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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

Schematic showing the internalization of 14C-Bp4eT/PIH or 14C-Dp44mT with or without HSA, 125I-HSA or 59Fe-125I-Tf into the cell(A) 14C-Bp4eT and 14C-PIH are transported by diffusion in the absence of HSA [34]. In the presence of HSA, HSA-binding inhibits the uptake of 14C-Bp4eT and 14C-PIH, irrespective of the cell-type. This is due to the high affinity of Bp4eT or PIH for HSA, relative to Dp44mT, reducing the levels of free drug available to diffuse into cells. (B) A different mechanism is demonstrated by the structurally similar ligand, Dp44mT. 14C-Dp44mT is taken up by cells via a receptor/carrier in the absence of HSA. This uptake process is saturable, energy- and temperature-dependent and subject to inhibition by excess unlabeled Dp44mT [34]. (C) In the presence of HSA, 14C-Dp44mT uptake occurs through a second, high capacity, saturable process that is cell-type specific and inhibited in the presence of excess unlabeled HSA. This process may be facilitated by: (i) a specific HSA-binding site; (ii) the fact most cellular HSA is bound to the cell membrane (rather than internalized); and (iii) the relatively low affinity of Dp44mT for HSA. These three properties facilitate Dp44mT delivery to cells and create a concentration gradient at the cell surface to enable enhanced uptake via dissociation and passive diffusion. The enhanced delivery of 14C-Dp44mT by HSA increases apoptosis and cytotoxicity. (D) 125I-HSA uptake by cells was saturable, temperature-dependent, inhibited by excess unlabeled HSA, and sensitive to glucose starvation and inhibitors of energy metabolism or endocytosis. Hence, this process was consistent with HSA endocytosis that is known to occur [74, 92]. (E) 59Fe-125I-Tf uptake occurs following the binding to its receptor, Tf receptor 1 (TfR1). This process was temperature- and energy-dependent, and subject to inhibition by excess unlabeled Tf, an endocytosis inhibitor, and in addition, lysosomotropic agents. Hence, 59Fe-125I-Tf uptake occurs through the well characterized process of receptor-mediated endocytosis that requires endosomal acidification [52, 68, 71, 93].
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Figure 7: Schematic showing the internalization of 14C-Bp4eT/PIH or 14C-Dp44mT with or without HSA, 125I-HSA or 59Fe-125I-Tf into the cell(A) 14C-Bp4eT and 14C-PIH are transported by diffusion in the absence of HSA [34]. In the presence of HSA, HSA-binding inhibits the uptake of 14C-Bp4eT and 14C-PIH, irrespective of the cell-type. This is due to the high affinity of Bp4eT or PIH for HSA, relative to Dp44mT, reducing the levels of free drug available to diffuse into cells. (B) A different mechanism is demonstrated by the structurally similar ligand, Dp44mT. 14C-Dp44mT is taken up by cells via a receptor/carrier in the absence of HSA. This uptake process is saturable, energy- and temperature-dependent and subject to inhibition by excess unlabeled Dp44mT [34]. (C) In the presence of HSA, 14C-Dp44mT uptake occurs through a second, high capacity, saturable process that is cell-type specific and inhibited in the presence of excess unlabeled HSA. This process may be facilitated by: (i) a specific HSA-binding site; (ii) the fact most cellular HSA is bound to the cell membrane (rather than internalized); and (iii) the relatively low affinity of Dp44mT for HSA. These three properties facilitate Dp44mT delivery to cells and create a concentration gradient at the cell surface to enable enhanced uptake via dissociation and passive diffusion. The enhanced delivery of 14C-Dp44mT by HSA increases apoptosis and cytotoxicity. (D) 125I-HSA uptake by cells was saturable, temperature-dependent, inhibited by excess unlabeled HSA, and sensitive to glucose starvation and inhibitors of energy metabolism or endocytosis. Hence, this process was consistent with HSA endocytosis that is known to occur [74, 92]. (E) 59Fe-125I-Tf uptake occurs following the binding to its receptor, Tf receptor 1 (TfR1). This process was temperature- and energy-dependent, and subject to inhibition by excess unlabeled Tf, an endocytosis inhibitor, and in addition, lysosomotropic agents. Hence, 59Fe-125I-Tf uptake occurs through the well characterized process of receptor-mediated endocytosis that requires endosomal acidification [52, 68, 71, 93].

Mentions: In this investigation, studies were performed to determine whether Dp44mT, Bp4eT or PIH were able to directly bind to albumin using fluorescence spectroscopy and equilibrium dialysis studies. These experiments demonstrated that all the ligands bind to HSA, although with different avidities (Fig. 1-2). In fact, equilibrium dialysis experiments indicated that 14C-Bp4eT became bound to HSA with similar avidity to 14C-PIH, while 14C-Dp44mT was most weakly bound to the protein (Fig. 2A). Molecular docking studies also supported these conclusions (Supplementary Fig. 1). Importantly, these findings indicating the avid binding of Bp4eT and PIH to HSA could explain the decreased uptake of these agents by cells in the presence of this protein (Fig. 3B-D). In fact, in the absence of HSA, Bp4eT and PIH are known to enter cells via passive diffusion [35]. Considering this, HSA may act as an extracellular ‘sink’, preventing the passive diffusion of Bp4eT and PIH into cells (Fig. 7A). Consequently, HSA did not enhance 14C-Bp4eT or 14C-PIH uptake or anti-proliferative activity, but conversely, decreased it (Fig. 3B-D, 7A).


