Overexpression Of Transferrin Receptor Research Articles

METTL3 silenced inhibited the ferroptosis development via regulating the TFRC levels in the Intracerebral hemorrhage progression

Intracerebral hemorrhage (ICH) refers to the hemorrhage caused by the increase and rupture of vascular brittleness in non traumatic brain parenchyma, which has been demonstrated to be closely related to ferroptosis. This study aimed to examine the effects of methyltransferase like 3 (METTL3) on the ferroptosis in the ICH progression. The PC12 cells was stimulated by hemin to establish a ICH model. The cell viability was tested by CCK8 assay. The Fe2+, reactive oxygen species (ROS), and malondialdehyde (MDA) levels were determined by the corresponding commercial kits. The cell death was analyzed by propidium Iodide (PI) staining. The lactylation levels were detected by western blot. M6A dot blot assay was performed to detected the total m6A levels and MeRIP assay was conducted to determine the m6A levels of transferrin receptor (TFRC). We found that the METTL3 and m6A levels were increased in the hemin treated PC12 cells. METTL3 knockdown increased the cell viability and decreased Fe2+, ROS and MDA levels in the hemin treated PC12 cells. The role of METTL3 knockdown in the hemin treated PC12 cells was reversed after TFRC overexpression. Mechanistically, the METTL3 lactylation was increased in the hemin treated PC12 cells, which further enhanced the protein stability and expression of METTL3. The up-regulated METTL3 increased the m6A levels and mRNA expressions of TFRC, which further induced the ferroptosis of the PC12 cells. In conclusion, the up-regulation of METTL3 lactylation enhanced the METTL3 protein stability and expression levels in hemin treated PC12 cells. METTL3 silenced suppressed the ferroptosis development through regulating the m6A levels of TFRC mRNA.

Identification of Transferrin Receptor 1 (TfR1) Overexpressed in Lung Cancer Cells, and Internalization of Magnetic Au-CoFe2O4 Core-Shell Nanoparticles Functionalized with Its Ligand in a Cellular Model of Small Cell Lung Cancer (SCLC).

Lung cancer is, currently, one of the main malignancies causing deaths worldwide. To date, early prognostic and diagnostic markers for small cell lung cancer (SCLC) have not been systematically and clearly identified, so most patients receive standard treatment. In the present study, we combine quantitative proteomics studies and the use of magnetic core-shell nanoparticles (mCSNP’s), first to identify a marker for lung cancer, and second to functionalize the nanoparticles and their possible application for early and timely diagnosis of this and other types of cancer. In the present study, we used label-free mass spectrometry in combination with an ion-mobility approach to identify 220 proteins with increased abundance in small cell lung cancer (SCLC) cell lines. Our attention was focused on cell receptors for their potential application as mCSNP’s targets; in this work, we report the overexpression of Transferrin Receptor (TfR1) protein, also known as Cluster of Differentiation 71 (CD71) up to a 30-fold increase with respect to the control cell. The kinetics of endocytosis, evaluated by a flow cytometry methodology based on fluorescence quantification, demonstrated that receptors were properly activated with the transferrin supported on the magnetic core-shell nanoparticles. Our results are important in obtaining essential information for monitoring the disease and/or choosing better treatments, and this finding will pave the way for future synthesis of nanoparticles including chemotherapeutic drugs for lung cancer treatments.

Exosomes derived from hepatitis B virus-infected hepatocytes promote liver fibrosis via miR-222/TFRC axis.

