PrPC Aptamer Conjugated-Gold Nanoparticles for Targeted Delivery of Doxorubicin to Colorectal Cancer Cells.

Anticancer drugs, such as fluorouracil (5-FU), oxaliplatin, and doxorubicin (Dox) are commonly used to treat colorectal cancer (CRC); however, owing to their low response rate and adverse effects, the development of efficient drug delivery systems (DDSs) is required. The cellular prion protein PrPC, which is a cell surface glycoprotein, has been demonstrated to be overexpressed in CRC, however, there has been no research on the development of PrPC-targeting DDSs for targeted drug delivery to CRC. In this study, PrPC aptamer (Apt)-conjugated gold nanoparticles (AuNPs) were synthesized for targeted delivery of Dox to CRC. Thiol-terminated PrPC-Apt was conjugated to AuNPs, followed by hybridization of its complementary DNA for drug loading. Finally, Dox was loaded onto the AuNPs to synthesize PrPC-Apt-functionalized doxorubicin-oligomer-AuNPs (PrPC-Apt DOA). The PrPC-Apt DOA were spherical nanoparticles with an average diameter of 20 nm. Treatment of CRC cells with PrPC-Apt DOA induced reactive oxygen species generation by decreasing catalase and superoxide dismutase activities. In addition, treatment with PrPC-Apt DOA inhibited mitochondrial functions by decreasing the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha, complex 4 activity, and oxygen consumption rates. Compared to free Dox, PrPC-Apt DOA decreased proliferation and increased apoptosis of CRC cells to a greater degree. In this study, we demonstrated that PrPC-Apt DOA targeting could effectively deliver Dox to CRC cells. PrPC-Apt DOA can be used as a treatment for CRC, and have the potential to replace existing anticancer drugs, such as 5-FU, oxaliplatin, and Dox.

Knockdown of CK2α reduces P-cresol-induced fibrosis in human renal proximal tubule epithelial cells via the downregulation of profilin-1.

Renal fibrosis is one of the main causes of chronic kidney disease. Many studies have focused on fibroblasts and myofibroblasts involved in renal fibrogenesis. Recently, several studies have reported that renal proximal tubule epithelial cells are possible initiators of renal fibrosis. However, the mechanism through which cells induce renal fibrosis is poorly understood. In this study, we found that CK2α induces fibrosis in renal proximal tubule epithelial cells (TH1) by regulating the expression of profilin-1 (Pfn1). CKD mouse model and TH1 cells treated with P-cresol also showed an increased level of Pfn1. The knockdown of CK2α suppressed fibrosis in TH1 cells via the downregulation of Pfn1. In particular, CK2α knockdown inhibited the expression of stress fibers and fibrosis-related proteins in P-cresol-treated TH1 cells. Furthermore, the knockdown of CK2α inhibited mitochondrial dysfunction and restored cellular senescence and cell cycle in P-cresol-treated TH1 cells. These results indicate that CK2α induces renal fibrosis through Pfn1, which makes CK2α a key target molecule in the treatment of fibrosis related to chronic kidney disease.

Melatonin Protects Human Renal Proximal Tubule Epithelial Cells Against High Glucose-Mediated Fibrosis via the Cellular Prion Protein-TGF-ß-Smad Signaling Axis.

Diabetes-mediated hyperglycemia is a major risk factor for renal fibrosis, resulting in the development of chronic kidney diseases. To address this issue, the effect of melatonin, which has an antioxidative potential, on renal fibrosis in human renal proximal tubule epithelial cells under high glucose conditions was investigated. Under high glucose conditions, the generation of reactive oxygen species was drastically increased in human renal proximal tubule epithelial cells, which lead to the inhibition of cell proliferation, enlargement of cell size, reduction of cell survival, and suppression of antioxidant enzyme activities. High glucose also increased the expression of transforming growth factor-ß, leading to an increase in Smad2 phosphorylation. These fibrotic phenotype changes increased the expression of fibrosis-mediated extracellular matrix proteins, such as fibronectin, collagen I, and α-smooth muscle actin. In addition, the level of cellular prion protein (PrPC), which is associated with several biological processes, was decreased by exposure to high glucose conditions. Melatonin recovered the expression levels of PrPC under high glucose conditions via phosphorylation of Akt, resulting in the prevention of high glucose-induced fibrosis. In particular, overexpression of PrPC blocked the high glucose-mediated fibrotic phenotype change. These findings indicate that melatonin could be a powerful agent for treating hyperglycemia-induced renal fibrosis.