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)

Schematic showing the internalization of 14C-Bp4eT/PIH or 14C-Dp44mT with or without HSA, 125I-HSA or 59Fe-125I-Tf into the cell(A) 14C-Bp4eT and 14C-PIH are transported by diffusion in the absence of HSA [34]. In the presence of HSA, HSA-binding inhibits the uptake of 14C-Bp4eT and 14C-PIH, irrespective of the cell-type. This is due to the high affinity of Bp4eT or PIH for HSA, relative to Dp44mT, reducing the levels of free drug available to diffuse into cells. (B) A different mechanism is demonstrated by the structurally similar ligand, Dp44mT. 14C-Dp44mT is taken up by cells via a receptor/carrier in the absence of HSA. This uptake process is saturable, energy- and temperature-dependent and subject to inhibition by excess unlabeled Dp44mT [34]. (C) In the presence of HSA, 14C-Dp44mT uptake occurs through a second, high capacity, saturable process that is cell-type specific and inhibited in the presence of excess unlabeled HSA. This process may be facilitated by: (i) a specific HSA-binding site; (ii) the fact most cellular HSA is bound to the cell membrane (rather than internalized); and (iii) the relatively low affinity of Dp44mT for HSA. These three properties facilitate Dp44mT delivery to cells and create a concentration gradient at the cell surface to enable enhanced uptake via dissociation and passive diffusion. The enhanced delivery of 14C-Dp44mT by HSA increases apoptosis and cytotoxicity. (D) 125I-HSA uptake by cells was saturable, temperature-dependent, inhibited by excess unlabeled HSA, and sensitive to glucose starvation and inhibitors of energy metabolism or endocytosis. Hence, this process was consistent with HSA endocytosis that is known to occur [74, 92]. (E) 59Fe-125I-Tf uptake occurs following the binding to its receptor, Tf receptor 1 (TfR1). This process was temperature- and energy-dependent, and subject to inhibition by excess unlabeled Tf, an endocytosis inhibitor, and in addition, lysosomotropic agents. Hence, 59Fe-125I-Tf uptake occurs through the well characterized process of receptor-mediated endocytosis that requires endosomal acidification [52, 68, 71, 93].
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Related In: Results  -  Collection

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

Figure 7: Schematic showing the internalization of 14C-Bp4eT/PIH or 14C-Dp44mT with or without HSA, 125I-HSA or 59Fe-125I-Tf into the cell(A) 14C-Bp4eT and 14C-PIH are transported by diffusion in the absence of HSA [34]. In the presence of HSA, HSA-binding inhibits the uptake of 14C-Bp4eT and 14C-PIH, irrespective of the cell-type. This is due to the high affinity of Bp4eT or PIH for HSA, relative to Dp44mT, reducing the levels of free drug available to diffuse into cells. (B) A different mechanism is demonstrated by the structurally similar ligand, Dp44mT. 14C-Dp44mT is taken up by cells via a receptor/carrier in the absence of HSA. This uptake process is saturable, energy- and temperature-dependent and subject to inhibition by excess unlabeled Dp44mT [34]. (C) In the presence of HSA, 14C-Dp44mT uptake occurs through a second, high capacity, saturable process that is cell-type specific and inhibited in the presence of excess unlabeled HSA. This process may be facilitated by: (i) a specific HSA-binding site; (ii) the fact most cellular HSA is bound to the cell membrane (rather than internalized); and (iii) the relatively low affinity of Dp44mT for HSA. These three properties facilitate Dp44mT delivery to cells and create a concentration gradient at the cell surface to enable enhanced uptake via dissociation and passive diffusion. The enhanced delivery of 14C-Dp44mT by HSA increases apoptosis and cytotoxicity. (D) 125I-HSA uptake by cells was saturable, temperature-dependent, inhibited by excess unlabeled HSA, and sensitive to glucose starvation and inhibitors of energy metabolism or endocytosis. Hence, this process was consistent with HSA endocytosis that is known to occur [74, 92]. (E) 59Fe-125I-Tf uptake occurs following the binding to its receptor, Tf receptor 1 (TfR1). This process was temperature- and energy-dependent, and subject to inhibition by excess unlabeled Tf, an endocytosis inhibitor, and in addition, lysosomotropic agents. Hence, 59Fe-125I-Tf uptake occurs through the well characterized process of receptor-mediated endocytosis that requires endosomal acidification [52, 68, 71, 93].
Mentions: In this investigation, studies were performed to determine whether Dp44mT, Bp4eT or PIH were able to directly bind to albumin using fluorescence spectroscopy and equilibrium dialysis studies. These experiments demonstrated that all the ligands bind to HSA, although with different avidities (Fig. 1-2). In fact, equilibrium dialysis experiments indicated that 14C-Bp4eT became bound to HSA with similar avidity to 14C-PIH, while 14C-Dp44mT was most weakly bound to the protein (Fig. 2A). Molecular docking studies also supported these conclusions (Supplementary Fig. 1). Importantly, these findings indicating the avid binding of Bp4eT and PIH to HSA could explain the decreased uptake of these agents by cells in the presence of this protein (Fig. 3B-D). In fact, in the absence of HSA, Bp4eT and PIH are known to enter cells via passive diffusion [35]. Considering this, HSA may act as an extracellular ‘sink’, preventing the passive diffusion of Bp4eT and PIH into cells (Fig. 7A). Consequently, HSA did not enhance 14C-Bp4eT or 14C-PIH uptake or anti-proliferative activity, but conversely, decreased it (Fig. 3B-D, 7A).

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