Exosomal miRNAs activates hepatic stellate cell (HSC) and promote fibrosis. miR-222 was found to be increased in hepatitis B virus (HBV)-infected hepatocytes, and ferroptosis was reported to ameliorate liver fibrosis (LF). Although miR-222 and ferroptosis have been implicated in LF, the association between miR-222 and ferroptosis and how they coordinate to regulate LF are still not explicit. This study investigates the roles of miR-222 and transferrin receptor (TFRC) in LF. Lipid reactive oxygen species (ROS) level was analyzed by flow cytometry. FerroOrange staining was used to measure intracellular iron level. Luciferase reporter assay was adopted to confirm the binding of miR-222 and TFRC. Real-time quantitative PCR and immunoblots were applied to analyze gene and protein expression. The results showed that supplementation of exosomes derived from HBV-infected LO2 cells remarkably enhanced LX-2 cell activation, evidenced by elevated hydroxyprolin (Hyp) secretion and α-SMA and COL1A2 expression. miR-222 was significantly increased in HBV-Exo. Overexpressing miR-222 upregulated cell viability, secretion of Hpy, and expression of α-SMA and COL1A2, which were all blocked by overexpression of TFRC. Further study showed that TFRC was a target of miR-222, and miR-222 promoted LX-2 cell activation through suppressing TFRC-induced ferroptosis in LX-2 cells. Exosomal miR-222 derived from HBV-infected hepatocytes promoted LF through inhibiting TFRC and TFRC-induced ferroptosis. This study emphasizes the significance of miR-222/TFRC axis in LF and suggests new insights in clinical decision making while treating LF. Exosomes derived from HBV-infected LO2 cells promote LX-2 cell activation and liver fibrosis in mouse Exosomal miR-222 derived from HBV-infected LO2 cells promotes LX-2 cell activation TFRC is a target of miR-222 and inhibits LX-2 cell activation induced by miR-222 miR-222 promotes LX-2 cell activation through inhibiting TFRC-induced ferroptosis.

Probing Transferrin Receptor Overexpression in Gastric Cancer Mice Models.

Exposure to carcinogenic chemicals, Helicobacter pylori infection, and high dietary salt are the risk factors associated with gastric cancer. Mice models of gastric cancer are key to understanding the cancer mechanism, to discerning the role played by different factors, and to determining therapeutic effects of different treatments. The goal has been to find targets which are only expressed with cancer so that they can be targeted specifically without harming normal cells. One such target could be the transferrin receptor, a glycoprotein receptor that is expressed many-folds on rapidly growing cells due to the greater demand of iron. In this study, gastric cancer was developed in mice (BALB/c) with human cancer-associated risk factors by feeding them with tumor-inducing concentration of methyl nitrosourea, dietary salt, and H. pylori along with normal feed and water. Three strategies were adopted to induce gastric cancer; (1) use of N-methyl-N-nitrosourea (MNU) with high dietary salt (NaCl), (2) infection with H. pylori (isolated from human gastric tissue), and (3) use of MNU along with high concentration of NaCl after H. pylori infection. Mice were dissected after induction, and histological study of gastric tissue was done with Hematoxylin and Eosin staining. A diagnostic probe comprising transferrin conjugated with cadmium sulfide quantum dots was prepared and characterized. It was used to study the transferrin receptor overexpression in gastric tissue of cancer-induced mice relative to the normal mice. Mice of group 3 showed the highest rate of the cancer incidence ratio (96%) along with a high expression of transferrin receptors among the three groups. Histochemical studies showed that different types of gastric cancer depend upon the cancer-induction conditions. The mouse model of group 3 has the potential to be used in the future to study the therapeutic effects of cancer medicines, and overexpression of transferrin receptors could be identified through the designed probe to be used as diagnostics.

Iron regulatory protein 1 promotes ferroptosis by sustaining cellular iron homeostasis in melanoma.

Melanoma, the most aggressive skin cancer, is mainly treated with BRAF inhibitors or immunotheareapy. However, most patients who initially responded to BRAF inhibitors or immunotheareapy become resistant following relapse. Ferroptosis is a form of regulated cell death characterized by its dependence on iron ions and the accumulation of lipid reactive oxygen species (ROS). Recent studies have demonstrated that ferroptosis is a good method for tumor treatment, and iron homeostasis is closely associated with ferroptosis. Iron regulatory protein (IRP)1 and 2 play important roles in maintaining iron homeostasis, but their functions in ferroptosis have not been investigated. The present study reported that the expression of IRP1 and IRP2 was increased by the ferroptosis inducers erastin and RSL3 in melanoma cells. Depletion of IRP1 significantly suppressed erastin- and RSL3-induced ferroptosis. IRP2 had a weak effect but could enhance the promoting function of IRP1 on ferroptosis. Further, erastin and RSL3 promoted the transition of aconitase 1 to IRP1, which regulated downstream iron metabolism proteins, including transferrin receptor (TFRC), ferroportin (FPN) and ferritin heavy chain 1 (FTH1). Moreover, overexpression of TFRC and knockdown of FPN and FTH1 significantly promoted erastin- and RSL3-induced ferroptosis in IRP1 knockdown melanoma cells. Collectively, the present findings indicate that IRP1 plays an essential role in erastin- and RSL3-induced ferroptosis by regulating iron homeostasis.

