Transferrin Receptor Expression Research Articles

Immunophenotypic features of molecular subtypes of breast cancer

Introduction. Currently, immunotropic drugs are used in the modern strategy of cancer treatment. Importance is given to immunological markers of the tumor, which may be associated with the prognosis of the disease, the effectiveness of treatment. Therefore, the study of tumor immunophenotype is one of the leading scientific directions. Of particular interest is the study of the immunophenotypic characteristics of breast cancer depending on its biological subtype.Purpose. To evaluate the frequency of expression of HLA-I, HLA-II, CD71, MUC1, Pgp170 molecules by breast cancer cells and determine their relationship with the molecular biological subtype of the tumor.Materials and methods. This study included 120 patients with breast cancer who received treatment at the National Medical Research Centre of Oncology n. a. N. N. Blokhin (Moscow, Russia). Tumor stages II and III prevailed: 56.7 % and 33.4 %, respectively. A moderate degree of differentiation (G2) was more often noted. The luminal subtype was 58.3 % (n = 70), non-luminal – in 41.7 % (n = 50). Immunophenotyping of the primary tumor was performed by immunofluorescence on cryostat sections. The reaction was evaluated using a Zeiss luminescent microscope (Axioskop, Germany). The frequency of expression of HLA-I and class II molecules was studied depending on the clinical and morphological characteristics of breast cancer. The frequency of expression of HLA-I, HLA-II, CD71, MUC1, Pgp170 molecules depending on the molecular subtype of breast cancer was studied.Results. The absence of molecules of the major histocompatibility complex of class I and II on breast cancer cells was found in 89.6 % of the samples. The monomorphic expression was observed in 23.4 % of cases. In the luminal subtype, HLA-II class molecules were expressed more often: in total, mosaic and monomorphic types of reactions were observed in 30.5 % (20/65) of cases. With non-luminal – 20.0 % (10/47) of cases. The frequency of expression of the transferrin receptor is significantly higher in the luminal subtype than in the non-luminal subtype: 85.9 % (n = 5) and 65.2 % (n = 30), p = 0.011. Luminal breast cancer cells express transferrin receptors predominantly monomorphically: 75.4 % (n = 49) vs 43.5 % (n = 20) in the non-luminal subtype, p = 0.003. The MUC 1 expressing monomorphically tumors is higher in luminal cancer: 83.3 % (n = 35) versus 65 % (n = 26) in the non-luminal subtype. Monomorphic expression of Pgp170 is more often observed in luminal breast cancer.Conclusion. Luminal breast cancer is characterized by unfavorable prognostic immunophenotypic features. In the luminal subtype, expression of CD71 is more often observed, predominantly monomorphic. In the non-luminal subtype, expression of Pgp170 is observed less frequently. No statistically significant differences between the molecular subtypes in terms of the level of expression of HLA-I and class II molecules were found.

Differential Effects of Monoferric Forms of Transferrin on Mouse Models of Anemia of Inflammation and Congenital Anemia

Transferrin (TF) is a bilobed 80kD glycoprotein with N- and C-lobe iron binding sites. TF circulates as four forms: unbound to iron (apo-TF), iron bound to the N-lobe, the C-lobe or to both lobes (diferric-TF). Most circulating TF under physiological conditions is monoferric (Dirusso et al., 1985). The TF forms interact with TF receptor-1 (TFR1), which is ubiquitously expressed and serves as the main mechanism for cellular iron delivery. TF also interacts with TF receptor-2 (TFR2) which is expressed on hepatocytes, erythroblasts, and other cells of the bone marrow and is thought to primarily influence cellular signaling events regulating hepcidin expression and erythropoiesis. In erythrocytes, loss of TFR2 results in abnormally high red blood cell production under conditions of iron-deficiency (Artuso et al., 2018), suggesting it functions as an iron sensor. Additional evidence suggests TFR2 modulates erythropoiesis via the erythropoietin (EPO) receptor (EPOR) (Forejtnikovà et al., 2010). To understand the role of monoferric TF forms in vivo, we previously generated N-lobe blocked (TfN-bl) or C-lobe blocked (TfC-bl) TF mutant mice. Characterization of these mice demonstrated dramatic differences in RBC levels, hepcidin expression relative to iron status, and EPO sensitivity (Parrow et al., 2019). More recently, we have become interested in characterizing the immune compartment of these mice. Recent studies point to iron being an important regulator of immune responses, which has important implications worldwide in understanding how to treat iron-deficient patients without exacerbating latent infections. Cells belonging to the immune branch have also been shown to express TFR2 and EPOR (Cantarelli et al., 2019; Roetto et al., 2011; Kawabata et al., 2001), however their function is largely unexplored. Therefore, in this work, we investigate the role of the two monoferric forms of TF on ineffective erythropoiesis (IE) using a mouse model of β-thalassemia (BT) intermedia (Hbbth3/+) and chronic anemia of inflammation using heat-killed Brucella Abortus (HKBA). Based on observations in BT mice treated with exogenous TF (Li et al., 2010), which led to reduced erythroid iron intake, reduced EPO requirement (as in TfC-bl mutant mice) and improved anemia, we hypothesized that Hbbth3/+ mice crossed with TfC-bl would demonstrate improved erythropoietic and iron parameters compared with Hbbth3/+TfN-bl. We measured effects on hematologic parameters, IE, and expression of relevant iron regulatory serum proteins. Hbbth3/+TfC-bl mice demonstrated significantly increased RBC counts, elevated hemoglobin, improved erythrocyte morphology, decreased splenomegaly, fewer bone marrow erythroblasts, and improvement of IE. Additionally, serum erythroferrone (ERFE) was significantly reduced and hepcidin levels were increased in Hbbth3/+TfC-bl relative to Hbbth3/+Tf+/+ controls. However, hematological parameters from Hbbth3/+TfN-bl mice did not show these improvements. In our studies examining the immune compartments of TfC-bl and TfN-bl mice we found striking differences in their expression of TFR1 and response to immunological challenge by HKBA. Our preliminary data shows that expression levels of TFR1 were elevated in both TF mutant mice relative to control at baseline on lymphocyte, monocyte and granulocyte populations of the bone marrow. When challenged with a single intraperitoneal injection of HKBA, TfC-bl-HKBA mice suffered a more severe anemic phenotype with a 30% probability of survival following HKBA treatment. On the contrary, TfN-bl-HKBA mice displayed a blunted response to the immunological challenge, with faster recovery and no death. Taken together, our work provides additional evidence that TF is not only delivering iron cargo to cells, but also functioning as a signaling molecule via the lobe that is bound to iron. Despite each mouse only possessing a TF capable of binding one atom of iron, the two mouse models show profound differences in the pathophysiology of BT and AI. We hypothesize that the monoferric TF forms exert their influence via TFR2. We are currently characterizing a new loxp flanked TFR2-FLAG mouse model generated by our lab, which has been crossed to the TF monoferric mutant mice to test this hypothesis. Additionally, we are also exploring the translation potential of the two TF forms to treat anemia in BT and AI.

