Distinct cellular responses induced by saporin and a transferrin-saporin conjugate in two different human glioblastoma cell lines.

Glioblastoma multiforme (GBM) is the most common primary brain tumour in adults, with a median survival of ~12-18 months post-diagnosis. GBM usually recurs within 12 months post-resection, with poor prognosis. Thus, novel therapeutic strategies to target and kill GBM cells are urgently needed. The marked difference of tumour cells with respect to normal brain cells renders glioblastoma a good candidate for selective targeted therapies. Recent experimental strategies focus on over expressed cell surface receptors. Targeted toxins represent a new class of selective molecules composed by a potent protein toxin and a carrier ligand. Targeted toxins approaches against glioblastoma were under investigation in phase I and II clinical trials with several immunotoxins (IT)/ligand toxins such as IL4-Pseudomonas aeruginosa exotoxin A (IL4-PE, NBI-3001), tumour growth factor fused to PE38, a shorter PE variant, (TGF)alpha-TP-38, IL13-PE38, and a transferrin-C diphtheriae toxin mutant (Tf-CRM107). In this work, we studied the effects of the plant ribosome-inactivating saporin and of its chimera transferrin-saporin against two different GBM cell lines. The data obtained here indicate that cell proliferation is affected by the toxin treatments but that different mechanisms are used, directly linked to the presence of an active or inactive p53. A model is proposed for these alternative intracellular pathways.

Robust Strategy for Antibody-Polymer-Drug Conjugation: Significance of Conjugating Orientation and Linker Charge on Targeting Ability.

Antibody-drug conjugates have shown great promise in active targeting for cancer therapy. The existing chemical techniques for antibody conjugation generally lack efficiency or universality. In this article, a site-specific antibody conjugation was developed by using a mild reaction between a benzoboroxole (BB) functionality and cis-diol moiety of sugar units in the antibody fragment crystallizable region under neutral pH conditions. A BB/PEG/ICG-grafted poly(aspartic acid) comb-like functional polymer was first synthesized and conjugated with transferrin (Tf) to form a transferrin-polymer-drug conjugate [Tf-P(BB)], which showed 120% increase in HepG2 hepatoma (Tf receptor overexpression) cell uptake compared to a nontargeting protein-polymer-drug conjugate [HRP-P(BB)]. The universality of this method was further demonstrated by the enhanced uptake of trastuzumab (anti-Her2 antibody)-polymer-drug conjugates in MCF-7 (295%) and MDA-MB-435S (66.4%) (Her2 positive) cells. The positive charge of the linker had great influence on the targeting ability of the antibody-polymer-drug conjugates. The in vivo studies demonstrated the distinct targeting ability of Tf-P(BB) in the HepG2 xenograft tumor, and the tumor accumulation of the Tf-P(BB) testing group increased by 92% with respect to the control group [HRP-P(BB)]. More significantly, the HepG2 cell uptake amount of the antibody-oriented conjugate [Tf-P'(BB)] was 2.4-fold higher than that of the controlled group [Tf-P'(Hex)]. On the basis of this facile site-specific conjugation method, the conjugates are able to change the antibody species easily against various cancers, while maintaining the antibody integrity and targeting ability.

A cation exchange strategy to construct a targeting nanoprobe for enhanced T1-weighted MR imaging of tumors.

Excellent imaging performance and good biocompatibility of contrast agents are considered as prerequisites for accurate tumor diagnosis. In this study, a novel imaging nanoprobe with actively targeting performance based on ultrasmall paramagnetic iron oxide (USPIO) nanoparticles was constructed by a facile cation exchange strategy followed by conjugation with transferrin (Tf). The stable gadolinium (Gd3+) chelation endows the nanoparticles (NPs) with a low value of r2/r1 (1.28) and a relatively high r1 value of 3.2 mM-1 s-1, enabling their use for T1-weighted positive magnetic resonance (MR) imaging. This constructed transferrin modified gadolinium-iron chelate nanoprobe, named as TUG, shows high biocompatibility within a given dose range. More importantly, compared with clinically used Gd-based small molecule contrast agents, the obtained TUG can be more engulfed by breast cancer cells, showing much enhanced T1-weighted positive MR imaging in both subcutaneous and orthotopic tumor models of breast cancer. This novel nanoprobe holds great promise to be utilized as a targeting contrast agent with high efficacy for T1-weighted positive MR imaging.

