[Show abstract][Hide abstract] ABSTRACT: The objectives of these studies were to investigate and compare solid lipid nanoparticles (SLNs) of two anthracyclines, idarubicin (IDA) and doxorubicin (DOX), against Pgp-mediated multiple drug resistance (MDR) in-vitro and in-vivo using different human and murine cancer cell models. IDA and DOX SLNs were developed from warm microemulsion precursors comprising emulsifying wax as the oil phase, and polyoxyl 20-stearyl ether (Brij 78) and D-alpha-tocopheryl polyethylene glycol succinate (Vitamin E TPGS) as the surfactants. Anionic ion-pairing agents, sodium taurodeoxycholate (STDC) and sodium tetradecyl sulfate (STS), were used to neutralize the charges of the cationic anthracyclines and enhance entrapment of the drugs in the SLN. The in-vitro cytotoxicity results showed that the IC50 value of DOX NPs was 9-fold lower than that of free DOX solution in resistant P388/ADR cell line. In contrast, free IDA had comparable IC50 values as IDA NPs in Pgp-overexpressing P388/ADR and HCT-15 cells. In the in-vivo P388/ADR leukemia mouse model, the median survival time of DOX NPs was significantly greater than that of free DOX, and controls. In contrast, free IDA was equally as effective as IDA NPs in P388 and Pgp-overexpressing HCT-15 mouse tumor models. The cell uptake of IDA formulated as free IDA and IDA NPs was comparable in Pgp-overexpressing cells. In conclusion, DOX NPs could overcome Pgp-mediated MDR both in-vitro in P388/ADR leukemia cells and in-vivo in the murine leukemia mouse model. The present study suggests that our SLNs may offer potential to deliver anticancer drugs for the treatment of Pgp-mediated MDR in leukemia; however, selection of target drug may be very important.
[Show abstract][Hide abstract] ABSTRACT: The purpose of these studies was to investigate and compare the composition, stability, antioxidant and anticancer properties and mechanisms of anthocyanin-containing blackberry extracts (ACEs) from selected cultivars and using different extraction methods. ACEs were analyzed for total anthocyanin and phenolics content, polymeric color, and total antioxidant capacity (TAC). The influence of water content in the extraction system was evaluated. A 90-day stability study of the extract and a 48-h stability study of the extract in biologically relevant buffers were completed. The cytotoxic effects of ACEs on HT-29, MCF-7, and HL-60 cells were determined. H2O2 production in culture medium was measured and intracellular ROS levels were quantified. As compared to powder-derived ACEs, puree-derived ACEs contained similar amounts of anthocyanins, but greater levels of phenolics, increased TAC, significantly enhanced production of H2O2, and significantly enhanced cytotoxicity in all cell lines. Catalase could not protect cells from ACE-induced cell death. Cyanidin 3-glucoside exerted anticancer effect by acting synergistically or additively with other active components in the extracts. These data suggest that anthocyanins and non-anthocyanin phenolics in ACEs act synergistically or additively in producing anticancer effects. These studies also provide essential information for the development of fruit-derived ACEs as potential Botanical Drug Products.
Food and chemical toxicology: an international journal published for the British Industrial Biological Research Association 02/2009; 47(4-47):837-847. DOI:10.1016/j.fct.2009.01.016 · 2.90 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: As we gain a better understanding of the factors affecting cancer etiology, we can design improved treatment strategies. Over the past three to four decades, there have been numerous successful efforts in recognizing important cellular proteins essential in cancer growth and therefore these proteins have been targeted for cancer treatment. However, studies have shown that targeting one or two proteins in the complex cancer cascade may not be sufficient in controlling and/or inhibiting cancer growth. Therefore, there is a need to examine features which are potentially involved in multiple facets of cancer development. In this review we discuss the targeting of the elevated copper (both in serum and tumor) and oxidative stress levels in cancer with the aid of a copper chelator d-penicillamine (d-pen) for potential cancer treatment. Numerous studies in the literature have reported that both the serum and tumor copper levels are elevated in a variety of malignancies, including both solid tumor and blood cancer. Further, the elevated copper levels have been shown to be directly correlated to cancer progression. Enhanced levels of intrinsic oxidative stress has been shown in variety of tumors, possibly due to the combination of factors such as elevated active metabolism, mitochondrial mutation, cytokines, and inflammation. The cancer cells under sustained ROS stress tend to heavily utilize adaptation mechanisms and may exhaust cellular ROS-buffering capacity. Therefore, the elevated copper levels and increased oxidative stress in cancer cells provide for a prospect of selective cancer treatment.