Ferroportin Q248H Mutation Leads to a Moderate Iron Load in Mutant Slc40a1Q248H/Q248H Mice

BACKGROUND: In Africans, ferroportin (Scl40a1 or FPN) Q248H mutation is associated with increased iron load. FPN Q248H has high allele frequency (2.2-13.4%) and is linked to reduction of inflammation and reduced anemia in Africa. We previously showed that FPN Q248H resists degradation by physiologic hepcidin concentrations and thus may facilitate higher iron uptake. OBJECTIVES: to analyze the effect of FPN Q248H mutation in vivo in a knock-in Slc40a1Q248H mouse model on normal and high iron diet. METHODS: The study was approved by Howard University IACUC committee. The Slc40a1Q248H/WT mice were generated by Flp- FRT recombination on the C57BL/6 background and homozygous Slc40a1Q248H/Q248H mutant mice were produced in Howard University animal facility by breeding. Twenty-three weeks old homozygous and wild type C57BL/6 (WT) females were fed normal or 2% carbonyl iron diet. Total iron concentration and ferritin were measured in serum by ELISA. Expression of FPN, transferrin receptor (TFR), ferritin heavy chain (FHC), divalent metal transporter 1 (DMT1), duodenal cytochrome B (Dcytb), hepcidin, and IL-1β, IL-6, and IL-18 cytokines were measured by real-time RT-PCR. Iron deposition was determined by Perl's staining. Hepatic oxidative stress was measured by malondialdehyde ELISA. RESULTS: On normal iron diet, Slc40a1Q248H/Q248H mice grew normally and showed a tendency to increased serum ferritin, decreased serum iron and reduced hematocrit compared to WT mice. Levels of FPN,Dcytb and DMT1 mRNA were increased in duodenum of mutant mice. In the spleen of Slc40a1Q248H/Q248H mice, FPN,FTH1, TFR and DMT1 mRNAs were also elevated. In contrast, in the liver of Slc40a1Q248H/Q248H mice, levels of FPN and TFR mRNA were decreased, but hepcidin levels were increased compared to WT mice. There was a trend towards hepatic FTH1 level increase in the mutant mice. Hepatic expression of pro-inflammatory IL-1β, IL-6, and IL-18 cytokines mRNA was similar between mutant and WT mice. No iron accumulation was found in the liver, and iron accumulation in the spleen was comparable between WT and Slc40a1Q248H/Q248H mice. On high iron diet, Slc40a1Q248H/Q248H mice showed significant increase in serum ferritin compared to WT mice on normal diet. Levels of FPN mRNA remained increased whereas Dcytb and DMT1 mRNA levels were decreased in duodenum of the mutant mice. In the liver, FPN mRNA levels were further decreased in the mutant mice, and FTH1 levels were equally increased in WT and Slc40a1Q248H/Q248H mice. In the spleen, FPN mRNA levels were equally increased for WT and Slc40a1Q248H/Q248H mice. Spleen FTH1 mRNA levels were further elevated in mutant mice compared to WT mice. Higher iron accumulation was found in the liver and spleen of Slc40a1Q248H/Q248H mice compared to WT mice. Elevated levels of hepatic oxidative stress were observed in the liver of Slc40a1Q248H/Q248H mice. Expression of pro-inflammatory IL-1β, IL-6, and IL-18 cytokines mRNA remained low in the liver of Slc40a1Q248H/Q248H mice. CONCLUSION: Upregulation of duodenal FPN, Dcytb and DMT1 and splenic FPN,FTH1, TFR and DMT1 expression indicated increased iron absorption and recycling in female Slc40a1Q248H/Q248H mice. The mutantmice showed tendency to mild iron load in tissues as indicated by increased serum ferritin and higher ferritin levels in the spleen and liver. High iron diet led to significant iron accumulation in the liver and increased hepatic stress compared to WT mice. Surprisingly, hepatic levels of pro-inflammatory cytokines were reduced in the mutant mice. Taken together, our study of the first in vivo model of FPNQ248H mutation indicates that it alters iron metabolism and might reduce liver inflammation. ACKNOWLEDGEMENTS: This work was supported by NIH grants 1R01HL125005, 5U54MD007597, 1P30AI117970-06 and 1SC1HL150685.