The construction of a novel kind of non-viral gene delivery vector based on protein as core backbone.

BACKGROUND AND OBJECTIVES: A novel kind of non-viral gene delivery vector based on transferrin (Tf) as the core component was constructed with high transfection efficiency and low toxicity. MATERIALS AND METHODS: The synthesis vector of Tf-PEI600 was confirmed by different physicochemical methods, including (1)H nuclear magnetic resonance, gel permeation chromatography, X-ray and thermogravimetric analysis. The cytotoxicity and gene delivery efficiency of the synthesized vector were verified by in vitro experiments. RESULTS: The agarose gel electrophoresis assay indicated that the novel copolymer Tf-PEI600 could efficiently condense plasmid DNA and the condensed nanoparticles exhibited a spherical shape. As the weight ratio of Tf-PEI600 to DNA reached 15.0, the particle size (about 200 nm) and the zeta potential (about 20 mV) of the nanoparticles became optimal for gene delivery. The methylthiazolyl tetrazolium (MTT) assay showed the cytotoxicity of Tf-PEI600 to be similar to that of PEI600 and much lower than that of PEI25kDa. In gene-delivery experiments with COS-7 cells and HepG2 cells, the Tf-PEI600 showed about a 30- to 53-fold higher efficiency than PEI600 and nearly equal to that of PEI25kDa. CONCLUSIONS: These data suggest that Tf-PEI600, with the advantages of low toxicity and high gene-delivery efficiency, might have great prospects in the practice of gene delivery. The core-shell structure of Tf-PEI600 also provided a novel strategy for the construction of non-viral gene delivery vectors.

Characteristics comparison before and after lyophilization of transferrin modified Procationic-Liposome-Protamine-DNA complexes (Tf-PLPD).

A novel non-viral gene delivery system, Procationic-Liposome-Protamine-DNA complexes (PLPD) which could further adsorb transferrin on the surface as a targeting ligand to form Tf-PLPD, was prepared and characterized before and after lyophilization. The size distribution of Tf-PLPD was in the range of 240 +/- 12 nm and the zeta potential was -24.10 +/- 2.5 mV. The transfection efficiencies of PLPD and Tf-PLPD were 12.18 +/- 3.8 and 24.26 +/- 2.6 mU beta-galactosidase/mg protein respectively. The lyophilization and the presence of serum didn't affect the tansfectivities of PLPD or Tf-PLPD. Compared to Lipofectamine 2000 (Invitrogen, U.S.A.), the procationic liposomes had less cytotoxicity to cells. In summary the procationic lipoplex described here, combining the condensing effect of protamine and the targeting capability of Tf, was a perspective non-viral vector for gene delivery system.

Tumor-promoting effect of urinary epidermal growth factor in rat urinary bladder carcinogenesis.

We previously demonstrated that the specific component of rat urine designated as Fraction I (Fr.I), which has been known to enhance carcinogenesis in the rat urinary bladder, contains epidermal growth factor (EGF) and transferrin (TF). The present study was designed to determine whether EGF or TF is responsible for the tumor-enhancing effect of Fr.I. The heterotopically transplanted rat urinary bladder (HTB), which has been developed in our laboratory, was used for the study. Fr.I was prepared from normal rat urine by a method published previously. Fr.I deficient in EGF or TF was prepared by passing this fraction through an Affi-Gel Hz column coupled with anti-rat EGF or TF antibodies, respectively. EGF and TF eluted from the column (designated as eluted EGF and eluted TF) were also tested for tumor-enhancing activity. Fr.I passed through the column coupled with nonimmune rabbit IgG served as control (Fr.I column control). After initiation of carcinogenesis in HTBs by instillation of a single dose of 0.25 mg of N-methyl-N-nitrosourea, test materials were administered into these HTBs once a week for 30 weeks. The results showed that removal of EGF significantly reduced the tumor-enhancing effect of Fr.I (P less than 0.001 as compared to that of the Fr.I column control) and that eluted EGF by itself significantly enhanced the carcinogenesis as compared to that of the vehicle control (P less than 0.006). Removal of TF from Fr.I also reduced the tumor-enhancing effect of Fr.I (P less than 0.01). However, removal of both EGF and TF from Fr.I did not enhance the inhibitory effect demonstrated by the Fr.I which was deficient in EGF. Likewise, combined use of TF and EGF did not exceed the tumor-promoting effect of EGF. The results indicate that EGF in Fr.I may play a significant role in the promotion of bladder carcinogenesis by urine.