Transferrin Receptor Serves as a Potential Target for Cancer Therapy

Transferrin receptor (TfR) is a glycoprotein that transfers iron from the extracellular matrix to the intracellular environment. Due to the rapid proliferation need, the demand for iron in cancer cells is much greater than in normal cells, possibly explaining the upregulated TfR expression in cancer cells. The overexpression of TfR and its extracellular accessibility, ability to internalize, and central role in cancer cell make this receptor a potential target for antibody-mediated therapy. The TfR can be targeted indirectly by antibodies conjugated to anti-cancer agents or directly through the use of antibodies that disrupt the function of the receptor and induce Fc-mediated effector functions. This article reviews the developments of antibody-based cancer therapy targeting TfR.

Transferrin Coated d-penicillamine-Au-Cu Nanocluster PLGA Nanocomposite Reverses Hypoxia-Induced EMT and MDR of Triple-Negative Breast Cancers.

Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer, lacks effective targeted therapies due to negative expression of the targetable bioreceptors. Additionally, hypoxic condition in solid tumors contributes to the epithelial to mesenchymal transition (EMT), which aggravates cancer progression, multidrug resistance (MDR), migration, and stemness of the TNBC. A therapeutic module has been established in this regard by coating PLGA nanoparticle with d-penicillamine templated Au-Cu bimetallic nanoclusters. Further, the resultant nanomaterials were coated with recombinant transferrin protein to specifically target transferrin receptor overexpressing TNBC. The synthesized nanocomposites showed strong orange emission band at 630 nm with fluorescence quantum yield of 2%, rendering it suitable for theranostic applications. Experimental results demonstrated efficient cellular internalization and significant innate anti-cell proliferative potential of the nanocomposites. The fabricated nanocomposites were also able to induce cell death in spheroids, which was confirmed by live/dead dual staining results. Furthermore, when EMT-induced TNBC cells were treated with nanocomposites, they generated reactive oxygen species (ROS), depolarized the mitochondrial membrane potential, and induced apoptosis. Gene expression by real-time PCR indicated that treatment of EMT-induced TNBC cells with nanocomposites facilitated mesenchymal to epithelial transition (MET). In MDA-MB-468 cells, treatment with nanocomposites resulted in a 1.35-fold rise in E-cadherin an epithelial marker and a 1.36-fold decrease in vimentin a mesenchymal marker. Similarly, 2.87-fold and 1.76-fold decrease in stemness markers ALDH1A3 and EpCAM were observed in MDA-MB-231. Furthermore, 4.63-fold decrease in expression of ABCC1, a prominent contributor of MDR, was observed in MDA-MB-231. Protein expression studies revealed that nanocomposites reduced p-STAT-3 by 1.61-fold in MDA-MB-231 and by 7.8-fold in MDA-MB-468. Importantly, nanocomposites downregulated the expression of β-catenin by 3-fold in MDA-MB-231 and by 3.11-fold in MDA-MB-468. Downregulation of EMT with concomitant alteration of STAT-3 and β-catenin signaling pathways led to reduced migration ability of the TNBC cells.

Optimized Doxorubicin Chemotherapy for Diffuse Large B-cell Lymphoma Exploits Nanocarrier Delivery to Transferrin Receptors.

New treatments are needed to address persistent unmet clinical needs for diffuse large B-cell lymphoma (DLBCL). Overexpression of transferrin receptor 1 (TFR1) is common across cancer and permits cell-surface targeting of specific therapies in preclinical and clinical studies of various solid tumors. Here, we developed novel nanocarrier delivery of chemotherapy via TFR1-mediated endocytosis, assessing this target for the first time in DLBCL. Analysis of published datasets showed novel association of increased TFR1 expression with high-risk DLBCL cases. Carbon-nitride dots (CND) are emerging nanoparticles with excellent in vivo stability and distribution and are adaptable to covalent conjugation with multiple substrates. In vitro, linking doxorubicin (Dox) and transferrin (TF) to CND (CND-Dox-TF, CDT) was 10-100 times more potent than Dox against DLBCL cell lines. Gain- and loss-of-function studies and fluorescent confocal microscopy confirmed dependence of these effects on TFR1-mediated endocytosis. In contrast with previous therapeutics directly linking Dox and TF, cytotoxicity of CDT resulted from nuclear entry by Dox, promoting double-stranded DNA breaks and apoptosis. CDT proved safe to administer in vivo, and when incorporated into standard frontline chemoimmunotherapy in place of Dox, it improved overall survival by controlling patient-derived xenograft tumors with greatly reduced host toxicities. Nanocarrier-mediated Dox delivery to cell-surface TFR1, therefore, warrants optimization as a potential new therapeutic option in DLBCL. SIGNIFICANCE: Targeted nanoparticle delivery of doxorubicin chemotherapy via the TRF1 receptor presents a new opportunity against high-risk DLBCL tumors using potency and precision.