The Cellular Prion Protein: A Promising Therapeutic Target for Cancer.

Studies on the cellular prion protein (PrPC) have been actively conducted because misfolded PrPC is known to cause transmissible spongiform encephalopathies or prion disease. PrPC is a glycophosphatidylinositol-anchored cell surface glycoprotein that has been reported to affect several cellular functions such as stress protection, cellular differentiation, mitochondrial homeostasis, circadian rhythm, myelin homeostasis, and immune modulation. Recently, it has also been reported that PrPC mediates tumor progression by enhancing the proliferation, metastasis, and drug resistance of cancer cells. In addition, PrPC regulates cancer stem cell properties by interacting with cancer stem cell marker proteins. In this review, we summarize how PrPC promotes tumor progression in terms of proliferation, metastasis, drug resistance, and cancer stem cell properties. In addition, we discuss strategies to treat tumors by modulating the function and expression of PrPC via the regulation of HSPA1L/HIF-1α expression and using an anti-prion antibody.

The Dual Role of Autophagy in Cancer Development and a Therapeutic Strategy for Cancer by Targeting Autophagy.

Autophagy is a delicate intracellular degradation process that occurs due to diverse stressful conditions, including the accumulation of damaged proteins and organelles as well as nutrient deprivation. The mechanism of autophagy is initiated by the creation of autophagosomes, which capture and encapsulate abnormal components. Afterward, autophagosomes assemble with lysosomes to recycle or remove degradative cargo. The regulation of autophagy has bipolar roles in cancer suppression and promotion in diverse cancers. Furthermore, autophagy modulates the features of tumorigenesis, cancer metastasis, cancer stem cells, and drug resistance against anticancer agents. Some autophagy regulators are used to modulate autophagy for anticancer therapy but the dual roles of autophagy limit their application in anticancer therapy, and present as the main reason for therapy failure. In this review, we summarize the mechanisms of autophagy, tumorigenesis, metastasis, cancer stem cells, and resistance against anticancer agents. Finally, we discuss whether targeting autophagy is a promising and effective therapeutic strategy in anticancer therapy.

Melatonin Suppresses Renal Cortical Fibrosis by Inhibiting Cytoskeleton Reorganization and Mitochondrial Dysfunction through Regulation of miR-4516.

Renal fibrosis, a major risk factor for kidney failure, can lead to chronic kidney disease (CKD) and is caused by cytoskeleton reorganization and mitochondrial dysfunction. In this study, we investigated the potential of melatonin treatment to reduce renal fibrosis by recovering the cytoskeleton reorganization and mitochondrial dysfunction. We found that miR-4516 expression was downregulated in the renal cortex of CKD mice and P-cresol-treated TH1 cells. Decreased miR-4516 expression stimulated cytoskeleton reorganization and mitochondrial dysfunction, and induced renal fibrosis. Melatonin treatment suppressed fibrosis by inhibiting cytoskeleton reorganization and restoring mitochondrial function via increased miR-4516 expression. More specifically, melatonin treatment increased miR-4516 expression while decreasing ITGA9 expression, thereby inhibiting cytoskeleton reorganization. In addition, increased expression of miR-4516 by melatonin treatment reduced ROS formation and restored mitochondrial function. These findings suggest that melatonin may be a promising treatment for patients with CKD having renal fibrosis. Moreover, regulation of miR-4516 expression may be a novel strategy for the treatment of renal fibrosis.

EVpedia: a community web portal for extracellular vesicles research.