Vascular protection by chloroquine during brain tumor therapy with Tf-CRM107.

Tf-CRM107 is a conjugate of transferrin and a point mutant of diphtheria toxin that selectively kills cells expressing high levels of the transferrin receptor. Tf-CRM107 has been infused intratumorally into patients with malignant brain tumors. Although approximately half of the patients exhibit tumor responses, patients receiving higher doses of Tf-CRM107 may develop magnetic resonance image (MRI) evidence of toxicity indicative of small vessel thrombosis or petechial hemorrhage. Consistent with these clinical results we found that intracerebral injection of Tf-CRM107 into rats at total doses > or =0.025 microg causes brain damage detectable by MRI and histology. To widen the therapeutic window of Tf-CRM107, we explored ways to prevent this damage to the vasculature. We reasoned that the vasculature may be protected to a greater extent than tumor from Tf-CRM107 infused into brain parenchyma by i.v. injection of reagents with low blood-brain barrier permeability that block the toxicity of Tf-CRM107. Chloroquine, a well-characterized antimalarial drug, blocks the toxicity of diphtheria toxin and Tf-CRM107. Systemic administration of chloroquine blocked the toxicity of Tf-CRM107 infused intracerebrally in rats and changed the maximum tolerated dose of Tf-CRM107 from 0.2 to 0.3 microg. Moreover, chloroquine treatment completely blocked the brain damage detected by MRI caused by intracerebral infusion of 0.05 microg of Tf-CRM107. In nude mice bearing s.c. U251 gliomas, chloroquine treatment had little effect on the antitumor efficacy of Tf-CRM107. Thus, chloroquine treatment may be useful to reduce the toxicity of Tf-CRM107 for normal brain without inhibiting antitumor efficacy and increase the therapeutic window of Tf-CRM107 for brain tumor therapy.

Facile preparation of hyaluronic acid and transferrin co-modified Fe3O4 nanoparticles with inherent biocompatibility for dual-targeting magnetic resonance imaging of tumors in vivo.

Clinical diagnosis of malignant tumors using nanoprobes needs severe improvements in the aspects of sensitivity and biocompatibility. Integrating a dual-targeting strategy with the selection of human-inherent elements and molecules as raw materials shows great potential in the development of a biosafe and sensitive nanoplatform. To carry out the proposed design, we constructed a biocompatible, dual-targeting MR imaging nanoprobe, based on Fe3O4 nanoparticles (NPs) co-modified with inherently innoxious hyaluronic acid (HA) and transferrin (Tf). HA was used as both a template and a targeting molecule to form Fe3O4@HA NPs through a one-step co-precipitation method, which were then further modified with Tf to obtain the dual-targeting Fe3O4@HA@Tf NPs at room temperature. The excellent biocompatibility of the nanoprobe was demonstrated via toxicity assays in vitro and in vivo. The desirable dual-targeting ability towards tumor cells was confirmed by a cellular uptake test (Hela cells, overexpressing both CD44 and transferrin receptors), and the developed nanoprobe was successfully applied in tumor-targeted MR imaging in vivo. In summation, we developed a dual-targeting Fe3O4 nanoprobe, following a facile procedure at room temperature. The nanoprobe showed a high targeting ability towards tumor cells and excellent biocompatibility, which showed its great potential to be applied in the clinical diagnosis of tumors.

Mechanisms of Dihydroartemisinin and Dihydroartemisinin/Holotransferrin Cytotoxicity in T-Cell Lymphoma Cells.

