Multistep, sequential control of the trafficking and function of the multiple sulfatase deficiency gene product, SUMF1 by PDI, ERGIC-53 and ERp44.

Sulfatase modifying factor 1 (SUMF1) encodes for the formylglicine generating enzyme, which activates sulfatases by modifying a key cysteine residue within their catalytic domains. SUMF1 is mutated in patients affected by multiple sulfatase deficiency, a rare recessive disorder in which all sulfatase activities are impaired. Despite the absence of canonical retention/retrieval signals, SUMF1 is largely retained in the endoplasmic reticulum (ER), where it exerts its enzymatic activity on nascent sulfatases. Part of SUMF1 is secreted and paracrinally taken up by distant cells. Here we show that SUMF1 interacts with protein disulfide isomerase (PDI) and ERp44, two thioredoxin family members residing in the early secretory pathway, and with ERGIC-53, a lectin that shuttles between the ER and the Golgi. Functional assays reveal that these interactions are crucial for controlling SUMF1 traffic and function. PDI couples SUMF1 retention and activation in the ER. ERGIC-53 and ERp44 act downstream, favoring SUMF1 export from and retrieval to the ER, respectively. Silencing ERGIC-53 causes proteasomal degradation of SUMF1, while down-regulating ERp44 promotes its secretion. When over-expressed, each of three interactors favors intracellular accumulation. Our results reveal a multistep control of SUMF1 trafficking, with sequential interactions dynamically determining ER localization, activity and secretion

Progressive fibrosing interstitial lung diseases: A current perspective

Interstitial lung diseases (ILDs) are a large and diverse group of rare and chronic respiratory disorders, with idiopathic pulmonary fibrosis (IPF) being the most common and best-studied member. Increasing interest in fibrosis as a therapeutic target and the appreciation that fibrotic mechanisms may be a treatable target of IPF prompted the development and subsequent approval of the antifibrotics, pirfenidone and nintedanib. The management of ILDs has changed considerably following an understanding that IPF and some ILDs share similar disease behavior of progressive fibrosis, termed “progressive fibrosing phenotype”. Indeed, antifibrotic treatment has shown to be beneficial in ILDs characterized by the progressive fibrosing phenotype. This narrative review summarizes current knowledge in the field of progressive fibrosing ILDs. Here, we discuss the clinical characteristics and pathogenesis of lung fibrosis and highlight relevant literature concerning the mechanisms underlying progressive fibrosing ILDs. We also summarize current diagnostic approaches and the available treatments of progressive fibrosing ILDs and address the optimization of treating progressive fibrosing ILDs with antifibrotics in clinical practice

Glutathione limits Ero1-dependent oxidation in the endoplasmic reticulum

Many proteins of the secretory pathway contain disulfide bonds that are essential for structure and function. In the endoplasmic reticulum (ER), Ero1alpha and Ero1beta oxidize protein disulfide isomerase (PDI), which in turn transfers oxidative equivalents to newly synthesized cargo proteins. However, oxidation must be limited, as some reduced PDI is necessary for disulfide isomerization and ER-associated degradation. Here we show that in semipermeable cells, PDI is more oxidized, disulfide bonds are formed faster, and high molecular mass covalent protein aggregates accumulate in the absence of cytosol. Addition of reduced glutathione (GSH) reduces PDI and restores normal disulfide formation rates. A higher GSH concentration is needed to balance oxidative folding in semipermeable cells overexpressing Ero1alpha, indicating that cytosolic GSH and lumenal Ero1alpha play antagonistic roles in controlling the ER redox. Moreover, the overexpression of Ero1alpha significantly increases the GSH content in HeLa cells. Our data demonstrate tight connections between ER and cytosol to guarantee redox exchange across compartments: a reducing cytosol is important to ensure disulfide isomerization in secretory proteins

Everolimus Nanoformulation in Biological Nanoparticles Increases Drug Responsiveness in Resistant and Low-Responsive Breast Cancer Cell Lines

Everolimus (Eve) is an FDA approved drug that inhibits mammalian target of rapamycin (mTOR). It is employed in breast cancer treatment even if its responsiveness is controversial. In an attempt to increase Eve effectiveness, we have developed a novel Eve nanoformulation exploiting H-ferritin nanocages (HEve) to improve its subcellular delivery. We took advantage of the natural tumor targeting of H-Ferritin, which is mediated by the transferrin receptor-1 (TfR1). Breast cancer cells overexpressing TfR-1 were successfully recognized by H-Ferritin, displaying quick nanocage internalization. HEve has been tested and compared to Eve for in vitro efficacy in sensitive and resistant breast cancer cells. Nanoformulated Eve induced remarkable antiproliferative activity in vitro, making even resistant cell lines sensitive to Eve. Moreover, the antiproliferative activity of HEve is fully in accordance with cytotoxicity observed by cell death assay. Furthermore, the significant increase in anticancer efficacy displayed in HEve-treated samples is due to the improved drug accumulation, as demonstrated by UHPLC-MS/MS quantifications. Our findings suggest that optimizing Eve subcellular delivery, thanks to nanoformulation, determines its improved antitumor activity in a panel of Eve-sensitive or resistant breast cancer cell lines

On the use of superparamagnetic hydroxyapatite nanoparticles as an agent for magnetic and nuclear in vivo imaging