Angiotensin II type-1 receptor-associated protein interacts with transferrin receptor-1 and promotes its internalization

Kidney fibrosis is a common pathway that leads to chronic kidney disease. Angiotensin II type-1 receptor (AT1R)-associated protein (ATRAP) was originally identified as an AT1R-binding protein. Previously, we reported that systemic knockout of ATRAP exacerbates kidney fibrosis in aged mice. Although these effects of ATRAP appeared to be AT1R-independent actions, the molecular mechanism remains poorly understood. To elucidate the molecular mechanism of ATRAP independent of AT1R, we explored novel ATRAP-interacting proteins. Mass spectrometric analysis of the immunoprecipitants of a Flag-tagged ATRAP complex revealed 376 candidate proteins that potentially interact with ATRAP. Gene ontology analysis revealed that proteins related to vesicle trafficking, membrane transport, and many membrane proteins, including transferrin receptor 1 (TfR1), were enriched. Because TfR1 promotes cellular iron uptake and iron is a key factor involved in kidney fibrosis, we focused on TfR1 and confirmed that it interacts with ATRAP. In addition, our findings revealed that enhanced ATRAP expression decreased cell-surface TfR1 expression without altering the overall cellular TfR1 expression levels. Furthermore, enhanced ATRAP expression attenuated cellular iron levels. Together, our results highlight the role of ATRAP as a suppressor of TfR1 that functions by facilitating TfR1 internalization, which affects iron metabolism and oxidative stress signaling.

Host and microbiota derived extracellular vesicles: Crucial players in iron homeostasis.

Iron is a double-edged sword. It is vital for all that’s living, yet its deficiency or overload can be fatal. In humans, iron homeostasis is tightly regulated at both cellular and systemic levels. Extracellular vesicles (EVs), now known as major players in cellular communication, potentially play an important role in regulating iron metabolism. The gut microbiota was also recently reported to impact the iron metabolism process and indirectly participate in regulating iron homeostasis, yet there is no proof of whether or not microbiota-derived EVs interfere in this relationship. In this review, we discuss the implication of EVs on iron metabolism and homeostasis. We elaborate on the blooming role of gut microbiota in iron homeostasis while focusing on the possible EVs contribution. We conclude that EVs are extensively involved in the complex iron metabolism process; they carry ferritin and express transferrin receptors. Bone marrow-derived EVs even induce hepcidin expression in β-thalassemia. The gut microbiota, in turn, affects iron homeostasis on the level of iron absorption and possibly macrophage iron recycling, with still no proof of the interference of EVs. This review is the first step toward understanding the multiplex iron metabolism process. Targeting extracellular vesicles and gut microbiota-derived extracellular vesicles will be a huge challenge to treat many diseases related to iron metabolism alteration.