MOTIVATION: Extracellular vesicles (EVs) are spherical bilayered proteolipids, harboring various bioactive molecules. Due to the complexity of the vesicular nomenclatures and components, online searches for EV-related publications and vesicular components are currently challenging. RESULTS: We present an improved version of EVpedia, a public database for EVs research. This community web portal contains a database of publications and vesicular components, identification of orthologous vesicular components, bioinformatic tools and a personalized function. EVpedia includes 6879 publications, 172 080 vesicular components from 263 high-throughput datasets, and has been accessed more than 65 000 times from more than 750 cities. In addition, about 350 members from 73 international research groups have participated in developing EVpedia. This free web-based database might serve as a useful resource to stimulate the emerging field of EV research. AVAILABILITY AND IMPLEMENTATION: The web site was implemented in PHP, Java, MySQL and Apache, and is freely available at http://evpedia.info.

Proteomic analysis of extracellular vesicles derived from Mycobacterium tuberculosis.

The release of extracellular vesicles, also known as outer membrane vesicles, membrane vesicles, exosomes, and microvesicles, is an evolutionarily conserved phenomenon from bacteria to eukaryotes. It has been reported that Mycobacterium tuberculosis releases extracellular vesicles harboring immunologically active molecules, and these extracellular vesicles have been suggested to be applicable in vaccine development and biomarker discovery. However, the comprehensive proteomic analysis has not been performed for M. tuberculosis extracellular vesicles. In this study, we identified a total of 287 vesicular proteins by four LC-MS/MS analyses with high confidence. In addition, we identified several vesicular proteins associated with the virulence of M. tuberculosis. This comprehensive proteome profile will help elucidate the pathogenic mechanism of M. tuberculosis. The data have been deposited to the ProteomeXchange with identifier PXD001160 (http://proteomecentral.proteomexchange.org/dataset/PXD001160).

Inhibitory Effect of Oxaliplatin-loaded Engineered Milk Extracellular Vesicles on Tumor Progression.

BACKGROUND/AIM: Anti-cancer chemotherapy is an effective therapeutic approach. Milk extracellular vesicles (EVs) loaded with chemotherapeutics have a potential anticancer effect by acting as a drug delivery system. Thus, our study aimed to explore the effect of engineered milk extracellular vesicles. MATERIALS AND METHODS: To treat epidermal growth factor receptor (EGFR) expressing solid tumors, we established oxaliplatin-loaded milk EV conjugated with GE11 peptide (GE11Milk EVoxal), which has a high affinity to EGFR and assessed their anti-cancer effect in vitro and in vivo. RESULTS: Drug-loaded GE11Milk EVoxal showed significantly higher incorporation into EGFR expressing cancer cells compared with milk EV without GE11 conjugation (Milk EVoxal), leading to apoptosis of cancer cells. GE11Milk EVoxal also inhibited cell viability compared to milk EVoxal or oxaliplatin alone. In colorectal cancer xenograft murine model, GE11Milk EVoxal showed the maximum therapeutic effect on tumor progression. These findings indicate that GE11Milk EVoxal suppresses EGFR expressing cancer through GE11 peptide-mediated EGFR targeting and subsequently anti-cancer drug delivery. CONCLUSION: Anti-cancer drug-loaded engineered milk EVs might be a novel therapeutic approach for treating patients with EGFR expressing solid tumors.

Melatonin Protects Chronic Kidney Disease Mesenchymal Stem/Stromal Cells against Accumulation of Methylglyoxal via Modulation of Hexokinase-2 Expression.

Treatment options for patients with chronic kidney disease (CKD) are currently limited; therefore, there has been significant interest in applying mesenchymal stem/stromal cell (MSC)-based therapy to treat CKD. However, MSCs harvested from CKD patients tend to show diminished viability and proliferation due to sustained exposure to uremic toxins in the CKD environment, which limits their utility for cell therapy. The application of melatonin has been demonstrated to improve the therapeutic efficacy of MSCs derived from and engrafted to tissues in patients suffering from CKD, although the underlying biological mechanism has not been elucidated. In this study, we observed overexpression of hexokinase-2 (HK2) in serum samples of CKD patients and MSCs harvested from an adenine-fed CKD mouse model (CKD-mMSCs). HK2 upregulation led to increased production levels of methylglyoxal (MG), a toxic metabolic intermediate of abnormal glycolytic processes. The overabundance of HK2 and MG was associated with impaired mitochondrial function and low cell proliferation in CKD-mMSCs. Melatonin treatment inhibited the increases in HK2 and MG levels, and further improved mitochondrial function, glycolytic metabolism, and cell proliferation. Our findings suggest that identifying and characterizing metabolic regulators such as HK2 in CKD may improve the efficacy of MSCs for treating CKD and other kidney disorders.