The validated therapeutic effects of dihydroartemisinin (DHA) in solid tumors have encouraged us to explore its potential in treating T-cell lymphoma. We found that Jurkat cells (a T-cell lyphoma cell line) were sensitive to DHA treatment with a IC50 of dihydroartemisinin. The cytotoxic effect of DHA in Jurkat cells showed a dose- and time- dependent manner. Interestingly, the cytotoxic effect of DHA was further enhanced by holotransferrin (HTF) due to the high expression of transferrin receptors in T-cell lymphoma. Mechanistically, DHA significantly increased the production of intracellular reactive oxygen species, which led to cell cycle arrest and apoptosis. The DHA treatment also inhibited the expression of protumorgenic factors including VEGF and telomerase catalytic subunit. Our results have proved the therapeutic effect of DHA in T-cell lymphoma. Especially in combination with HTF, DHA may provide a novel efficient approach in combating the deadly disease.

Targeted lung cancer therapy: preparation and optimization of transferrin-decorated nanostructured lipid carriers as novel nanomedicine for co-delivery of anticancer drugs and DNA.

PURPOSE: Nanostructured lipid carriers (NLC) represent an improved generation of lipid nanoparticles. They have specific nanostructures to accommodate drugs/genes, and thus achieve higher loading capacity. The aim of this study was to develop transferrin (Tf)-decorated NLC as multifunctional nanomedicine for co-delivery of paclitaxel (PTX) and enhanced green fluorescence protein plasmid. METHODS: Firstly, Tf-conjugated ligands were synthesized. Secondly, PTX- and DNA-loaded NLC (PTX-DNA-NLC) was prepared. Finally, Tf-containing ligands were used for the surface decoration of NLC. Their average size, zeta potential, drug, and gene loading were evaluated. Human non-small cell lung carcinoma cell line (NCl-H460 cells) was used for the testing of in vitro transfection efficiency, and in vivo transfection efficiency of NLC was evaluated on mice bearing NCl-H460 cells. RESULTS: Tf-decorated PTX and DNA co-encapsulated NLC (Tf-PTX-DNA-NLC) were nano-sized particles with positive zeta potential. Tf-PTX-DNA-NLC displayed low cytotoxicity, high gene transfection efficiency, and enhanced antitumor activity in vitro and in vivo. CONCLUSION: The results demonstrated that Tf-PTX-DNA-NLC can achieve impressive antitumor activity and gene transfection efficiency. Tf decoration also enhanced the active targeting ability of the carriers to NCl-H460 cells. The novel drug and gene delivery system offers a promising strategy for the treatment of lung cancer.

Synthesis and in vitro efficacy of transferrin conjugates of the anticancer drug chlorambucil.

One strategy for improving the selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing soluble macromolecules or carrier proteins. Thus, five maleimide derivatives of chlorambucil were bound to thiolated human serum transferrin which differ in the stability of the chemical link between drug and spacer. The maleimide ester derivatives 1 and 2 were prepared by reacting 2-hydroxyethylmaleimide or 3-maleimidophenol with the carboxyl group of chlorambucil, and the carboxylic hydrazone derivatives 5-7 were obtained through reaction of 2-maleimidoacetaldehyde, 3-maleimidoacetophenone, or 3-maleimidobenzaldehyde with the carboxylic acid hydrazide derivative of chlorambucil. The alkylating activity of transferrin-bound chlorambucil was determined with the aid of 4-(4-nitrobenzyl)pyridine (NBP) demonstrating that on average 3 equivalents were protein-bound. Evaluation of the cytotoxicity of free chlorambucil and the respective transferrin conjugates in the MCF7 mammary carcinoma and MOLT4 leukemia cell line employing a propidium iodide fluorescence assay demonstrated that the conjugates in which chlorambucil was bound to transferrin through non-acid-sensitive linkers, i.e., an ester or benzaldehyde carboxylic hydrazone bond, were not, on the whole, as active as chlorambucil. In contrast, the two conjugates in which chlorambucil was bound to transferrin through acid-sensitive carboxylic hydrazone bonds were as active as or more active than chlorambucil in both cell lines. Especially, the conjugate in which chlorambucil was bound to transferrin through an acetaldehyde carboxylic hydrazone bond exhibited IC50 values which were approximately 3-18-fold lower than those of chlorambucil. Preliminary toxicity studies in mice showed that this conjugate can be administered at higher doses in comparison to unbound chlorambucil. The structure-activity relationships of the transferrin conjugates are discussed with respect to their pH-dependent acid sensitivity, their serum stability, and their cytotoxicity.

