Hemodialysis Removes Uremic Toxins That Alter the Biological Actions of Endothelial Cells

Chronic kidney disease is linked to systemic inflammation and to an increased risk of ischemic heart disease and atherosclerosis. Endothelial dysfunction associates with hypertension and vascular disease in the presence of chronic kidney disease but the mechanisms that regulate the activation of the endothelium at the early stages of the disease, before systemic inflammation is established remain obscure. In the present study we investigated the effect of serum derived from patients with chronic kidney disease either before or after hemodialysis on the activation of human endothelial cells in vitro, as an attempt to define the overall effect of uremic toxins at the early stages of endothelial dysfunction. Our results argue that uremic toxins alter the biological actions of endothelial cells and the remodelling of the extracellular matrix before signs of systemic inflammatory responses are observed. This study further elucidates the early events of endothelial dysfunction during toxic uremia conditions allowing more complete understanding of the molecular events as well as their sequence during progressive renal failure

Study of proteasome activity in myelodysplastic syndromes and investigation of proteasome inhibitors resistance mechanism

MDS is a group of clonal hematopoietic cell disorders with an increased risk of transformation into acute myelogenous leukemia. Proteasome inhibitors such as Bortezomib are a targeted therapy for various types of hematological malignancies, by blocking the ubiquitin-proteasome system crucial for cancer cells. The ineffectiveness of proteasome inhibitors in MDS is currently being investigated, although it is not clear whether they rapidly develop drug resistance or whether proteasome proteolytic activity is intrinsically lower in these cells, thus limiting the therapeutic effects of the drug. In this dissertation, patients with MDS were recruited and grouped according to their IPSS score into lower- or higher-risk groups and the proteasomal activity and levels of reactive oxygen species in blood and bone marrow mononuclear cells were determined. In parallel, in order to study the development of resistance to proteasomal inhibitors in a solid tumor model and to compare the two conditions, a resistant cell line based on the DU-145 prostate line was generated. Resistance-related cellular functions were studied with emphasis on signaling, reactive oxygen species levels and cell death. The results of the study demonstrated a close link between proteasomal activity, oxidative stress management and induction of autophagy as an alternative proteolytic mechanism.Τα ΜΔΣ είναι μια ομάδα κλωνικών αιμοποιητικών διαταραχών με αυξημένο κίνδυνο μετασχηματισμού σε οξεία μυελογενή λευχαιμία. Οι αναστολείς πρωτεασώματος όπως η Βορτεζομίμπη αποτελούν στοχευμένη θεραπεία για διάφορους τύπους αιματολογικών κακοηθειών, στοχεύοντας το καθοριστικής σημασίας για τα καρκινικά κύτταρα σύστημα ουβικιτίνης-πρωτεασώματος. Η αναποτελεσματικότητα των αναστολέων πρωτεασώματος στα ΜΔΣ διερευνάται επί του παρόντος, αν και δεν είναι σαφές εάν αναπτύσσουν γρήγορα αντίσταση στο φάρμακο ή εάν η πρωτεολυτική δραστηριότητα του πρωτεασώματος είναι ιδιοσυστατικά χαμηλότερη σε αυτά τα κύτταρα, περιορίζοντας έτσι τα θεραπευτικά αποτελέσματα του φαρμάκου. Στην παρούσα διατριβή, ασθενείς με ΜΔΣ στρατολογήθηκαν και ομαδοποιήθηκαν σύμφωνα με τη βαθμολογία IPSS τους σε ομάδες χαμηλότερου ή υψηλότερου κινδύνου και προσδιορίστηκε η πρωτεασωματική δραστηριότητα και τα επίπεδα δραστικών μορφών οξυγόνου στα μονοπύρηνα κύτταρα αίματος και μυελού των οστών. Παράλληλα, με σκοπό να μελετηθεί η ανάπτυξη ανθεκτικότητας στους πρωτεασωματικούς αναστολείς, σε μοντέλο συμπαγούς όγκου, και να συγκριθούν οι δύο καταστάσεις, δημιουργήθηκε ανθεκτική κυτταρική σειρά βασισμένη στην προστατική σειρά DU-145. Μελετήθηκαν οι σχετικές με την ανθεκτικότητα κυτταρικές λειτουργίες με έμφαση στη σηματοδότηση, τα επίπεδα δραστικών μορφών οξυγόνου και τον κυτταρικό θάνατο. Τα αποτελέσματα της μελέτης κατέδειξαν μια στενή σύνδεση μεταξύ της πρωτεασωματικής δραστικότητας, της διαχείρισης της οξειδωτικής καταπόνησης και της επαγωγής της αυτοφαγίας ως εναλλακτικό πρωτεολυτικό μηχανισμό

Autophagy and oxidative stress modulation mediate Bortezomib resistance in prostate cancer.