PrPC Regulates the Cancer Stem Cell Properties via Interaction With c-Met in Colorectal Cancer Cells.

BACKGROUND/AIM: Studies have reported that the expression of c-Met and PrPC improves tumor progression. However, not much is known about their relationship. We hypothesized that c-Met and PrPC interact with each other, and enhance cancer stem cell (CSC) characteristics. MATERIALS AND METHODS: Magnetic activated cell sorting was used to examine the interaction between c-Met and PrPC The effects of the interaction on downstream signals, stem cell marker expression, and sphere formation of colorectal cancer (CRC) cells were investigated. RESULTS: We demonstrated the increased expression and binding levels of c-Met and PrPC in CRC cells compared to normal colon epithelial cells. We revealed that the c-Met and PrPC interaction induced the ERK activation and Oct4 upregulation. The inhibition of c-Met by crizotinib reduced ERK activation and Oct4 expression and suppressed CSC properties. CONCLUSION: c-Met and PrPC interact with each other, and targeting c-Met using crizotinib could be a powerful strategy for CRC therapy.

Bovine milk extracellular vesicles induce the proliferation and differentiation of osteoblasts and promote osteogenesis in rats.

Bone is constantly balanced between the formation of new bone by osteoblasts and the absorption of old bone by osteoclasts. To promote bone growth and improve bone health, it is necessary to promote the proliferation and differentiation of osteoblasts. Although bovine milk is known to exert a beneficial effect on bone formation, the study on the effect of bovine milk extracellular vesicles (EVs) on osteogenesis in osteoblasts is limited. In this study, we demonstrated that bovine milk EVs promoted the proliferation of human osteogenic Saos-2 cells by increasing the expression of cell cycle-related proteins. In addition, bovine milk EVs also induced the differentiation of Saos-2 cells by increasing the expression of RUNX2 and Osterix which are key transcription factors for osteoblast differentiation. Oral administration of milk EVs did not cause toxicity in Sprague-Dawley rats. Furthermore, milk EVs promoted longitudinal bone growth and increased the bone mineral density of the tibia. Our findings suggest that milk EVs could be a safe and powerful applicant for enhancing osteogenesis. PRACTICAL APPLICATIONS: Until now, calcium and vitamin D have been prescribed to promote bone formation or to prevent bone diseases such as osteoporosis. Recently, several studies to find bioactive molecules that regulate cellular functions of osteoblasts or osteoclasts are actively underway. Milk basic proteins and lactoferrin present in milk are known to promote bone formation, but they exist in small quantities and the isolation of these proteins is complicated making mass production difficult. Recently, it has been found that milk contains large quantities of EVs, and that they promote bone formation. Studies on the effect of Milk EVs on osteoblasts during osteogenesis will help in the development of biomaterials for osteogenesis.

Harnessing the Physiological Functions of Cellular Prion Protein in the Kidneys: Applications for Treating Renal Diseases.

A cellular prion protein (PrPC) is a ubiquitous cell surface glycoprotein, and its physiological functions have been receiving increased attention. Endogenous PrPC is present in various kidney tissues and undergoes glomerular filtration. In prion diseases, abnormal prion proteins are found to accumulate in renal tissues and filtered into urine. Urinary prion protein could serve as a diagnostic biomarker. PrPC plays a role in cellular signaling pathways, reno-protective effects, and kidney iron uptake. PrPC signaling affects mitochondrial function via the ERK pathway and is affected by the regulatory influence of microRNAs, small molecules, and signaling proteins. Targeting PrPC in acute and chronic kidney disease could help improve iron homeostasis, ameliorate damage from ischemia/reperfusion injury, and enhance the efficacy of mesenchymal stem/stromal cell or extracellular vesicle-based therapeutic strategies. PrPC may also be under the influence of BMP/Smad signaling and affect the progression of TGF-ß-related renal fibrosis. PrPC conveys TNF-α resistance in some renal cancers, and therefore, the coadministration of anti-PrPC antibodies improves chemotherapy. PrPC can be used to design antibody-drug conjugates, aptamer-drug conjugates, and customized tissue inhibitors of metalloproteinases to suppress cancer. With preclinical studies demonstrating promising results, further research on PrPC in the kidney may lead to innovative PrPC-based therapeutic strategies for renal disease.