Cancer Treatment Reviews 10/2008; 35(1):32-46. DOI:10.1016/j.ctrv.2008.07.004 · 7.59 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: D-Penicillamine (D-pen) is an established copper chelator. We have recently shown that the copper-catalyzed D-pen oxidation generates concentration-dependent hydrogen peroxide (H 2O 2). Additionally, D-pen coincubated with cupric sulfate resulted in cytotoxicity in human leukemia and breast cancer cells due to the extracellular generation of reactive oxygen species (ROS). The inherent physicochemical properties of D-pen such as its short in vivo half-life, low partition coefficient, and rapid metal catalyzed oxidation limit its intracellular uptake and the potential utility as an anticancer agent in vivo. Therefore, to enhance the intracellular delivery and to protect the thiol moiety of D-pen, we designed, synthesized, and evaluated a novel gelatin-D-pen conjugate. D-pen was covalently coupled to gelatin with a biologically reversible disulfide bond with the aid of a heterobifunctional cross-linker ( N-succinimidyl-3-(2-pyridyldithio)-propionate) (SPDP). Additionally, fluorescein-labeled gelatin-D-pen conjugate was synthesized for cell uptake studies. D-pen alone was shown not to enter leukemia cells. In contrast, the qualitative intracellular uptake of the conjugate in human leukemia cells (HL-60) was shown with confocal microscopy. The conjugate exhibited slow cell uptake (over the period of 48 to 72 h). A novel HPLC assay was developed to simultaneously quantify both D-pen and glutathione in a single run. The conjugate was shown to completely release D-pen in the presence of glutathione (1 mM) in approximately 3 h in PBS buffer, pH 7.4. The gelatin-D-pen conjugate resulted in significantly greater cytotoxicity compared to free D-pen, gelatin alone, and a physical mixture of gelatin and D-pen in human leukemia cells. Further studies are warranted to assess the potential of D-pen conjugate in the delivery of D-pen as a ROS generating anticancer agent.
[Show abstract][Hide abstract] ABSTRACT: Serum and tumor copper levels are significantly elevated in a variety of malignancies including breast, ovarian, gastric, lung, and leukemia. D-Penicillamine (D-pen), a copper-chelating agent, at low concentrations in the presence of copper generates concentration-dependent cytotoxic hydrogen peroxide (H(2)O(2)). The purpose of these studies was to investigate the in vitro cytotoxicity, intracellular reactive oxygen species (ROS) generation, and the reduction in intracellular thiol levels due to H(2)O(2) and other ROS generated from copper-catalyzed D-pen oxidation in human breast cancer cells (BT474, MCF-7) and human leukemia cells (HL-60, HL-60/VCR, HL-60/ADR). D-pen (< or = 400 microM) in the presence of cupric sulfate (10 microM) resulted in concentration-dependent cytotoxicity. Catalase was able to completely protect the cells, substantiating the involvement of H(2)O(2) in cancer cell cytotoxicity. A linear correlation between the D-pen concentration and the intracellular ROS generated was shown in both breast cancer and leukemia cells. D-pen in the presence of copper also resulted in a reduction in intracellular reduced thiol levels. The H(2)O(2)-mediated cytotoxicity was greater in leukemia cells compared to breast cancer cells. These results support the hypothesis that D-pen can be employed as a cytotoxic copper-chelating agent based on its ROS-generating ability.
Free Radical Biology and Medicine 11/2007; 43(9):1271-8. DOI:10.1016/j.freeradbiomed.2007.07.003 · 5.74 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: D-Penicillamine is a potent copper (Cu) chelating agent. D-Pen reduces Cu(II) to Cu(I) in the process of chelation while at the same time being oxidized to D-penicillamine disulfide. It has been proposed that hydrogen peroxide is generated during this process. However, definitive experimental proof that hydrogen peroxide is generated remains lacking. Thus, the major aims of these studies were to confirm and quantitatively assess the in vitro production of hydrogen peroxide during copper catalyzed D-penicillamine oxidation. The potential cytotoxic effect of hydrogen peroxide generation was also investigated in vitro against MCF-7 human breast cancer cells. Cell cytotoxicity resulting from the incubation of D-penicillamine with copper was compared to that of D-penicillamine, copper and hydrogen peroxide. The mechanism of copper catalyzed D-penicillamine oxidation and simultaneous hydrogen peroxide production was investigated as a function of time, concentration of cupric sulfate or ferric chloride, temperature, pH, anaerobic condition and chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid. A simple, sensitive and rapid HPLC assay was developed to simultaneously detect D-penicillamine, its major oxidation product D-penicillamine disulfide, and hydrogen peroxide in a single run. Hydrogen peroxide was shown to be generated in a concentration dependent manner as a result of D-penicillamine oxidation in the presence of cupric sulfate. Chelators such as ethylenediaminetetraacetic acid and bathocuproinedisulfonic acid were able to inhibit D-penicillamine oxidation. The incubation of MCF-7 human breast cancer cells with D-penicillamine plus cupric sulfate resulted in the production of reactive oxygen species within the cell and cytotoxicity that was comparable to free hydrogen peroxide.
