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A putrescine-anthracene conjugate: a paradigm for selective drug delivery.

Palmer AJ, Ghani RA, Kaur N, Phanstiel O, Wallace HM - Biochem. J. (2009)

Bottom Line: Apoptosis was the predominant type of cell death, with mechanistic studies revealing that oxidative stress and DNA damage may have a part to play.For the first time, uptake of Ant 4 via the PTS was demonstrated both directly and indirectly in human cell lines.In addition, Ant 4 significantly reduced putrescine uptake, demonstrating that this conjugate not only used the PTS, but also could successfully compete with its native polyamine for uptake.

View Article: PubMed Central - PubMed

Affiliation: Divison of Applied Medicine, School of Medicine and Dentistry, University of Aberdeen, Aberdeen AB25 2ZD, UK.

ABSTRACT
Increased polyamine concentrations play an important role in the development of cancer at all stages, from initiation through to maintenance of the transformed phenotype. One way cancer cells accumulate increased concentrations of polyamines is by increased uptake of preformed polyamines via their PTS (polyamine transport system). The PTS is promiscuous and will transport a range of polyamine-based molecules. Therefore it may be that cytotoxic drugs could be attached to polyamine vectors and targeted selectively to cancer cells by utilizing the PTS. The aim of the present study was to investigate the potential of Ant 4, a putrescine-anthracene conjugate, to target cytotoxic agents to human cancer cells as a paradigm for a novel method of selective drug delivery. Ant 4 induced cytotoxicity after only 24 h exposure. Apoptosis was the predominant type of cell death, with mechanistic studies revealing that oxidative stress and DNA damage may have a part to play. For the first time, uptake of Ant 4 via the PTS was demonstrated both directly and indirectly in human cell lines. In addition, Ant 4 significantly reduced putrescine uptake, demonstrating that this conjugate not only used the PTS, but also could successfully compete with its native polyamine for uptake. However, the most interesting finding was the intracellular depletion of the polyamine pools, providing an additional mode of toxicity for Ant 4 and the possibility that this molecule may act as a 'double-edged sword': preventing cell growth by delivery of the toxic moiety and by depletion of intracellular polyamine content.

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Related in: MedlinePlus

Deconvolution microscopy of (a) untreated HL-60 cells, (b) Ant 4-treated HL-60 cells and (c) DFMO- and Ant 4-treated HL-60 cellsHL-60 cells were seeded at 6.8×104 cells/ml on 3.5-cm plates and grown for 48 h with (c) and without (a and b) 5 mM DFMO pre-treatment. Following this, cells were left untreated (a) or treated (b and c) with 15 μM Ant 4 for 30 min. After this time, cells were harvested and examined by deconvolution microscopy. Ant 4 localization is shown by the fluorescent purple colour. The experiment was repeated (n=3) with no replicates. Scale bar, 20 μm.
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Figure 4: Deconvolution microscopy of (a) untreated HL-60 cells, (b) Ant 4-treated HL-60 cells and (c) DFMO- and Ant 4-treated HL-60 cellsHL-60 cells were seeded at 6.8×104 cells/ml on 3.5-cm plates and grown for 48 h with (c) and without (a and b) 5 mM DFMO pre-treatment. Following this, cells were left untreated (a) or treated (b and c) with 15 μM Ant 4 for 30 min. After this time, cells were harvested and examined by deconvolution microscopy. Ant 4 localization is shown by the fluorescent purple colour. The experiment was repeated (n=3) with no replicates. Scale bar, 20 μm.

Mentions: In addition to inhibition of topoisomerase II, the anthracene component of Ant 4 has been shown to fluoresce with a wavelength maximum near 364 nm and emission maximum near 417 nm [12]. As such, the fluorescent properties of Ant 4 can be used to track its cellular localization. Therefore to see whether the decrease in the IC50 value was due to increased uptake of the conjugate and not due to synergy of DFMO and Ant 4, deconvolution microscopy was used to see whether Ant 4 gained entry to the HL-60 cells. Ant 4 was shown to localize within HL-60 cells (Figure 4b) after 30 min exposure in comparison with the untreated controls (Figure 4a). Furthermore, HL-60 cells grown for 48 h in 5 mM DFMO prior to Ant 4 exposure demonstrated greater accumulation of Ant 4 (Figure 4c) in comparison with cells treated with Ant 4 only (Figure 4b).


