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Potentiating the cellular targeting and anti-tumor activity of Dp44mT via binding to human serum albumin: two saturable mechanisms of Dp44mT uptake by cells.

Merlot AM, Sahni S, Lane DJ, Fordham AM, Pantarat N, Hibbs DE, Richardson V, Doddareddy MR, Ong JA, Huang ML, Richardson DR, Kalinowski DS - Oncotarget (2015)

Bottom Line: Considering albumin alters the uptake of many drugs, we examined the effect of human serum albumin (HSA) on the cellular uptake of Dp44mT, Bp4eT and PIH.Interestingly, HSA decreased Bp4eT and PIH uptake, potentially due to its high affinity for the ligands.This second saturable Dp44mT uptake process was inhibited by excess HSA and had characteristics suggesting it was mediated by a specific binding site.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, The University of Sydney, Sydney, NSW, Australia.

ABSTRACT
Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) demonstrates potent anti-cancer activity. We previously demonstrated that 14C-Dp44mT enters and targets cells through a carrier/receptor-mediated uptake process. Despite structural similarity, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and pyridoxal isonicotinoyl hydrazone (PIH) enter cells via passive diffusion. Considering albumin alters the uptake of many drugs, we examined the effect of human serum albumin (HSA) on the cellular uptake of Dp44mT, Bp4eT and PIH. Chelator-HSA binding studies demonstrated the following order of relative affinity: Bp4eT≈PIH>Dp44mT. Interestingly, HSA decreased Bp4eT and PIH uptake, potentially due to its high affinity for the ligands. In contrast, HSA markedly stimulated Dp44mT uptake by cells, with two saturable uptake mechanisms identified. The first mechanism saturated at 5-10 µM (B(max):1.20±0.04 × 10⁷ molecules/cell; K(d):33±3 µM) and was consistent with a previously identified Dp44mT receptor/carrier. The second mechanism was of lower affinity, but higher capacity (B(max):2.90±0.12 × 10⁷ molecules/cell; K(d):65±6 µM), becoming saturated at 100 µM and was only evident in the presence of HSA. This second saturable Dp44mT uptake process was inhibited by excess HSA and had characteristics suggesting it was mediated by a specific binding site. Significantly, the HSA-mediated increase in the targeting of Dp44mT to cancer cells potentiated apoptosis and could be important for enhancing efficacy.

No MeSH data available.


Related in: MedlinePlus

(A): Line drawings of the chemical structures of the iron chelators: di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and pyridoxal isonicotinoyl hydrazone (PIH)Asterisk (*) indicates position of the 14C-label. (B) Fluorescence emission spectrum of HSA (2 μM) excited at 295 nm in the presence of increasing concentrations (A→L; 0-3.67 μM) of: (i) Dp44mT; (ii) Bp4eT; or (iii) PIH in PBS at 37°C/pH 7.4. (C) Circular dichroism of HSA (2 μM) in the presence of: (i) Dp44mT, (ii) Bp4eT or (iii) PIH (10 μM) after a 2 h incubation at 37°C. Results shown are typical of 3 experiments performed.
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Figure 1: (A): Line drawings of the chemical structures of the iron chelators: di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and pyridoxal isonicotinoyl hydrazone (PIH)Asterisk (*) indicates position of the 14C-label. (B) Fluorescence emission spectrum of HSA (2 μM) excited at 295 nm in the presence of increasing concentrations (A→L; 0-3.67 μM) of: (i) Dp44mT; (ii) Bp4eT; or (iii) PIH in PBS at 37°C/pH 7.4. (C) Circular dichroism of HSA (2 μM) in the presence of: (i) Dp44mT, (ii) Bp4eT or (iii) PIH (10 μM) after a 2 h incubation at 37°C. Results shown are typical of 3 experiments performed.

Mentions: Thiosemicarbazone ligands are anti-cancer agents that bind metal ions and have shown anti-tumor activity in numerous investigations in vitro and in vivo, including many clinical trials [18-22]. As part of a specific strategy to generate selective and active anti-tumor agents, the di-2-pyridylketone thiosemicarbazones were developed [18, 19, 23-25]. In particular, the ligand, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT; Fig. 1A) and its analogs, were shown to have potent in vitro and in vivo anti-tumor activity [18, 24-26] and to possess marked anti-metastatic efficacy [27-29]. Additionally, the activity of Dp44mT was potentiated in drug-resistant cancer cells [24].


Potentiating the cellular targeting and anti-tumor activity of Dp44mT via binding to human serum albumin: two saturable mechanisms of Dp44mT uptake by cells.