Revealing the Dynamic Mechanism by Which Transferrin Promotes the Cellular Uptake of HAIYPRH Peptide-Conjugated Nanostructures by Force Tracing.

The HAIYPRH (T7) peptide has been widely used as a ligand for constructing tumor-targeted nanodrug delivery systems since it can target the transferrin receptor (TfR) and then enter cells easily with the help of transferrin (Tf). However, the dynamic mechanism by which transferrin promotes the entry of T7-conjugated nanostructures into cells remains unclear. Herein, a force tracing technique based on atomic force microscopy (AFM) was used to track the ultrafast dynamic process of a T7-conjugated gold nanoparticle (AuNP-T7) entering a cell at the single-particle level in real time. Tf helped decrease the endocytosis force and increase the endocytosis speed of AuNP-T7 in A549 cells. However, Tf only increased the endocytosis speed of AuNP-T7 in HeLa cells. In contrast, in Vero cells without TfR overexpression, Tf decreased the endocytosis speed. This report provides important insights for redesigning and developing T7-conjugated nanodrug carriers in targeted nanodrug delivery systems.

Transferrin Receptor and Cellular Iron are Potential Molecular Targets for Diagnosis and Treatment of Lung Cancer

Lung cancer is one of the leading causes of cancer death. Most current treatments have debilitating side effects with poor selectivity and pharmacodynamic properties. To develop more effective and safer anticancer drugs, we synthesized trioxane (DMR) and dioxazinane (HSM), both novel Artemisinin (ART) analogs. These analogs induced apoptosis in cancer but not normal lung cells and was reactive oxygen species (ROS) dependent. We also showed that cancer cells have higher transferrin receptor (TfR) expression compared to normal cells. We hypothesize high levels of TfR expression and [iron]i are responsible for the cancer specific effects of analogs. To study this, we confirmed iron’s role in ART analog‐induced apoptosis. We also knocked out TfR in cancer cells and overexpressed TfR in normal cells.Confluent normal (BEAS2B) and cancer (A549) human lung cells were treated (18 H) with 10μM of DMR, HSM ±Deferoxamine (DFO, iron chelator; 10 μM). [Iron]i was assessed in cell lysates using a colorimetric assay (Biovision, CA). Cell death was assessed by i) staining with FITC‐Annexin V (AV, apoptosis), propidium iodide (PI, cell death), followed by imaging, and quantification using flow cytometry and/or microscopy (Image J) and ii) measuring Lactate dehydrogenase (LDH) release. Cells were transfected with CRISPR/Cas9 overexpression (OE) or knockout (KO) plasmid (Santa Cruz, TX) containing Green Fluorescent Protein (GFP). Co‐transfection with HDR plasmid allowed for puromycin selection. Transfection efficiency was assessed using RT‐PCR, Western blot and GFP expression.A549 cells had 7‐fold more [iron]i than BEAS2B cells. This was essential for the apoptotic effects, as chelating iron with DFO prevented ART analog‐induced cell death (% AV+ A549 cells, 18 H: HSM: 54±4; HSM+DFO: 1±0.5; DMR: 32±3; DMR+DFO: 0.3±0.1, n≥3). KO of TfR in A549 cells yielded a low transfection efficiency (10%, Image J). Western blot of cell lysates did not show a significant reduction in TfR protein expression. Co‐transfection with HDR plasmid followed by puromycin selection increased transfection efficiency. However, there was increased cell death in TfR KO cells in culture (72 H, PI+ cells; mean pixel intensity (mpi), control: 15±0.2; GFP: 19±2, TfR KO: 49±42; n=3). Overexpression of TfR in BEAS2B cells yielded a higher transfection efficiency (~25%; Image J and RT‐PCR). Similar to TfR KO in A549 cells, after 72 H, there was a marked increase in cell death in TfR OE BEAS2B (PI+ cells (mpi), control: 18±2; GFP: 13±4, TfR OE: 83±20; n=3). TfR overexpression in normal lung cells possibly resulted in iron overload and ferroptosis.Our findings demonstrate that abnormal TfR expression and the associated changes in iron uptake induces death in normal and cancer cells, highlighting its importance in cell survival and proliferation. Understanding the role of TfR and iron in carcinogenesis will help develop potent therapeutic drugs to treat cancer, a disease that accounts for ~ 9 million deaths annually. Further, synthesizing novel analogs such as DMR‐tagged transferrin will provide specific and efficient drug delivery to cancer cells.Support or Funding InformationAPS‐STRIDE (Grant #1 R25 HL 115473‐01); NSSRP BenU Funds