Breast carcinoma and the role of iron metabolism. A cytochemical, tissue culture, and ultrastructural study.

Transferrin receptors on proliferating and malignant cells are well documented. Iron is an essential micronutrient for cell growth that plays an important role in energy metabolism and DNA synthesis. Malignant cells requiring more iron modulate a transferrin receptor. Iron-bound transferrin interacts with this receptor, facilitating the transport of iron across the cell membrane. Transferrin is a glycoprotein and is the chief iron transport protein in mammalian blood. The more aggressive the tumor, the higher the transferrin receptor levels and the greater the proliferative index. We have found by cytochemical and ultrastructural studies that ferritin, an iron storage protein, is increased in breast cancer tissue. Anaplastic tumors have higher tissue ferritin levels. Tissue ferritin concentration may be an indirect method of measuring transferrin receptors and thus might be an index of proliferation and a prognostic indicator. Transferrin may be used as a carrier to target toxic therapy selectively to tumor tissue. A platinum transferrin complex (MPTC-63) has been developed and shown to be cytostatic in tissue culture, animal, and human studies. It also sensitizes tissue to agents that produce free radicals, such as adriamycin, and thus is synergistic with other drugs and radiation. Other transferrin complexes and conjugates of gallium, indium, and daunorubicin have also shown growth inhibition in tissue culture and animals. Human studies are in progress. By studying iron metabolism in breast cancer, we may be able to selectively inhibit tumor growth without toxic effects, and with other tumor biologic data be better able to select the stage I patient for adjuvant therapy.

In vitro cytotoxicity of paclitaxel-transferrin conjugate on H69 cells.

Transferrin is a serum glycoprotein involved in iron transport. Transferrin acts also in cell growth regulation through membrane receptors. The number of transferrin receptors is increased in tumor and other rapidly dividing cells. This renders transferrin suitable for use in cytotoxic drugs targetting tumor cells. Paclitaxel was derivatized on 2' carbon and coupled with trasferrin using glutaraldehyde. The cytotoxicity of the conjugate was evaluated on small cell carcinoma of the lung cell line (H69). As compared to paclitaxel, the conjugate exhibited a slight decrease in cytotoxicity.

Selective toxicity of dihydroartemisinin and holotransferrin toward human breast cancer cells.

Artemisinin becomes cytotoxic in the presence of ferrous iron. Since iron influx is high in cancer cells, artemisinin and its analogs selectively kill cancer cells under conditions that increase intracellular iron concentrations. We report here that after incubation with holotransferrin, which increases the concentration of ferrous iron in cancer cells, dihydroartemisinin, an analog of artemisinin, effectively killed a type of radiation-resistant human breast cancer cell in vitro. The same treatment had considerably less effect on normal human breast cells. Since it is relatively easy to increase the iron content inside cancer cells in vivo, administration of artemisinin-like drugs and intracellular iron-enhancing compounds may be a simple, effective, and economical treatment for cancer.

Sensitivity of multidrug-resistant MCF-7 cells to a transferrin-doxorubicin conjugate.

Two multidrug-resistant breast cancer cell lines (MCF-7/AdrVp and MCF-7/D.40) each expressing a different membrane protein, involved in the drug resistance, have been treated with a transferrin-doxorubicin conjugate. Conjugates have shown an increase of activity over free doxorubicin on these resistant cell lines. Growth inhibition of doxorubicin-resistant cells, as evaluated by the MTT-assay, was higher for conjugates than for free doxorubicin especially for a 4-day contact period. I D 50 were twice and 10-fold lower for the conjugate than for free doxorubicin on resistant cells. MCF-7/AdrVp seemed to be particularly affected by the conjugate even if its intracellular content of doxorubicin was similar. With the Trf-Dox conjugate, an inverted correlation does exist between the drug-DNA content and the cytotoxicity of the conjugate. Verapamil influenced the uptake of free doxorubicin but not the uptake of Trf-Dox conjugate, thus showing a different mechanism of entry.