Immunophenotypic features of molecular subtypes of breast cancer

Currently,immunotropic drugs are used in the modern strategy of cancer treatment. Importance is given to immunological markers of the tumor, which may be associated with the prognosis of the disease, the effectiveness of treatment. Therefore, the study of tumor immunophenotype is one of the leading scientific directions. Of particular interest is the study of the immunophenotypic characteristics of breast cancer depending on its biological subtype. to evaluate the frequency of expression of HLA-I, HLA-II, CD71, MUC1,0 Pgp170 molecules by breast cancer cells and determine their relationship with the molecular biological subtype of the tumor. This study included 120 patients with breast cancer who received treatment at the Federal State Budgetary Institution “N.N. Blokhin" Ministry of Health of the Russian Federation. Tumor stages II and III prevailed: 56.7% and 33.4%, respectively. A moderate degree of differentiation (G2) was more often noted. The luminal subtype was 58.3% (n=70), non-luminal - in 41.7% (n=50). Immunophenotyping of the primary tumor was performed by immunofluorescence on cryostat sections. The reaction was evaluated using a ZEISS luminescent microscope (AXIOSKOP; Germany). The frequency of expression of HLA-I and class II molecules was studied depending on the clinical and morphological characteristics of breast cancer. The frequency of expression of HLA-I, HLA-II, CD71, MUC1.0 Pgp170 molecules depending on the molecular subtype of breast cancer was studied. Theabsence of molecules of the major histocompatibility complex of class I and II on breast cancer cells was found in 89.6% of the samples. In 23.4% of cases, their monomorphic expression was observed. In the luminal subtype, HLA-II class molecules were expressed somewhat more often: in total, mosaic and monomorphic types of reactions were observed in 30.5% (20/65) of cases. With non-luminal - 20.0% (10/47) of cases. The frequency of expression of the transferrin receptor is significantly higher in the luminal subtype than in the nonluminal subtype: 85.9% (n=5) and 65.2% (n=30), p=0.011. Luminal breast cancer cells express transferrin receptors predominantly monomorphically: 75.4% (n=49) versus 43.5% (n=20) in the non-luminal subtype, p=0.003. The percentage of monomorphically expressing MUC1 tumors is higher in luminal cancer: 83.3% (n=35) versus 65% (n=26) in the non-luminal subtype. Expression of Pgp70, namely monomorphic, is more often observed in luminal breast cancer. . Luminal breast cancer is characterized by unfavorable prognostic immunophenotypic features. In the luminal subtype, expression of CD71 is more often observed, predominantly monomorphic. In the non-luminal subtype, expression of Pgp 170 is observed less frequently. No statistically significant differences between the molecular subtypes in terms of the level of expression of HLA-I and class II molecules were found.

Role of Fe, Transferrin and Transferrin Receptor in Anti-Tumor Effect of Vitamin C.

Simple SummaryHigh-dose vitamin C (VC) inhibits cell proliferation in a variety of tumors, which is mediated by ROS. Iron is an important factor in the Fenton reaction, in which H2O2 reacts with ferrous iron to produce hydroxyl radicals, which may enhance the killing of tumor cells. In this study, we investigated the roles of iron level and the mainly pathway of iron uptake-TF/TFR system on VC-induced cytotoxicity. We conducted a preliminary evaluation of the potential value of 68Ga-citrate imaging in the anti-tumor therapy of VC combined with iron supplementation.High-dose vitamin C (VC) exhibits anti-tumor effects, and the cytotoxicity of VC is correlated with oxidative stress. However, iron, as a redox metal, plays an important effect in redox cycling and free radical formation in cells. This study addresses the role of iron ion in the cytotoxicity of VC. We found that iron supplementation increases the anti-tumor effect of VC, which was influenced by the cellular iron uptake pathway–transferrin (TF)/transferrin receptor (TFR) system. The TFR expression of tumors can be assessed by 68Ga-citrate PET imaging, and it would be helpful to screen out the tumor type which is more sensitive to VC combined with an iron supplementation treatment.

Immunoglobulin A promotes IL-6 and IL-8 production, proliferation, and migration by the human bronchial smooth muscle cells

Immunoglobulin A (IgA) is important in biological defense, mainly in the mucosal area, and plays pathogenic roles in various diseases by activating both inflammatory and structural cells. The current study aimed to validate the effects of IgA on the human bronchial smooth muscle cell (BSMC), which plays a major role in airway inflammation and remodeling. Serum IgA induced interleukin (IL)-6 and IL-8 production at both mRNA and protein levels, and enhanced cell proliferation and migration by the BSMCs. The synthetic phenotype markers were regulated and the contractile phenotype markers were downregulated by serum IgA. Mitogen-activated protein kinase, phosphatidylinositol 3-kinase/Akt, and nuclear factor-κB pathways were involved in IgA-induced IL-6 and IL-8 production. The BSMCs expressed transferrin receptor (TfR), and TfR siRNA transfection inhibited IL-6 and IL-8 production by serum IgA. In summary, serum IgA is a potent activator of the BSMCs at least partially via TfR.

Altered blood-brain barrier transport of nanotherapeutics in lysosomal storage diseases.

Treatment of neurological lysosomal storage disorders (LSDs) are limited because of impermeability of the blood-brain barrier (BBB) to macromolecules. Nanoformulations targeting BBB transcytosis are being explored, but the status of these routes in LSDs is unknown. We studied nanocarriers (NCs) targeted to the transferrin receptor (TfR), ganglioside GM1 or ICAM1, associated to the clathrin, caveolar or cell adhesion molecule (CAM) routes, respectively. We used brain endothelial cells and mouse models of acid sphingomyelinase-deficient Niemann Pick disease (NPD), and postmortem LSD patients' brains, all compared to respective controls. NC transcytosis across brain endothelial cells and brain distribution in mice were affected, yet through different mechanisms. Reduced TfR and clathrin expression were found, along with decreased transcytosis in cells and mouse brain distribution. Caveolin-1 expression and GM1 transcytosis were also reduced, yet increased GM1 levels seemed to compensate, providing similar NC brain distribution in NPD vs. control mice. A tendency to lower NHE-1 levels was seen, but highly increased ICAM1 expression in cells and human brains correlated with increased transcytosis and brain distribution in mice. Thus, transcytosis-related alterations in NPD and likely other LSDs may impact therapeutic access to the brain, illustrating the need for these mechanistic studies.