Transferrin-binding peptide functionalized polymersomes mediate targeted doxorubicin delivery to colorectal cancer in vivo

Transferrin receptor (TfR) is a promising target validated in the clinical trials for managing various malignancies. Transferrin (Tf) and single chain antibody fragment can target TfR and are typically conjugated to nanomedicines via post-modification, which poses significant production challenges. Here, we report that the polymersomes functionalized with a Tf-binding peptide CGGGHKYLRW (TBP-Ps) can selectively and stably bind Tf and subsequently mediate targeted doxorubicin (Dox) delivery to TfR over-expressing HCT-116 colorectal cancer cells in vitro and in vivo. The Tf surface density of the polymersomes could be controlled by the surface content of TBP. Interestingly, modifying Dox-loaded TBP-Ps with Tf led to greatly increased cellular uptake and inhibitory effect of HCT-116 cells. Tf-bound TBP-Ps demonstrated rapid accumulation in the tumor xenografts in nude mice following i.v. injection. More importantly, Dox-loaded Ps with Tf binding significantly enhanced the antitumor efficacy in mice bearing HCT-116 tumors compared to polymersomes without Tf binding. Surface functionalization of the nanoparticles with Tf-binding peptide provides an appealing strategy in formulating Tf-targeted nanomedicines.

Over-Expression of Transferrin Receptor (TFRC/CD71) and Low Expression of Innate and Adaptive Immune Cell Subsets in HIV-Associated, GCB-DLBCL By Digital Gene Expression Profiling

