction. C&D) Cells treated with different concentrations of DT(GSHMe)2 analyzed for cell cycle by flow cytometry. C) Gated population P2, P3, P4, P5 representing cells in G1/G0, S, G2/M and apoptotic cells respectively. B) Bar graph depicting fraction of cells in different phases (P2, P3, P4, P5) of cell cycle.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519333&req=5

pone.0134281.g002: In vitro cytotoxic evaluation of DT(GSHMe)2 A&B) Data is plotted for surviving fraction versus concentration for different intervals.A) Represents MTT and B) represents SRB assay showing similar trend in the surviving fraction. C&D) Cells treated with different concentrations of DT(GSHMe)2 analyzed for cell cycle by flow cytometry. C) Gated population P2, P3, P4, P5 representing cells in G1/G0, S, G2/M and apoptotic cells respectively. B) Bar graph depicting fraction of cells in different phases (P2, P3, P4, P5) of cell cycle.
Mentions: Cell viability was assessed by calculating surviving fraction for various drug concentrations (0.001 μM- 1000 μM) for different time intervals 24, 48 and 72 h. Fig 2A shows time dependent curve which depicts both concentration and time dependent toxicity. The drug was found to be nontoxic at lower concentration, but showed a fall in cell viability from concentration 100 μM and above. Significant cell death about 70 ± 4.38% was observed at 1000 μM at 72 h. IC50 of DT(GSHMe)2 was found to be approximately 500 μM.

Bottom Line: Gamma Glutamyl Transferase (GGT) is an important biomarker in malignant cancers.Preclinical in vitro evaluations on cell lines suggested minimal toxicity of DT(GSHMe)2 at 100 μM concentration.Kinetic analysis revealed transport of 99mTc-DT(GSHMe)2 occurs via a saturable high-affinity carrier with Michaelis constant (Km) of 2.25 μM and maximal transport rate velocity (Vmax) of 0.478 μM/min.

View Article: PubMed Central - PubMed

Affiliation: Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Delhi, India; Department of Biophysics, Biomedical Sciences Block, Panjab University, Chandigarh, India.

ABSTRACT
Gamma Glutamyl Transferase (GGT) is an important biomarker in malignant cancers. The redox processes ensuing from GGT-mediated metabolism of extracellular GSH are implicated in critical aspects of tumor cell biology. Reportedly, Glutathione monoethyl ester (GSHMe) is a substrate of GGT, which has been used for its rapid transport over glutathione. Exploring GGT to be an important target, a homobivalent peptide system, DT(GSHMe)2 was designed to target GGT-over expressing tumors for diagnostic purposes. DT(GSHMe)2 was synthesized, characterized and preclinically evaluated in vitro using toxicity, cell binding assays and time dependent experiments. Stable and defined radiochemistry with 99mTc and 68Ga was optimized for high radiochemical yield. In vivo biodistribution studies were conducted for different time points along with scintigraphic studies of radiolabeled DT(GSHMe)2 on xenografted tumor models. For further validation, in silico docking studies were performed on GGT (hGGT1, P19440). Preclinical in vitro evaluations on cell lines suggested minimal toxicity of DT(GSHMe)2 at 100 μM concentration. Kinetic analysis revealed transport of 99mTc-DT(GSHMe)2 occurs via a saturable high-affinity carrier with Michaelis constant (Km) of 2.25 μM and maximal transport rate velocity (Vmax) of 0.478 μM/min. Quantitative estimation of GGT expression from western blot experiments showed substantial expression with 41.6 ± 7.07 % IDV for tumor. Small animal micro PET (Positron Emission Tomography)/CT(Computed Tomography) coregistered images depicted significantly high uptake of DT(GSHMe)2 at the BMG-1 tumor site. ROI analysis showed high tumor to contra lateral muscle ratio of 9.33 in PET imaging studies. Avid accumulation of radiotracer was observed at tumor versus inflammation site at 2 h post i.v. injection in an Ehrlich Ascites tumor (EAT) mice model, showing evident specificity for tumor. We propose DT(GSHMe)2 to be an excellent candidate for prognostication and tumor imaging using PET/SPECT.

No MeSH data available.


Related in: MedlinePlus