Aberrant Expression TFR1/CD71 in Gastric Cancer Identifies a Novel Potential Prognostic Marker and Therapeutic Target.

Background Gastric cancer (GC) is one of the most common malignant tumors with poor prognosis. So far, other than the HER2, GC lacks effective therapeutic targets. Transferrin receptor 1 (TFR1) expressions are abnormally upregulated in various cancers for the satisfaction of iron demand increased. This study aimed to explore the expression and clinical value of TFR1 in GC. Methods A tissue microarray including GC tissues and matched noncancerous tissues from 155 GC patients were collected. Moreover, the level of TFR1 expression was detected by immunohistochemistry, and we also evaluated the relationship between TFR1 expression and the clinicopathologic characteristics. What is more, univariate analysis and multivariate analysis were used to evaluate the risk factors and independent risk factors affecting the prognosis of GC. Results We found that TFR1 was overexpressed in GC tissues compared with noncancerous tissues, and a significant relationship was found between TFR1 expression and age (P=0.001), Lauren type (P=0.008), T stage (P=0.003), HER2 (P=0.003), PD-L1 (P < 0.001), and the level of CA72-4 (P=0.028). Survival analysis confirmed that GC patients with positive TFR1 expression had a poorer OS than that with negative TFR1 expression, and TFR1 expression was an independent risk factor in GC. Furthermore, we also found that there was a significant difference between the TFR1-PD-L1− group and the TFR1+PD-L1+ group (P=0.023), while there was no significant difference between the TFR1-PD-L1− group and the TFR1+PD-L1− group (P=0.119), or between the TFR1-PD-L1− group and the TFR1-PD-L1+ group (P=0.396). Conclusions TFR1 was overexpressed in GC and its aberrant expression identifies a novel potential prognostic marker and therapeutic target. In addition, TFR1 expression may be associated with the immune microenvironment and suppress the immune response via regulating the PD-L1 expression.

Electroacupuncture Reduces Cerebral Hemorrhage Injury in Rats by Improving Cerebral Iron Metabolism.

Objective To study the effects of electroacupuncture at Baihui and Dazhui points on the expression of hepcidin (Hepc), transferrin (Tf), transferrin receptor (TfR), and ferritin (Ft) in rats with cerebral hemorrhage to provide a theoretical basis for the treatment of cerebral hemorrhage with acupuncture. Method The model of cerebral hemorrhage in rats was established by autologous blood injection method and treated by electroacupuncture (EA) at the acupoints of Baihui and Dazhui. Hepc siRNA was injected into the lateral ventricle 30 min before model preparation to produce the cerebral hemorrhage model. The modified neurological severity score (mNSS) was used to assess the neurological function, and the total iron content in brain tissue was determined using atomic absorption spectrometry; the expression of Hepc, Ft, Tf, and TfR in perihematoma tissue was detected using immunohistochemistry; the interference efficiency of Hepc siRNA was detected using western blot and reverse transcription polymerase chain reaction (RT-PCR). Results The degree of neurological deficit showed a downward trend at 3 days, 7 days, and 14 days, and electroacupuncture significantly reduced the neurological deficit score at each time point (P < 0.01). Regarding total iron content in brain tissue, on the 3rd day, the 7th day, and the 14th day, the iron content of the hematoma tissue after intracerebral hemorrhage was reduced by electroacupuncture (P < 0.01). Regarding immunohistochemical results. Hepc, Ft, Tf, and TfR protein expressions on day 14 were significantly higher after cerebral hemorrhage (P < 0.01). After electroacupuncture, the expression of Hepc, Ft, Tf, and TfR protein was significantly reduced (P < 0.01). Western blot and RT-PCR revealed that the interference efficiency of Hepc siRNA was statistically significant (P < 0.01). Conclusion Electroacupuncture can reduce neurological severity scores in rats with cerebral hemorrhage and may exert cerebral protective effects by reducing Hepc protein and gene expression; lowering Ft, Tf, and TfR protein expression; and promoting iron metabolism in the brain of rats with cerebral hemorrhage.