Introduction: HIV infected individuals are 17x more likely to receive a diagnosis of Diffuse Large B cell Lymphoma (DLBCL) compared to their uninfected counterparts. Moreover, DLBCL is more aggressive in HIV infected individuals. However, the molecular pathology driving the aggressive nature of HIV related DLBCL is poorly understood. Previously, we demonstrated that HIV-associated [HIV(+)] germinal center B-cell (GCB) DLBCL tumors are more proliferative, with enhanced genomic stability and increased expression of DNA repair genes compared to their HIV-not associated [HIV(-)] counterparts (Maguire et al, Int J Cancer,2019). Given the immunocompromised nature of these patients, herein we assessed whether specific immunological signaling pathways are also altered in HIV(+) GCB DLBCL tumors, that may work in conjunction with, or independently of, increased DNA repair, to drive the enhanced aggressive nature of HIV related DLBCL. Methods: We used our previously studied cohort of patient samples, which included a total of 39 molecularly defined GCB DLBCL cases. Of these, 18 were HIV(+) cases from the AIDS Cancer Specimen Resource Network (https://acsr.ucsf.edu/) and 21 were HIV(-) institutional cases. Samples underwent hematopathologist review for diagnosis confirmation and tumor content assessment. Samples with <60% tumor content were macrodissected, with a total of 4x 5µm FFPE sections per sample used for RNA/DNA extraction. Digital gene expression profiling was used to assess differential expression of 579 immune related genes using the NanoString PanCancer Immune Profiling panel. The data was normalized using nSolver, and differential expression analysis was performed using the R statistical software package NanoStringDiff. The resulting expression data were then analyzed using Gene Set Enrichment Analysis (GSEA), and GSEA Molecular Signatures Database (MSigDB) overlap analyses. Results: A total of 110 genes were differentially expressed between the HIV(+) and HIV(-) cohorts at p<0.05. As expected due to immunosuppression in HIV(+) individuals, 100 genes (91%) were significantly reduced and 10 genes (9%) were significantly increased in the HIV(+) cohort compared to the HIV(-) cohort. GSEA and MSigDB analyses revealed that the genes with reduced expression in the HIV(+) cohort were associated with losses in both adaptive and innate immune signaling including cytokine interactions, the IL1 pathway and B-cell receptor signaling amongst others. MSigDB overlap analyses of the 10 genes upregulated in the HIV(+) cohort identified 3 Gene Ontology molecular functions, all of which were receptor signaling related; including Receptor Binding (CD8A, CD160, CCL8, C1QBP, MIF), Receptor Activity (CD8A, CD160, CCRL1, TFRC, KLRC2), and Cargo Receptor Activity (CCRL1, TFRC). Gene overlaps between the subsets include genes associated with NK and cytolytic T cells (CD8A, CD160), as well as the transferrin receptor TRFC/CD71, which with a fold increase of 3.9, was the most significantly increased gene in the HIV(+) cohort (p=0.002, FDR=0.0025). Conclusions: As expected, the results demonstrate a loss in both adaptive and innate immune signaling in the HIV(+) cohort compared to the HIV negative cohort as well as alterations in receptor signaling. In line with our previous observation that HIV(+) GCB DLBCL tumors have higher expression of proliferation genes and the Ki67 antigen than their HIV(-) counterparts, the results of this study also reveal overexpression of TRFC/CD71 mRNA in HIV(+) GCB DLBCL tumors compared to the HIV(-) cases. TFRC/CD71 is well known as a mediator of iron uptake in erythroid cells. Iron uptake through TFRC/CD71 is also necessary for, and positively regulates T and B cell proliferation; and is commonly expressed on aggressive B cell lymphomas. We suggest that TFRC/CD71 may play a role in the enhanced proliferative capabilities of HIV(+) GCB DLBCL. These effects may be mediated through the HIV protein Nef, which has known effects on TFRC/CD71 protein recycling in lymphoid populations (Madrid et al, J Biol Chem 2005), and has been shown to be transferred from HIV infected macrophages to uninfected B-cells via contact-dependent cellular conduits (Xu et al, Nat Immunol 2009). Nef transfer between cells has also been described to occur via microvesicle transfer and trogocytosis (reviewed in Ellwanger et al, Infect Genet Evol 2017). Further analysis of these pathways is ongoing. Disclosures Rimsza: NanoSting: Patents & Royalties: Named inventor on a patent licensed to NanoSting [Institution].

Inducing cell death in vitro in cancer cells by targeted delivery of cytochrome c via a transferrin conjugate.

One of the major drawbacks of many of the currently used cancer drugs are off-target effects. Targeted delivery is one method to minimize such unwanted and detrimental events. To actively target lung cancer cells, we have developed a conjugate of the apoptosis inducing protein cytochrome c with transferrin because the transferrin receptor is overexpressed by many rapidly dividing cancer cells. Cytochrome c and transferrin were cross-linked with a redox sensitive disulfide bond for the intra-cellular release of the protein upon endocytosis by the transferrin receptor. Confocal results demonstrated the cellular uptake of the cytochrome c-transferrin conjugate by transferrin receptor overexpressing A549 lung cancer cells. Localization studies further validated that this conjugate escaped the endosome. Additionally, an in vitro assay showed that the conjugate could induce apoptosis by activating caspase-3. The neo-conjugate not only maintained an IC50 value similar to the well known drug cisplatin (50 μM) in A549 cancer cells but also was nontoxic to the normal lung (MRC5) cells. Our neo-conjugate holds promise for future development to target cancers with enhanced transferrin receptor expression.

Transferrin-Conjugated Nanocarriers as Active-Targeted Drug Delivery Platforms for Cancer Therapy.

A lot of effort has been devoted to achieving active targeting for cancer therapy in order to reach the right cells. Hence, increasingly it is being realized that active-targeted nanocarriers notably reduce off-target effects, mainly because of targeted localization in tumors and active cellular uptake. In this context, by taking advantage of the overexpression of transferrin receptors on the surface of tumor cells, transferrin-conjugated nanodevices have been designed, in hope that the biomarker grafting would help to maximize the therapeutic benefit and to minimize the side effects. Notably, active targeting nanoparticles have shown improved therapeutic performances in different tumor models as compared to their passive targeting counterparts. In this review, current development of nano-based devices conjugated with transferrin for active tumor-targeting drug delivery are highlighted and discussed. The main objective of this review is to provide a summary of the vast types of nanomaterials that have been used to deliver different chemotherapeutics into tumor cells, and to ultimately evaluate the progression on the strategies for cancer therapy in view of the future research.