Merlot AM, Sahni S, Lane DJ, Fordham AM, Pantarat N, Hibbs DE, Richardson V, Doddareddy MR, Ong JA, Huang ML, Richardson DR, Kalinowski DS - Oncotarget (2015)

(A): Line drawings of the chemical structures of the iron chelators: di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and pyridoxal isonicotinoyl hydrazone (PIH)Asterisk (*) indicates position of the 14C-label. (B) Fluorescence emission spectrum of HSA (2 μM) excited at 295 nm in the presence of increasing concentrations (A→L; 0-3.67 μM) of: (i) Dp44mT; (ii) Bp4eT; or (iii) PIH in PBS at 37°C/pH 7.4. (C) Circular dichroism of HSA (2 μM) in the presence of: (i) Dp44mT, (ii) Bp4eT or (iii) PIH (10 μM) after a 2 h incubation at 37°C. Results shown are typical of 3 experiments performed.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496362&req=5

Figure 1: (A): Line drawings of the chemical structures of the iron chelators: di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT), 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and pyridoxal isonicotinoyl hydrazone (PIH)Asterisk (*) indicates position of the 14C-label. (B) Fluorescence emission spectrum of HSA (2 μM) excited at 295 nm in the presence of increasing concentrations (A→L; 0-3.67 μM) of: (i) Dp44mT; (ii) Bp4eT; or (iii) PIH in PBS at 37°C/pH 7.4. (C) Circular dichroism of HSA (2 μM) in the presence of: (i) Dp44mT, (ii) Bp4eT or (iii) PIH (10 μM) after a 2 h incubation at 37°C. Results shown are typical of 3 experiments performed.
Mentions: Thiosemicarbazone ligands are anti-cancer agents that bind metal ions and have shown anti-tumor activity in numerous investigations in vitro and in vivo, including many clinical trials [18-22]. As part of a specific strategy to generate selective and active anti-tumor agents, the di-2-pyridylketone thiosemicarbazones were developed [18, 19, 23-25]. In particular, the ligand, di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT; Fig. 1A) and its analogs, were shown to have potent in vitro and in vivo anti-tumor activity [18, 24-26] and to possess marked anti-metastatic efficacy [27-29]. Additionally, the activity of Dp44mT was potentiated in drug-resistant cancer cells [24].

Bottom Line: Considering albumin alters the uptake of many drugs, we examined the effect of human serum albumin (HSA) on the cellular uptake of Dp44mT, Bp4eT and PIH.Interestingly, HSA decreased Bp4eT and PIH uptake, potentially due to its high affinity for the ligands.This second saturable Dp44mT uptake process was inhibited by excess HSA and had characteristics suggesting it was mediated by a specific binding site.

View Article: PubMed Central - PubMed

Affiliation: Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, The University of Sydney, Sydney, NSW, Australia.

ABSTRACT
Di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone (Dp44mT) demonstrates potent anti-cancer activity. We previously demonstrated that 14C-Dp44mT enters and targets cells through a carrier/receptor-mediated uptake process. Despite structural similarity, 2-benzoylpyridine 4-ethyl-3-thiosemicarbazone (Bp4eT) and pyridoxal isonicotinoyl hydrazone (PIH) enter cells via passive diffusion. Considering albumin alters the uptake of many drugs, we examined the effect of human serum albumin (HSA) on the cellular uptake of Dp44mT, Bp4eT and PIH. Chelator-HSA binding studies demonstrated the following order of relative affinity: Bp4eT≈PIH>Dp44mT. Interestingly, HSA decreased Bp4eT and PIH uptake, potentially due to its high affinity for the ligands. In contrast, HSA markedly stimulated Dp44mT uptake by cells, with two saturable uptake mechanisms identified. The first mechanism saturated at 5-10 µM (B(max):1.20±0.04 × 10⁷ molecules/cell; K(d):33±3 µM) and was consistent with a previously identified Dp44mT receptor/carrier. The second mechanism was of lower affinity, but higher capacity (B(max):2.90±0.12 × 10⁷ molecules/cell; K(d):65±6 µM), becoming saturated at 100 µM and was only evident in the presence of HSA. This second saturable Dp44mT uptake process was inhibited by excess HSA and had characteristics suggesting it was mediated by a specific binding site. Significantly, the HSA-mediated increase in the targeting of Dp44mT to cancer cells potentiated apoptosis and could be important for enhancing efficacy.

No MeSH data available.


Related in: MedlinePlus