The identification of alternative biocompatible magnetic NPs for advanced clinical application is becoming an important need due to raising concerns about iron accumulation in soft issues associated to the administration of superparamagnetic iron oxide nanoparticles (NPs). Here, we report on the performance of previously synthetized iron-doped hydroxyapatite (FeHA) NPs as contrast agent for magnetic resonance imaging (MRI). The MRI contrast abilities of FeHA and Endorem® (dextran coated iron oxide NPs) were assessed by 1H nuclear magnetic resonance relaxometry and their performance in healthy mice was monitored by a 7 Tesla scanner. FeHA applied a higher contrast enhancement, and had a longer endurance in the liver with respect to Endorem® at iron equality. Additionally, a proof of concept of FeHA use as scintigraphy imaging agent for positron emission tomography (PET) and single photon emission computed tomography (SPECT) was given labeling FeHA with 99mTc-MDP by a straightforward surface functionalization process. Scintigraphy/x-ray fused imaging and ex vivo studies confirmed its dominant accumulation in the liver, and secondarily in other organs of the mononuclear phagocyte system. FeHA efficiency as MRI-T2 and PET-SPECT imaging agent combined to its already reported intrinsic biocompatibility qualifies it as a promising material for innovative nanomedical applications. STATEMENT OF SIGNIFICANCE: The ability of iron-doped hydroxyapatite nanoaprticles (FeHA) to work in vivo as imaging agents for magnetic resonance (MR) and nuclear imaging is demonstrated. FeHA applied an higher MR contrast in the liver, spleen and kidneys of mice with respect to Endorem®. The successful radiolabeling of FeHA allowed for scintigraphy/X-ray and ex vivo biodistribution studies, confirming MR results and envisioning FeHA application for dual-imaging

Combined Ferritin Nanocarriers with ICG for Effective Phototherapy Against Breast Cancer

Leopoldo Sitia,1,* Paola Saccomandi,2,* Leonardo Bianchi,2 Marta Sevieri,1 Cristina Sottani,3 Raffaele Allevi,1 Elena Grignani,3 Serena Mazzucchelli,1 Fabio Corsi1,4 1Department of Biomedical and Clinical Sciences, Università degli studi di Milano, Milan, Italy; 2Department of Mechanical Engineering, Politecnico di Milano, Milan, Italy; 3Environmental Research Center, Istituti Clinici Scientifici Maugeri IRCCS, Pavia, Italy; 4Istituti Clinici Scientifici Maugeri IRCCS, Pavia, Italy*These authors contributed equally to this workCorrespondence: Serena Mazzucchelli, Department of Biomedical and Clinical Sciences, Università degli studi di Milano, via G.B. Grassi 74, Milan, 20157, Italy, Email [email protected] Fabio Corsi, Istituti Clinici Scientifici Maugeri IRCCS, via S. Maugeri 10, Pavia, 27100, Italy, Email [email protected]: Photodynamic Therapy (PDT) is a promising, minimally invasive treatment for cancer with high immunostimulatory potential, no reported drug resistance, and reduced side effects. Indocyanine Green (ICG) has been used as a photosensitizer (PS) for PDT, although its poor stability and low tumor-target specificity strongly limit its efficacy. To overcome these limitations, ICG can be formulated as a tumor-targeting nanoparticle (NP).Methods: We nanoformulated ICG into recombinant heavy-ferritin nanocages (HFn-ICG). HFn has a specific interaction with transferrin receptor 1 (TfR1), which is overexpressed in most tumors, thus increasing HFn tumor tropism. First, we tested the properties of HFn-ICG as a PS upon irradiation with a continuous-wave diode laser. Then, we evaluated PDT efficacy in two breast cancer (BC) cell lines with different TfR1 expression levels. Finally, we measured the levels of intracellular endogenous heavy ferritin (H-Fn) after PDT treatment. In fact, it is known that cells undergoing ROS-induced autophagy, as in PDT, tend to increase their ferritin levels as a defence mechanism. By measuring intracellular H-Fn, we verified whether this interplay between internalized HFn and endogenous H-Fn could be used to maximize HFn uptake and PDT efficacy.Results: We previously demonstrated that HFn-ICG stabilized ICG molecules and increased their delivery to the target site in vitro and in vivo for fluorescence guided surgery. Here, with the aim of using HFn-ICG for PDT, we showed that HFn-ICG improved treatment efficacy in BC cells, depending on their TfR1 expression. Our data revealed that endogenous H-Fn levels were increased after PDT treatment, suggesting that this defence reaction against oxidative stress could be used to enhance HFn-ICG uptake in cells, increasing treatment efficacy.Conclusion: The strong PDT efficacy and peculiar Trojan horse-like mechanism, that we revealed for the first time in literature, confirmed the promising application of HFn-ICG in PDT. Keywords: heavy-ferritin nanocages, indocyanine green, photodynamic therapy, breast cancer, tumor-targeted nanoparticle

Co-administration of H-ferritin-doxorubicin and Trastuzumab in neoadjuvant setting improves efficacy and prevents cardiotoxicity in HER2 + murine breast cancer model

Neoadjuvant chemotherapy has been established as the standard of care for HER2-positive breast cancer since it allows cancer down-staging, up to pathological complete response. The standard of care in the neoadjuvant setting for HER2-positive breast cancer is a combination of highly cytotoxic drugs such as anthracyclines and the anti-HER2 monoclonal antibody. Despite this cocktail allows a pathological complete response in up to 50%, their co-administration is strongly limited by intrinsic cardiotoxicity. Therefore, only a sequential administration of anthracyclines and the anti-HER2 treatment is allowed. Here, we propose the anthracycline formulation in H-Ferritin nanocages as promising candidate to solve this unmet clinical need, thanks to its capability to increase anthracyclines efficacy while reducing their cardiotoxicity. Treating a murine model of HER2-positive breast cancer with co-administration of Trastuzumab and H-Ferritin anthracycline nanoformulation, we demonstrate an improved tumor penetration of drugs, leading to increased anticancer efficacy and reduced of cardiotoxicity