Correction: Yun et al. Prion Protein of Extracellular Vesicle Regulates the Progression of Colorectal Cancer. Cancers 2021, 13, 2144.

The authors wish to make the following corrections to this paper [...].

Prion Protein of Extracellular Vesicle Regulates the Progression of Colorectal Cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer-related death due to its aggressive metastasis in later stages. Although there is a growing interest in the tumorigenic role of cellular prion protein (PrPC) in the process of metastasis, the precise mechanism behind the cellular communication involving prion proteins remains poorly understood. This study found that hypoxic tumor microenvironment increased the PrPC-expressing exosomes from CRC, and these exosomes regulate the CRC cell behavior and tumor progression depending on the expression of PrPC. Hypoxic exosomes from CRC cells promoted sphere formation, the expression of tumor-inducing genes, migration, invasion, and tumor growth. Furthermore, these exosomes increased endothelial permeability, migration, invasion, and angiogenic cytokine secretion. These effects were associated with PrPC expression. Application of anti-PrPC antibody with 5-fluorouracil significantly suppressed the CRC progression in a murine xenograft model. Taken together, these findings indicate that PrP-expressing exosomes secreted by hypoxic CRC cells are a key factor in the tumorigenic CRC-to-CRC and CRC-to-endothelial cell communication. Significance: These findings suggest that inhibiting PrPC in hypoxic exosomes during chemotherapy may be an effective therapeutic strategy in colorectal cancer.

Melatonin Treatment Improves Renal Fibrosis via miR-4516/SIAH3/PINK1 Axis.

Dysregulation in mitophagy, in addition to contributing to imbalance in the mitochondrial dynamic, has been implicated in the development of renal fibrosis and progression of chronic kidney disease (CKD). However, the current understanding of the precise mechanisms behind the pathogenic loss of mitophagy remains unclear for developing cures for CKD. We found that miR-4516 is downregulated and its target SIAH3, an E3 ubiquitin protein ligase that reduces PINK1 accumulation to damaged mitochondria, is upregulated in the renal cortex of CKD mice. Here, we demonstrated that melatonin injection induces miR-4516 expression and suppresses SIAH3, and promotes PINK1/Parkin-mediated mitophagy. Furthermore, we demonstrated that melatonin injection attenuates the pathological features of CKD by improving mitochondrial homeostasis. Our data supports that mitochondrial autophagy regulation by activating miR-4516/SIAH3/PINK1 mitophagy signaling axis can be a viable new strategy for treating CKD.

Topical Administration of Melatonin-Loaded Extracellular Vesicle-Mimetic Nanovesicles Improves 2,4-Dinitrofluorobenzene-Induced Atopic Dermatitis.

Atopic dermatitis (AD) is caused by multiple factors that trigger chronic skin inflammation, including a defective skin barrier, immune cell activation, and microbial exposure. Although melatonin has an excellent biosafety profile and a potential to treat AD, there is limited clinical evidence from controlled trials that support the use of melatonin as an AD treatment. The delivery of melatonin via the transdermal delivery system is also a challenge in designing melatonin-based AD treatments. In this study, we generated melatonin-loaded extracellular vesicle-mimetic nanoparticles (MelaNVs) to improve the transdermal delivery of melatonin and to evaluate their therapeutic potential in AD. The MelaNVs were spherical nanoparticles with an average size of 100 nm, which is the optimal size for the transdermal delivery of drugs. MelaNVs showed anti-inflammatory effects by suppressing the release of TNF-α and ß-hexosaminidase in LPS-treated RAW264.7 cells and compound 48/80-treated RBL-2H3 cells, respectively. MelaNVs showed a superior suppressive effect compared to an equivalent concentration of free melatonin. Treating a 2,4-dinitrofluorobenzene (DNCB)-induced AD-like mouse model with MelaNVs improved AD by suppressing local inflammation, mast cell infiltration, and fibrosis. In addition, MelaNVs effectively suppressed serum IgE levels and regulated serum IFN-γ and IL-4 levels. Taken together, these results suggest that MelaNVs are novel and efficient transdermal delivery systems of melatonin and that MelaNVs can be used as a treatment to improve AD.