Proteasome inhibitors such as Bortezomib represent an established type of targeted treatment for several types of hematological malignancies, including multiple myeloma, Waldenstrom's macroglobulinemia, and mantle cell lymphoma, based on the cancer cell's susceptibility to impairment of the proteasome-ubiquitin system. However, a major problem limiting their efficacy is the emergence of resistance. Their application to solid tumors is currently being studied, while simultaneously, a wide spectrum of hematological cancers, such as Myelodysplastic Syndromes show minimal or no response to Bortezomib treatment. In this study, we utilize the prostate cancer cell line DU-145 to establish a model of Bortezomib resistance, studying the underlying mechanisms. Evaluating the resulting resistant cell line, we observed restoration of proteasome chymotrypsin-like activity, regardless of drug presence, an induction of pro-survival pathways, and the substitution of the Ubiquitin-Proteasome System role in proteostasis by induction of autophagy. Finally, an estimation of the oxidative condition of the cells indicated that the resistant clones reduce the generation of reactive oxygen species induced by Bortezomib to levels even lower than those induced in non-resistant cells. Our findings highlight the role of autophagy and oxidative stress regulation in Bortezomib resistance and elucidate key proteins of signaling pathways as potential pharmaceutical targets, which could increase the efficiency of proteasome-targeting therapies, thus expanding the group of molecular targets for neoplastic disorders

Apoptosis and cell cycle analysis of DU-145 RB60U Cells.

(A, B) The DU-145 RB60U cell clone maintains the apoptosis evasion observed on the DU-145 RB60 cells after a 24-week drug deprivation with a slight increase of its apoptotic rate. (C, D) Following the same procedures as in naïve and DU-145 RB60 cells, the cell cycle of DU-145 RB60U cells was analyzed using PI and indicated a mild G2 arrest following incubation with 60 nM of Bortezomib for the first time after 24 weeks. The subsequent cell cycle inhibition does not result in apoptosis as has been shown by the rest experiments assessing apoptosis compared to the naïve DU-145 that within 48–72 h undergo apoptosis. (TIF)</p

Autophagy and oxidative stress assays using western blots, flow cytometry and confocal microscopy.

(A) Western blot analyses of key proteins regulating autophagy (LC3, Beclin-1, p62) and stress markers (Hsp70 and phosphor-p38). The experiments were conducted after 24 h of Bortezomib incubation following a 48 h Bortezomib deprivation of RB60 cells, as previously noted. The + corresponds to the low dose of 20 nM Bortezomib, and the ++ corresponds to the medium dose of 60 nM. (B) Staining of naïve DU-145 cells with Lysotracker RED, which stains acidic proteins, following treatment with 20 nM Bortezomib. The cells were cultured on coverslips and stained (without fixation) with Lysotracker RED for 15 min at 37°C, followed by confocal imaging. (C) Flow cytometry analysis of Lysotracker RED inside naïve and resistant live cells. The cells were trypsinized and subsequently stained with Lysotracker RED for 45 min followed by analysis using a FACS Calibur flow cytometer. (D) Flow cytometry analysis of ROS generation using H2DCFDA. The cells were incubated for 24 h with Bortezomib and then trypsinized. During the staining procedure, they were maintained inside the culture medium to avoid heat shock and starvation stress. Staining was performed at 37°C and the samples were rinsed with PBS and analyzed using a FACS Calibur flow cytometer.</p

S1 Raw images -

Proteasome inhibitors such as Bortezomib represent an established type of targeted treatment for several types of hematological malignancies, including multiple myeloma, Waldenstrom’s macroglobulinemia, and mantle cell lymphoma, based on the cancer cell’s susceptibility to impairment of the proteasome-ubiquitin system. However, a major problem limiting their efficacy is the emergence of resistance. Their application to solid tumors is currently being studied, while simultaneously, a wide spectrum of hematological cancers, such as Myelodysplastic Syndromes show minimal or no response to Bortezomib treatment. In this study, we utilize the prostate cancer cell line DU-145 to establish a model of Bortezomib resistance, studying the underlying mechanisms. Evaluating the resulting resistant cell line, we observed restoration of proteasome chymotrypsin-like activity, regardless of drug presence, an induction of pro-survival pathways, and the substitution of the Ubiquitin-Proteasome System role in proteostasis by induction of autophagy. Finally, an estimation of the oxidative condition of the cells indicated that the resistant clones reduce the generation of reactive oxygen species induced by Bortezomib to levels even lower than those induced in non-resistant cells. Our findings highlight the role of autophagy and oxidative stress regulation in Bortezomib resistance and elucidate key proteins of signaling pathways as potential pharmaceutical targets, which could increase the efficiency of proteasome-targeting therapies, thus expanding the group of molecular targets for neoplastic disorders.</div

Chemotactic assay of DU-145 naive and DU-145 RB60 cells using Boyden chambers.