Doxorubicin-gallium-transferrin conjugate overcomes multidrug resistance: evidence for drug accumulation in the nucleus of drug resistant MCF-7/ADR cells.

Development of multidrug resistance (MDR) in cancer cells decreases net doxorubicin (ADR) uptake as a result of increased efflux, increased intracellular sequestration, and decreased membrane permeability. In this study, we investigated whether conjugation of ADR to transferrin (Tf) could overcome MDR in breast cancer cells. The multidrug resistant MCF-7/ADR breast cancer cell line was over 1000-fold more resistant to ADR, than its parental MCF-7 cell line, as determined by 3[H]-thymidine assay. The MCF-7/ADR cell line also expressed both MDR1 and MRP genes, as detected by RT-PCR. The ADR was coupled using a glutaraldehyde technique to human transferrin saturated with either ferric chloride (Fe-Tf) or gallium nitrate (Ga-Tf). These conjugates were tested for cytotoxicity on both MCF-7 and MCF-7/ADR cells after 6 days of incubation. The doxorubicin-gallium-transferrin conjugate (ADR-Ga-Tf) exhibited approximately the same inhibitory effect as ADR on MCF-7 cells with IC50s of 2.34 x 10(-3) microM and 1.42 x 10(-3) microM, respectively. However in MCF-7/ADR cells ADR-Ga-Tf reversed resistance to free ADR and decreased 100-fold the IC50 from 8.98 microM with free ADR to 9.52 x 10(-2) microM. ADR-Fe-Tf was 10-fold more inhibitory to MCF-7/ADR cells than free ADR. Compared to Ga-Tf, ADR-Ga-Tf was 500- and 3000-fold more inhibitory to MCF-7 and MCF-7/ADR cells, respectively. These results demonstrated that ADR-Ga-Tf reverses resistance to free ADR and Ga-Tf in MCF-7/ADR cells. The distribution of ADR in both cell lines was examined by fluorescence microscopy. It was noted that ADR mainly accumulated in the cytoplasm around the nucleus in MCF-7/ADR cells, but in both the cytoplasm and nucleus of MCF-7 cells. However the conjugate of ADR-Ga-Tf allowed ADR to accumulate in the cytoplasm and nucleus of both the MCF-7/ADR and MCF-7 cells. Further investigation of MDR1 and MRP genes expression by RT-PCR demonstrated that Ga-Tf decreased expression of the MRP more than the MDR1 gene. Therefore the reversal of resistance to ADR by the ADR-Ga-Tf conjugate is mediated by the transferrin receptor transmembrane transport mechanism, redistribution of ADR into the nucleus of ADR resistant MCF-7/ADR cells and inhibition of MRP gene expression.

In vivo cytotoxicity and antineoplastic activity of a transferrin-daunorubicin conjugate.

Transferrin, the major iron-binding protein in the plasma of vertebrate species, is an essential growth factor for proliferating malignant cells. The specific receptor binds diferric transferrin and is endocytosed into the cell. We would like to take advantage of this physiologic phenomenon using an active transferrin-daunorubicin conjugate to target cancer cells. We have compared the in vitro and the in vivo activity of free daunorubicin and of daunorubicin transferrin conjugate on cancer cells. The results suggest that our conjugate is less toxic and more active upon malignant cells than the free drug, daunorubicin, while being less toxic for normal cells.

Mechanism of free and conjugated neocarzinostatin activity: studies on chromophore and protein uptake using a transferrin-neocarzinostatin conjugate.