A novobiocin derivative, XN4, triggers ferroptosis in gastric cancer cells via the activation of NOX4

Context A novobiocin derivative, XN4, has been shown to promote cell apoptosis in chronic myeloid leukaemia. Objective This study explores the mechanism by which XN4 promotes ferroptosis of gastric cancer (GC) cells. Materials and methods Human GC SGC-7901 and BGC-823 cells were treated with different XN4 concentrations (0, 0.1, 0.5, 1.0, 5.0, and 10.0 μmol/L) to evaluate effects of XN4. Additionally, cells were pre-treated for 24 h with si-NOX4, for 1 h with the iron chelator deferoxamine mesylate (DFO) or for 1 h with the lipid peroxidation inhibitor liproxstatin-1 before being treated with XN4 to analyse the mechanism of XN4. Results XN4 increased cell death (IC50 values of XN4 on SGC-7901 and BGC-823 cells: 1.592 ± 0.14 μmol/L and 2.022 ± 0.19 μmol/L) and Fe2+ levels in SGC-7901 and BGC-823 cells. These effects of 2.0 μmol/L XN4 were abolished by 100 μmol/L DFO treatment. XN4 enhanced transferrin and transferrin receptor expression to induce Fe2+ accumulation. XN4 decreased mitochondrial membrane potentials in GC cells, similar to erastin. Additionally, XN4 increased MDA, hydrogen peroxide, and ROS levels, but diminished total glutathione levels. Liproxstatin-1 (200 nmol/L) nullified the effects of XN4 (2.0 μmol/L) on MDA levels and cell death. Moreover, GPX4 levels decreased, but NOX4 and ferroptosis-related protein PTGS2 levels increased in GC cells following XN4 treatment, which was nullified by NOX4 knockdown. Discussion and conclusions The pro-ferroptotic role of XN4 in GC might enable it to become a promising drug for GC treatment in the future despite the need for extensive research.

Hepcidin regulates neuronal ferroptosis: A mechanism for postoperative cognitive dysfunction.

Toll-like receptor 4 (TLR4) is a signaling molecule responsible for the expression of hepcidin (Hepc), while myeloid differentiation protein 2 (MD2) is one major accessory protein of TLR4. This study focuses on exploring the neurocyte ferroptosis mediated through the regulation of Hepc expression by MD2, which is also one of the mechanisms for postoperative cognitive dysfunction (POCD).An experimental study was carried out using aged wild-type (Wt) and MD2 transgenic (Tg) mice. The neurocyte ferroptosis and POCD in the mice were assessed following splenectomy. Morris water maze was utilized to assess the neurocognitive abilities, hematoxylin and eosin (H&E) assay was performed to examine histopathology, and Nissl staining was used to evaluate the neurocyte damage. The Fe2+ , superoxide dismutase(SOD), malondialdehyde(MDA), glutathione(GSH), and glutathioneperoxidase 4(GPX4) levels were determined with kits. The expressions of transferrin receptor(TFR), Hepc, and MD2 were measured by Western blotting, while the expressions of TFR and GPX4 were measured by immunohistochemical staining. In Tg mice, we observed neurocyte ferroptosis and POCD following treatment with anMD2 inhibitor. PC12 cells were used as a neurocyte model. Ferroptosis was induced after treatment with anMD2 inhibitor, and the cell viability was assayed by Cell Counting Kit-8. Immunofluorescent staining was used to measure the TFR and GPX4 expressions. Meanwhile, the intracellular levels of Fe2+ , SOD, MDA, GSH, GPX4, and Hepc were also measured.POCD occurred among aged Wt and Tg mice. The Tg-POCD mice had more apparent POCD than the Wt-POCD mice. Nissl and H&E staining revealed neurocyte damage in brain tissues. Besides this, the Fe2+ and MDA expressions were upregulated, while the SOD, GSH, and GPX4 expressions were downregulated. Elevations in tissue levels of TFR, Hepc, and MD2 were observed, which were higher than those of Wt-POCD mice. After treatment with anMD2 inhibitor, the POCD could be prominently ameliorated in Tg-POCD mice, the Fe2+ and MDA levels could be reduced, while the SOD, GSH, and GPX4 levels could be elevated. In the PC12 model, ferroptosis could be suppressed by inhibiting the expression of MD2.MD2 is capable of regulating neurocyte ferroptosis by promoting Hepc expression, which has great potential as a novel target for POCD therapy.

Discovery of a Transferrin Receptor 1-Binding Aptamer and Its Application in Cancer Cell Depletion for Adoptive T-Cell Therapy Manufacturing.

The clinical manufacturing of chimeric antigen receptor (CAR) T cells includes cell selection, activation, gene transduction, and expansion. While the method of T-cell selection varies across companies, current methods do not actively eliminate the cancer cells in the patient's apheresis product from the healthy immune cells. Alarmingly, it has been found that transduction of a single leukemic B cell with the CAR gene can confer resistance to CAR T-cell therapy and lead to treatment failure. In this study, we report the identification of a novel high-affinity DNA aptamer, termed tJBA8.1, that binds transferrin receptor 1 (TfR1), a receptor broadly upregulated by cancer cells. Using competition assays, high resolution cryo-EM, and de novo model building of the aptamer into the resulting electron density, we reveal that tJBA8.1 shares a binding site on TfR1 with holo-transferrin, the natural ligand of TfR1. We use tJBA8.1 to effectively deplete B lymphoma cells spiked into peripheral blood mononuclear cells with minimal impact on the healthy immune cell composition. Lastly, we present opportunities for affinity improvement of tJBA8.1. As TfR1 expression is broadly upregulated in many cancers, including difficult-to-treat T-cell leukemias and lymphomas, our work provides a facile, universal, and inexpensive approach for comprehensively removing cancerous cells from patient apheresis products for safe manufacturing of adoptive T-cell therapies.