Cells were transferred into a chamber containing serum-free medium with or without Bortezomib. The chambers were placed inside microplates’ wells containing medium supplemented with 20% FBS and left to migrate for 24 h. The DU-145 cells, when exposed to Bortezomib (20 nM), decreased their migration rate. Inhibition of migration was also observed when Bortezomib was added to the lower compartment, indicating a chemorepellent role. The DU-145 RB60 cells were also repelled by Bortezomib (60 nM), while the presence of the drug in the upper compartment induced migration towards the other side of the membrane, where Bortezomib was absent. (TIF)</p

Main cell function assays of naïve DU-145, DU-145 RB60, and DU-145 RB60U cells.

(A, B) Equal numbers of cells were seeded on 24-well plates, and after 24 h of attachment, various doses of Bortezomib (A) or Carfilzomib (B) were added. Following 72 h of incubation, the cells were fixed and subsequently stained with crystal violet. The proliferation rate of naïve DU-145, DU-145 RB60 resistant cells after 24 weeks of drug presence, DU-145 RB60U cells after 24 weeks of drug absence, and DU-145 RB60 cells after 48 weeks of drug presence (60 nM) was assessed by a spectrophotometrical determination of the crystal violet solution’s O.D. at 595 nm. The analysis was performed using the built-in tools provided with the GraphPad Prism 8 software. (C) Cells were transferred into a chamber containing serum-free medium with or without Bortezomib. The chambers were placed inside microplates’ wells containing medium supplemented with 20% FBS and left to migrate for 24 h. The cells crossing the porous filter were then fixed and stained, and after photographing, they were counted using the Cell Counter tool by ImageJ. (D) Cells were seeded on 6-well plates and left to form monolayers. After reaching the desired confluency, wounds were scratched, and the Bortezomib-free media were replaced with medium containing 10% FBS and Bortezomib. The naïve cells were assessed using 20 nM of Bortezomib, and the DU-145 RB60 cells were assessed under the influence of 60 nM Bortezomib. After replacement of the media, photographs were taken, and the wound closure rate was determined by capturing images at the specific time-points of 0, 24, 48, and 72 h. The photographs were analyzed using the ImageJ Manual Wound Healing Size tool and the subsequent analysis was performed using Microsoft Office Excel and GraphPad Prism 8.</p

List of primary antibodies used for western blot analyses.

List of primary antibodies used for western blot analyses.</p

Ubiquitin-Proteasome system assessment in naïve DU-145, DU-145 RB60U, and DU-145 RB60 resistant cells.

We examined the function of the UPS with Western blots of polyubiquitinated proteins. (A) An initial dose-response experiment was conducted on all three clones (naïve DU-145, DU-145 RB60U, and DU-145 RB60) following 24 h of incubation with Bortezomib. (B) Time-course experiments verified the stable ubiquitination levels at the key intervals of 24, 36, and 48 h following incubation with 20 nM Bortezomib, validating the 24-hour time point as an adequate time point to study effects on main signaling pathways. (C) Further dose-response experiments verified the susceptibility of DU-145 RB60 cells to Bortezomib at concentrations greater than 180 nM. (D) PSMB5 is the most ubiquitous proteasome subunit exhibiting chymotrypsin-like (ChT-L) activity. Following treatment with Bortezomib, we used Western blot analysis to estimate the PSMB5 protein levels. (E) The results were quantified using ImageJ, and to compare the groups, two-tailed t-tests were performed in GraphPad Prism 8. Between naïve and resistant cells, statistically significant differences were documented, regarding PSMB5 accumulation (PF) We estimated the ChT-L activity of naïve DU-145 and DU-145 RB60 cells using fluorometry. The resistant cells exhibited almost 3-fold increased proteasome activity compared to the naïve clone (PA-E) were performed in triplicate, and wherever bar charts are shown; the bars represent the means, and the error bars are the SEM.</p