Mathematical modeling of mutant transferrin-CRM107 molecular conjugates for cancer therapy

The transferrin (Tf) trafficking pathway is a promising mechanism for use in targeted cancer therapy due to the overexpression of transferrin receptors (TfRs) on cancerous cells. We have previously developed a mathematical model of the Tf/TfR trafficking pathway to improve the efficiency of Tf as a drug carrier. By using diphtheria toxin (DT) as a model toxin, we found that mutating the Tf protein to change its iron release rate improves cellular association and efficacy of the drug. Though this is an improvement upon using wild-type Tf as the targeting ligand, conjugated toxins like DT are unfortunately still highly cytotoxic at off-target sites. In this work, we address this hurdle in cancer research by developing a mathematical model to predict the efficacy and selectivity of Tf conjugates that use an alternative toxin. For this purpose, we have chosen to study a mutant of DT, cross-reacting material 107 (CRM107). First, we developed a mathematical model of the Tf-DT trafficking pathway by extending our Tf/TfR model to include intracellular trafficking via DT and DT receptors. Using this mathematical model, we subsequently investigated the efficacy of several conjugates in cancer cells: DT and CRM107 conjugated to wild-type Tf, as well as to our engineered mutant Tf proteins (K206E/R632A Tf and K206E/R534A Tf). We also investigated the selectivity of mutant Tf-CRM107 against non-neoplastic cells. Through the use of our mathematical model, we predicted that (i) mutant Tf-CRM107 exhibits a greater cytotoxicity than wild-type Tf-CRM107 against cancerous cells, (ii) this improvement was more drastic with CRM107 conjugates than with DT conjugates, and (iii) mutant Tf-CRM107 conjugates were selective against non-neoplastic cells. These predictions were validated with in vitro cytotoxicity experiments, demonstrating that mutant Tf-CRM107 conjugates is indeed a more suitable therapeutic agent. Validation from in vitro experiments also confirmed that such whole-cell kinetic models can be useful in cancer therapeutic design.

Targeted Delivery of siRNA to Transferrin Receptor Overexpressing Tumor Cells via Peptide Modified Polyethylenimine.

The use of small interference RNA (siRNA) to target oncogenes is a promising treatment approach for cancer. However, siRNA cancer therapies are hindered by poor delivery of siRNA to cancer cells. Transferrin receptor (TfR) is overexpressed in many types of tumor cells and therefore is a potential target for the selective delivery of siRNA to cancer cells. Here, we used the TfR binding peptide HAIYPRH (HAI peptide) conjugated to cationic polymer branched polyethylenimine (bPEI), optimized the coupling strategy, and the TfR selective delivery of siRNA was evaluated in cells with high (H1299) and low TfR expression (A549 and H460). The HAI-bPEI conjugate exhibited chemico-physical properties in terms of size, zeta-potential, and siRNA condensation efficiency similar to unmodified bPEI. Confocal microscopy and flow cytometry results revealed that HAI-bPEI selectively delivered siRNA to H1299 cells compared with A549 or H460 cells. Moreover, HAI-bPEI achieved more efficient glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene knockdown in H1299 cells compared with bPEI alone. However, despite optimization of the targeting peptide and coupling strategy, HAI-bPEI can only silence reporter gene enhanced green fluorescent protein (eGFP) at the protein level when chloroquine is present, indicating that further optimization of the conjugate is required. In conclusion, the HAI peptide may be useful to target TfR overexpressing tumors in targeted gene and siRNA delivery approaches.

Enhanced Efficacy of Artemisinin Loaded in Transferrin-Conjugated Liposomes versus Stealth Liposomes against HCT-8 Colon Cancer Cells.