[Show abstract][Hide abstract] ABSTRACT: Metal ions accumulate in the brain with aging and in several neurodegenerative diseases. Aside from the copper storage disease, Wilson's disease, recent attention has focused on the accumulation of zinc, copper and iron in the Alzheimer's disease (AD) brain and the accumulation of iron in Parkinson's disease. In particular, the parenchymal deposition of beta-amyloid (Abeta) and its interaction with metal ions has been postulated to play a role in the progression of AD. Thus, the strategy of lowering brain metal ions and targeting the interaction of Abeta peptide and metal ions through the administration of chelators has merit. Our recent finding that nanoparticle delivery systems can cross the blood-brain barrier has led us to investigate whether chelators delivered conjugated to nanoparticles could act to reverse metal ion induced protein precipitation. In the present studies, the Cu (I) chelator D-penicillamine was covalently conjugated to nanoparticles via a disulfide bond or a thioether bond. Nanoparticle-chelator conjugates were stable between pH 6-8 in aqueous suspension if stored at 4 degrees C, and did not aggregate when challenged with salts and serum. Release of D-penicillamine from the nanoparticles was achieved using reducing agents such as dithiothreitol (as a model for glutathione). Nanoparticles treated only under reducing conditions that released the conjugated D-penicillamine were able to effectively resolubilize copper-Abeta (1-42) aggregates. These results indicate that nanoparticles have potential to deliver D-penicillamine to the brain for the prevention of Abeta (1-42) accumulation, as well as to reduce metal ion accumulation in other CNS diseases.
European Journal of Pharmaceutics and Biopharmaceutics 03/2005; 59(2):263-72. DOI:10.1016/j.ejpb.2004.07.009 · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The significantly increased copper and oxidative stress levels are characteristic hallmarks of cancer cells. These differences provide a unique opportunity for selective targeting of cancer cells. D-penicillamine (D-pen) has been proposed to generate reactive oxygen species (ROS) in presence of copper. Therefore, these studies were aimed at investigating the potential application of a currently marketed copper chelator, D-pen, as a novel cytotoxic anti-cancer agent. D-pen was shown to produce ROS, specifically hydrogen peroxide (H2O2), in the presence of cupric sulfate through a copper catalyzed oxidation reaction. During this process D-pen was converted to D-pen disulfide. The experimental proof of the H2O2 generation was conclusively shown with the aid of a novel High Performance Liquid Chromatography (HPLC) assay. The in-vitro cytotoxicity of D-pen co-incubated with cupric sulfate was examined in human beast cancer (MCF-7 and BT474) and leukemia cells (HL-60, HL-60/VCR, and HL-60/ADR). D-pen was shown to cause concentration dependent cytotoxicity in both leukemia and breast cancer cells. A direct correlation between the detection of intracellular ROS and cytotoxicity was established. The treatment of D-pen plus cupric sulfate resulted in a significant reduction in the intracellular thiol content. D-pen is highly hydrophilic and is rapidly eliminated from the body; therefore to improve the intracellular uptake and to protect the thiol group of D-pen, we carried out the synthesis and the in-vitro characterization of a novel gelatin-D-pen conjugate. It was shown that D-pen alone does not enter cells. Confocal microscopy was employed to exhibit the uptake of the novel gelatin-D-pen conjugate by cancer cells. As the cancer cells in-vitro do not accumulate the same levels of copper as reported for cancer cells in-vivo, cancer cells were pre-treated with cupric sulfate to simulate the elevated copper levels. The cupric sulfate pretreatment resulted in reduced thiol level and significantly increased cellular copper content compared to untreated cells. Whereas both free D-pen and gelatin-D-pen conjugate lacked cytotoxicity in un-treated cells, both agents caused concentration dependent cytotoxicity in cupric sulfate pre-treated leukemia cells. Therefore, it was shown that the administration of D-pen as polymer conjugate would potentially provide cytotoxicity and specificity in the treatment of cancer.