A putrescine-anthracene conjugate: a paradigm for selective drug delivery.

Palmer AJ, Ghani RA, Kaur N, Phanstiel O, Wallace HM - Biochem. J. (2009)

Deconvolution microscopy of (a) untreated HL-60 cells, (b) Ant 4-treated HL-60 cells and (c) DFMO- and Ant 4-treated HL-60 cellsHL-60 cells were seeded at 6.8×104 cells/ml on 3.5-cm plates and grown for 48 h with (c) and without (a and b) 5 mM DFMO pre-treatment. Following this, cells were left untreated (a) or treated (b and c) with 15 μM Ant 4 for 30 min. After this time, cells were harvested and examined by deconvolution microscopy. Ant 4 localization is shown by the fluorescent purple colour. The experiment was repeated (n=3) with no replicates. Scale bar, 20 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2805923&req=5

Figure 4: Deconvolution microscopy of (a) untreated HL-60 cells, (b) Ant 4-treated HL-60 cells and (c) DFMO- and Ant 4-treated HL-60 cellsHL-60 cells were seeded at 6.8×104 cells/ml on 3.5-cm plates and grown for 48 h with (c) and without (a and b) 5 mM DFMO pre-treatment. Following this, cells were left untreated (a) or treated (b and c) with 15 μM Ant 4 for 30 min. After this time, cells were harvested and examined by deconvolution microscopy. Ant 4 localization is shown by the fluorescent purple colour. The experiment was repeated (n=3) with no replicates. Scale bar, 20 μm.
Mentions: In addition to inhibition of topoisomerase II, the anthracene component of Ant 4 has been shown to fluoresce with a wavelength maximum near 364 nm and emission maximum near 417 nm [12]. As such, the fluorescent properties of Ant 4 can be used to track its cellular localization. Therefore to see whether the decrease in the IC50 value was due to increased uptake of the conjugate and not due to synergy of DFMO and Ant 4, deconvolution microscopy was used to see whether Ant 4 gained entry to the HL-60 cells. Ant 4 was shown to localize within HL-60 cells (Figure 4b) after 30 min exposure in comparison with the untreated controls (Figure 4a). Furthermore, HL-60 cells grown for 48 h in 5 mM DFMO prior to Ant 4 exposure demonstrated greater accumulation of Ant 4 (Figure 4c) in comparison with cells treated with Ant 4 only (Figure 4b).

Bottom Line: Apoptosis was the predominant type of cell death, with mechanistic studies revealing that oxidative stress and DNA damage may have a part to play.For the first time, uptake of Ant 4 via the PTS was demonstrated both directly and indirectly in human cell lines.In addition, Ant 4 significantly reduced putrescine uptake, demonstrating that this conjugate not only used the PTS, but also could successfully compete with its native polyamine for uptake.

View Article: PubMed Central - PubMed

Affiliation: Divison of Applied Medicine, School of Medicine and Dentistry, University of Aberdeen, Aberdeen AB25 2ZD, UK.

ABSTRACT
Increased polyamine concentrations play an important role in the development of cancer at all stages, from initiation through to maintenance of the transformed phenotype. One way cancer cells accumulate increased concentrations of polyamines is by increased uptake of preformed polyamines via their PTS (polyamine transport system). The PTS is promiscuous and will transport a range of polyamine-based molecules. Therefore it may be that cytotoxic drugs could be attached to polyamine vectors and targeted selectively to cancer cells by utilizing the PTS. The aim of the present study was to investigate the potential of Ant 4, a putrescine-anthracene conjugate, to target cytotoxic agents to human cancer cells as a paradigm for a novel method of selective drug delivery. Ant 4 induced cytotoxicity after only 24 h exposure. Apoptosis was the predominant type of cell death, with mechanistic studies revealing that oxidative stress and DNA damage may have a part to play. For the first time, uptake of Ant 4 via the PTS was demonstrated both directly and indirectly in human cell lines. In addition, Ant 4 significantly reduced putrescine uptake, demonstrating that this conjugate not only used the PTS, but also could successfully compete with its native polyamine for uptake. However, the most interesting finding was the intracellular depletion of the polyamine pools, providing an additional mode of toxicity for Ant 4 and the possibility that this molecule may act as a 'double-edged sword': preventing cell growth by delivery of the toxic moiety and by depletion of intracellular polyamine content.

Show MeSH
Related in: MedlinePlus