Knockdown of CBX7 inhibits ferroptosis in rats with cerebral ischemia and improves cognitive dysfunction by activating the Nrf2/HO-1 pathway

Iron-dependent lipid peroxidation-induced ferroptosis has been implicated in the pathogenesis of cerebral ischemia. Ischemia-reperfusion (I/R) contributes to cognitive dysfunction. Chromobox7 (CBX7) was upregulated in neural progenitor cells, followed by hypoxia and ischemia, and the suppression of CBX7 protected against renal and cerebral I/R injuries. However, the role of CBX7 in ferroptosis and cognitive dysfunction in rats with cerebral ischemia has not yet been reported. In our study, first, rats with cerebral I/R were established through middle cerebral artery occlusion (MCAO). Intracerebroventricular injection of shRNA targeting CBX7 (sh-CBX7) enhanced brain water content, and reduced neurological deficit scores and infarct size in rats post MCAO. Second, rats were subjected to 4 trials per day for 4 consecutive days in a Morris water maze. The time to find the platform in with 1 min was recorded as escape latency. On day 5, the platform was removed to conduct a spatial probe test. Rats were given 1 min to find the position of the platform, and the times for crossing the target within 1 min were recorded. Crossing the platform time is the time spent in each quadrant. Data from the Morris water maze showed that cognitive dysfunction in ischemic rats was ameliorated by the injection of sh-CBX7, which decreased the swimming distance and escape latency of ischemic rats, increased the times for crossing the target and platform in the maze. Moreover, injection of sh-CBX7 down-regulated levels of MDA and ROS, while it up-regulated SOD and GSH-Px to repress the oxidative stress. Third, knockdown of CBX7 in ischemic rats decreased the concentration of the ferric ions and the protein expression of transferrin receptor 1 (TFR1), but enhanced the solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), resulting in the inhibition of ferroptosis. Lastly, expressions of Nrf2, HO-1 and NAD(P)H:quinone oxidoreductase 1 (NQO1) were up-regulated in ischemic rats post sh-CBX7 injection. In conclusion, suppression of CBX7 attenuated I/R injury, improved cognitive dysfunction and inhibited ferroptosis through the activation of the Nrf2/HO-1 signaling pathway.

Norcantharidin down-regulates iron contents in the liver and spleen of lipopolysaccharide-treated mice

ABSTRACT Objective The inhibiting effect of Norcantharidin (NCTD) on IL-6 (interleukin-6) and STAT3 and the involvement of the IL-6/STAT3 pathway in hepcidin expression prompted us to speculate that NCTD could affect iron metabolism. Methods We examined the effects of NCTD on serum iron (SI) and transferrin (Tf) saturation, iron and ferritin light chain (FTL), transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (Fpn1), iron regulatory protein 1 (IRP1) and hepcidin, as well as IL-6 and STAT3 in the liver, spleen and duodenum of mice treated with lipopolysaccharide (LPS) in vivo, using RT-PCR, Western blotting and immunofluorescence analysis. Results NCTD could increase SI and Tf saturation and reduce tissue iron and FTL content by affecting expression of cell-iron transport proteins TfR1, DMT1 and Fpn1. The impact of NCTD on TfR1, DMT1 and Fpn1 expression is mediated by up-regulating IRP1 and down-regulating hepcidin expression, while NCTD-induced down-regulation of hepcidin is mediated by the IL-6/STAT3 signalling pathway in LPS-treated mice. Conclusions NCTD affects iron metabolism by modifying the expression of IL-6/JAK2/STAT3/hepcidin and IRP1 and suggest that the ability of NCTD to reduce tissue iron contents may be a novel mechanism associated with the anti-cancer effects of NCTD.

Ferroptosis participates in neuron damage in experimental cerebral malaria and is partially induced by activated CD8+ T cells

Cerebral malaria is the most serious complication of malaria infection, with 26% of surviving children having neurological sequelae, which may be caused by neuron damage, but the mechanism is not clear. Ferroptosis has been reported to play an important role in neuron damage in several nervous system diseases. However, the occurrence of ferroptosis in experimental cerebral malaria (ECM) pathogenesis is still unknown. In this study, we firstly detected increased levels of malondialdehyde (MDA) and iron, which are indicators of ferroptosis, in the cerebrum of ECM mice. Some important regulators of ferroptosis, including upregulated expression of transferrin receptor 1 (TfR1) and acyl-CoA synthetase long-chain family member 4 (ACSL4), and downregulation of glutathione peroxidase 4 (GPX4) levels, were also confirmed in ECM mice. Consistently, neuron damage, which was detected in the cerebrum of ECM mice, was positively correlated with reduced GPX4 expression and furtherly rescued by administration of the ferroptosis inhibitor ferrostatin-1 (Fer-1). In addition, primary neurons were damaged by activated CD8+ T cells, an effect that was also partially rescued by Fer-1 on amyloid precursor protein expression and mitochondrial membrane potential levels in vitro. Activated CD8+ T cells were also shown to infiltrate the cerebrum of ECM mice and upregulate TfR1 expression in primary neurons, which may be an important event for inducing ferroptosis in ECM. Altogether, we show that ferroptosis contributes to neuron damage in ECM pathogenesis, and activated CD8+ T cells may be important inducers of neuronal ferroptosis. Hence, targeting ferroptosis may be a promising adjuvant therapeutic strategy for neurological sequelae in patients with cerebral malaria.