Targeting studies using the anti-cancer agent neocarzinostatin (NCS), conjugated to anti-bodies have shown relatively poor specificity. From the literature, it is unclear whether NCS mediates its effects either in conjugated or unconjugated form. In the present work we have used a conjugate of NCS with transferrin, a biological ligand with a well defined endocytic route, to probe these mechanisms. NCS was covalently coupled to transferrin using the heterobifunctional reagent sulfo-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC) and 2-iminothiolane to give a stable thioether-linked conjugate with a ratio of 1.6 mol of NCS per mole of transferrin. The binding activity of transferrin was completely retained. Conjugation of NCS to transferrin resulted in an apparent enhancement of cytotoxicity. However, incubation with excess transferrin had no influence on the observed enhanced toxicity, indicating that endocytosis is not responsible. Further experiments demonstrated that the apparent enhancement was dependent on incubation conditions and not an effect due to endocytosis of ligand. Studies where apo-NCS competed with holo-NCS and transferrin strongly indicated that the cytotoxicity of both NCS and conjugate is mediated by direct entry of the dissociated chromophore into the cell.

Transferrin as a drug carrier: Cytotoxicity, cellular uptake and transport kinetics of doxorubicin transferrin conjugate in the human leukemia cells.

Leukemias are one of most common malignancies worldwide. There is a substantial need for new chemotherapeutic drugs effective against this cancer. Doxorubicin (DOX), used for treatment of leukemias and solid tumors, is poorly efficacious when it is administered systemically at conventional doses. Therefore, several strategies have been developed to reduce the side effects of this anthracycline treatment. In this study we compared the effect of DOX and doxorubicin-transferrin conjugate (DOX-TRF) on human leukemia cell lines: chronic erythromyeloblastoid leukemia (K562), sensitive and resistant (K562/DOX) to doxorubicin, and acute lymphoblastic leukemia (CCRF-CEM). Experiments were also carried out on normal cells, peripheral blood mononuclear cells (PBMC). We analyzed the chemical structure of DOX-TRF conjugate by using mass spectroscopy. The in vitro growth-inhibition assay XTT, indicated that DOX-TRF is more cytotoxic for leukemia cells sensitive and resistant to doxorubicin and significantly less sensitive to normal cells compared to DOX alone. During the assessment of intracellular DOX-TRF accumulation it was confirmed that the tested malignant cells were able to retain the examined conjugate for longer periods of time than normal lymphocytes. Comparison of kinetic parameters showed that the rate of DOX-TRF efflux was also slower in the tested cells than free DOX. The results presented here should contribute to the understanding of the differences in antitumor activities of the DOX-TRF conjugate and free drug.

Killing of K562 cells with conjugates between human transferrin and a ribosome-inactivating protein (SO-6).

Cellular iron uptake is mediated by binding of transferrin with specific surface receptors and internalization of the Fe-transferrin-receptor complex. This has been examined as a possible pathway for carrying into leukaemic cells a ribosome-inactivating protein (RIP), SO-6, derived from Saponaria officinalis. Purified human differic transferrin was conjugated with SO-6 and a pool of proteins was obtained, with variable numbers of SO-6 molecules linked to a single transferrin molecule. Human erythroleukaemic K562 cells were grown in the presence of human transferrin, SO-6 and human transferrin conjugated with SO-6. The conjugate was found to be internalized via binding with transferrin receptor. Whereas the presence of unconjugated human transferrin and SO-6 in the medium did not significantly influence K562 cell growth, the conjugated proteins displayed an inhibitory activity on cell proliferation. This was maximal after 72 h at a transferrin concentration of 10(-9) M, with about 50% of cells being killed. Bovine transferrin, present in fetal calf serum, did not appear to compete with human diferric transferrin in binding to K562 cells in suspension culture. In a clonogenic assay, colony formation by leukaemic cells was not influenced by free SO-6 or transferrin, whereas the conjugated proteins were markedly inhibitory (about 100% at 10(-9) M). Our findings indicate that SO-6 can be efficiently carried into mammalian cells via the transferrin-transferrin receptor cycle and exert its ribosome inactivating activity. This is in keeping with the existence of an alternative pathway of transferrin endocytosis in addition to the classic acidic endosome pathway. From a practical viewpoint, conjugates between transferrin and SO-6 can be useful tools for studying the expression of transferrin receptors, and deserve also to be investigated for a possible use in cancer therapy.