Artemisinin (ART) is a unique sesquiterpene lactone isolated from Artemisia annua that is well known for antimalarial properties and was recently reported as a promising anticancer drug. The aim of our work was to develop a novel nanocarrier for enhanced ART delivery and activation in cancer tissues, because transferrin receptors are largely expressed in cancer cells and the iron content is higher than in normal cells. ART was loaded in transferrin-conjugated liposomes (ART-L-Tf), and the performance was compared with ART loaded in stealth liposomes (ART-L). All of the liposomes were fully characterized in terms of size, drug-entrapment efficiency, transferrin coupling moieties, and stability. Both cell uptake and cytotoxicity studies of the developed nanocarriers were tested in the HCT-8 cell line, selected among several cell lines because of transferrin receptor overexpression. The results confirmed the enhanced delivery of ART-L-Tf in comparison with ART-L in the targeting of the HCT-8 cell line and an improved cytotoxicity as a result of the presence of iron ions, which resulted in concomitant synergism derived from the increased expression of transferrin receptors on the surface of the tumor cells.

The Oral Iron Chelator, Deferasirox, Reverses the Age-Dependent Alterations in Iron and Amyloid-β Homeostasis in Rat Brain: Implications in the Therapy of Alzheimer's Disease.

The altered metabolism of iron impacts the brain function in multiple deleterious ways during normal aging as well as in Alzheimer's disease. We have shown in this study that chelatable iron accumulates in the aged rat brain along with overexpression of transferrin receptor 1 (TfR1) and ferritin, accompanied by significant alterations in amyloid-β (Aβ) peptide homeostasis in the aging brain, such as an increased production of the amyloid-β protein precursor, a decreased level of neprilysin, and increased accumulation of Aβ42. When aged rats are given daily the iron chelator, deferasirox, over a period of more than 4 months starting from the 18th month, the age-related accumulation of iron and overexpression of TfR1 and ferritin in the brain are significantly prevented. More interestingly, the chelator treatment also considerably reverses the altered Aβ peptide metabolism in the aging brain implying a significant role of iron in the latter phenomenon. Further, other results indicate that iron accumulation results in oxidative stress and the activation of NF-κB in the aged rat brain, which are also reversed by the deferasirox treatment. The analysis of the results together suggests that iron accumulation and oxidative stress interact at multiple levels that include transcriptional and post-transcriptional mechanisms to bring about changes in the expression levels of TfR1 and ferritin and also alterations in Aβ peptide metabolism in the aging rat brain. The efficacy of deferasirox in preventing age-related changes in iron and Aβ peptide metabolism in the aging brain, as shown here, has obvious therapeutic implications for Alzheimer's disease.

MicroRNA-152-mediated dysregulation of hepatic transferrin receptor 1 in liver carcinogenesis.

Over-expression of transferrin receptor 1 (TFRC) is observed in hepatocellular carcinoma (HCC); however, there is a lack of conclusive information regarding the mechanisms of this dysregulation. In the present study, we demonstrated a significant increase in the levels of TFRC mRNA and protein in preneoplastic livers from relevant experimental models of human hepatocarcinogenesis and in human HCC cells. Additionally, using the TCGA database, we demonstrated an over-expression of TFRC in human HCC tissue samples and a markedly decreased level of microRNA-152 (miR-152) when compared to non-tumor liver tissue. The results indicated that the increase in levels of TFRC in human HCC cells and human HCC tissue samples may be attributed, in part, to a post-transcriptional mechanism mediated by a down-regulation of miR-152. This was evidenced by a strong inverse correlation between the level of TFRC and the expression of miR-152 in human HCC cells (r = -0.99, p = 4. 7 × 10-9), and was confirmed by in vitro experiments showing that transfection of human HCC cell lines with miR-152 effectively suppressed TFRC expression. This suggests that miR-152-specific targeting of TFRC may provide a selective anticancer therapeutic approach for the treatment of HCC.

An innovative pre-targeting strategy for tumor cell specific imaging and therapy.

A programmed pre-targeting system for tumor cell imaging and targeting therapy was established based on the "biotin-avidin" interaction. In this programmed functional system, transferrin-biotin can be actively captured by tumor cells with the overexpression of transferrin receptors, thus achieving the pre-targeting modality. Depending upon avidin-biotin recognition, the attachment of multivalent FITC-avidin to biotinylated tumor cells not only offered the rapid fluorescence labelling, but also endowed the pre-targeted cells with targeting sites for the specifically designed biotinylated peptide nano-drug. Owing to the successful pre-targeting, tumorous HepG2 and HeLa cells were effectively distinguished from the normal 3T3 cells via fluorescence imaging. In addition, the self-assembled peptide nano-drug resulted in enhanced cell apoptosis in the observed HepG2 cells. The tumor cell specific pre-targeting strategy is applicable for a variety of different imaging and therapeutic agents for tumor treatments.