Role of PrPC in Cancer Stem Cell Characteristics and Drug Resistance in Colon Cancer Cells.

BACKGROUND/AIM: Cancer stem cell characteristics and drug resistance of colorectal cancer are associated with failure of cancer treatment. In this study, we investigated the effects of PrPC on cancer stem cell characteristics, migration, invasion, and drug resistance of 5FU-resistant CRC cells. MATERIALS AND METHODS: PrPC negative and PrPC positive cells were isolated from 5FU-resistant CRC cells using magnetic activated cell sorting. Sphere formation, cancer stem cell marker expression, migration, invasion, and drug resistance were analyzed. RESULTS: PrPC positive cells showed increased sphere formation capacity and increased expression of cancer stem cell markers compared to PrPC negative cells. In addition, PrPC positive cells showed increased migration, invasion and drug resistance compared to PrPC negative cells. Furthermore, knockdown of PrPC abolished these effects. CONCLUSION: PrPC expression is important in CRC cell behavior, such as sphere formation, migration, invasion, and drug resistance. PrPC is an important therapeutic target for the treatment of CRC.

Quantitative proteomic analysis of trypsin-treated extracellular vesicles to identify the real-vesicular proteins.

Extracellular vesicles (EVs) are nano-sized vesicles surrounded by a lipid bilayer and released into the extracellular milieu by most of cells. Although various EV isolation methods have been established, most of the current methods isolate EVs with contaminated non-vesicular proteins. By applying the label-free quantitative proteomic analyses of human colon cancer cell SW480-derived EVs, we identified trypsin-sensitive and trypsin-resistant vesicular proteins. Further systems biology and protein-protein interaction network analyses based on their cellular localization, we classified the trypsin-sensitive and trypsin-resistant vesicular proteins into two subgroups: 363 candidate real-vesicular proteins and 151 contaminated non-vesicular proteins. Moreover, the protein interaction network analyses showed that candidate real-vesicular proteins are mainly derived from plasma membrane (46.8%), cytosol (36.6%), cytoskeleton (8.0%) and extracellular region (2.5%). On the other hand, most of the contaminated non-vesicular proteins derived from nucleus, Golgi apparatus, endoplasmic reticulum and mitochondria. In addition, ribosomal protein complexes and T-complex proteins were classified as the contaminated non-vesicular proteins. Taken together, our trypsin-digested proteomic approach on EVs is an important advance to identify the real-vesicular proteins that could help to understand EV biogenesis and protein cargo-sorting mechanism during EV release, to identify more reliable EV diagnostic marker proteins, and to decode pathophysiological roles of EVs.

Indoor dust extracellular vesicles promote cancer lung metastasis by inducing tumour necrosis factor-α.

Indoor pollutants are important problems to public health. Among indoor pollutants, indoor dust contains extracellular vesicles (EVs), which are associated with pulmonary inflammation. However, it has not been reported whether indoor dust EVs affect the cancer lung metastasis. In this study, we isolated indoor dust EVs and investigated their roles in cancer lung metastasis. Upon intranasal administration, indoor dust EVs enhanced mouse melanoma lung metastasis in a dose-dependent manner in mice. Pre-treatment or co-treatment of indoor dust EVs significantly promoted melanoma lung metastasis, whereas post-treatment of the EVs did not. In addition, the lung lysates from indoor dust EV-treated mice significantly increased tumour cell migration in vitro. We observed that tumour necrosis factor-α played important roles in indoor dust EV-mediated promotion of tumour cell migration in vitro and cancer lung metastasis in vivo. Furthermore, Pseudomonas EVs, the main components of indoor dust EVs, and indoor dust EVs showed comparable effects in promoting tumour cell migration in vitro and cancer lung metastasis in vivo. Taken together, our results suggest that indoor dust EVs, at least partly contributed by Pseudomonas EVs, are potential promoting agents of cancer lung metastasis.