On demand expression control of endogenous genes with DExCon, DExogron and LUXon reveals differential dynamics of Rab11 family members.

CRISPR technology has made generation of gene knock-outs widely achievable in cells. However, once inactivated, their re-activation remains difficult, especially in diploid cells. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation), and LUXon (light responsive DExCon) approaches which combine one-step CRISPR-Cas9-mediated targeted knockin of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene expression with the ability to re-activate expression on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knock-out/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene, and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon), or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, transferrin receptor recycling, expression kinetics, and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in ovarian cancer cell lines.

Abstract 362: MYCN-amplified neuroblastoma is addicted to iron and vulnerable to ferroptosis

Abstract MYCN is amplified in 20% to 25% of neuroblastoma, and MYCN-amplified neuroblastoma contributes to a large percent of pediatric cancer-related deaths. Therapy improvements for this subtype of cancer are a high priority. Ferroptosis is an iron-dependent, oxidative form of cell death that is counteracted mainly by the production of Glutathione Peroxidase 4 (GPX4), a phospholipid hydroperoxidase that is produced through the glutathione pathway. The identification of cancers that may benefit from ferroptosis inducers are just emerging. Here we uncover a MYCN-dependent therapeutic vulnerability in neuroblastoma. Namely, amplified MYCN rewires the cell through expression of key receptors, ultimately enhancing iron influx through increased expression of the iron import transferrin receptor 1 (TFR1). Accumulating iron causes reactive oxygen species (ROS) production, and MYCN-amplified neuroblastomas show enhanced reliance on the system Xc- cystine/glutamate antiporter for ROS detoxification through increased transcription of this receptor. By performing metabolomics, we demonstrate that the transsulfuration pathway is also activated by MYCN. The increased activation of both pathways leads to cysteine accumulation that results in inhibition of lipid peroxidation. Utilizing drugs that target the main components of the glutathione and transsulfuration pathway we sensitize the MYCN neuroblastomas to ferroptotic cell death. These data provide novel insights into how MYCN alters the transcriptome in neuroblastoma to confer growth and survival advantages and simultaneously sheds light on the mechanism of action of ferroptosis inducers with potential application in other types of cancer. Citation Format: Konstantinos V. Floros, Mia O. Johnson-Berro, Richard Kurupi, Carter K. Fairchild, Krista Dalton, Bin Hu, Madhavi Puchalapalli, Mikhail G. Dozmorov, Jennifer E. Koblinski, James A. Olzmann, Lauren A. Cowart, Anthony C. Faber. MYCN-amplified neuroblastoma is addicted to iron and vulnerable to ferroptosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 362.

Apoferritin-Encapsulated Jerantinine A for Transferrin Receptor Targeting and Enhanced Selectivity in Breast Cancer Therapy.

The O-acetyl (or acetate) derivative of the Aspidosperma alkaloid Jerantinine A (JAa) elicits anti-tumor activity against cancer cell lines including mammary carcinoma cell lines irrespective of receptor status (0.14 < GI50 < 0.38 μM), targeting microtubule dynamics. By exploiting breast cancer cells’ upregulated transferrin receptor 1 (TfR1) expression and apoferritin (AFt) recognition, we sought to develop an AFt JAa-delivery vehicle to enhance tumor-targeting and reduce systemic toxicity. Optimizing pH-mediated reassembly, ∼120 JAa molecules were entrapped within AFt. Western blot and flow cytometry demonstrate TfR1 expression in cancer cells. Enhanced internalization of 5-carboxyfluorescein-conjugated human AFt in SKBR3 and MDA-MB-231 cancer cells is observed compared to MRC5 fibroblasts. Accordingly, AFt–JAa delivers significantly greater intracellular JAa levels to SKBR3 and MDA-MB-231 cells than naked JAa (0.2 μM) treatment alone. Compared to naked JAa (0.2 μM), AFt–JAa achieves enhanced growth inhibition (2.5–14-fold; <0.02 μM < GI50 < 0.15 μM) in breast cancer cells; AFt–JAa treatment results in significantly reduced clonal survival, more profound cell cycle perturbation including G2/M arrest, greater reduction in cell numbers, and increased apoptosis compared to the naked agent (p < 0.01). Decreased PLK1 and Mcl-1 expression, together with the appearance of cleaved poly (ADP-ribose)-polymerase, corroborate the augmented potency of AFt–JAa. Hence, we demonstrate that AFt represents a biocompatible vehicle for targeted delivery of JAa, offering potential to minimize toxicity and enhance JAa activity in TfR1-expressing tumors.