Overcoming bortezomib resistance in human B cells by anti-CD20/rituximab-mediated complement-dependent cytotoxicity and epoxyketone-based irreversible proteasome inhibitors

UNLABELLED: BACKGROUND: In clinical and experimental settings, antibody-based anti-CD20/rituximab and small molecule proteasome inhibitor (PI) bortezomib (BTZ) treatment proved effective modalities for B cell depletion in lymphoproliferative disorders as well as autoimmune diseases. However, the chronic nature of these diseases requires either prolonged or re-treatment, often with acquired resistance as a consequence. METHODS: Here we studied the molecular basis of acquired resistance to BTZ in JY human B lymphoblastic cells following prolonged exposure to this drug and examined possibilities to overcome resistance by next generation PIs and anti-CD20/rituximab-mediated complement-dependent cytotoxicity (CDC). RESULTS: Characterization of BTZ-resistant JY/BTZ cells compared to parental JY/WT cells revealed the following features: (a) 10-12 fold resistance to BTZ associated with the acquisition of a mutation in the PSMB5 gene (encoding the constitutive $β$5 proteasome subunit) introducing an amino acid substitution (Met45Ile) in the BTZ-binding pocket, (b) a significant 2-4 fold increase in the mRNA and protein levels of the constitutive $β$5 proteasome subunit along with unaltered immunoproteasome expression, (c) full sensitivity to the irreversible epoxyketone-based PIs carfilzomib and (to a lesser extent) the immunoproteasome inhibitor ONX 0914. Finally, in association with impaired ubiquitination and attenuated breakdown of CD20, JY/BTZ cells harbored a net 3-fold increase in CD20 cell surface expression, which was functionally implicated in conferring a significantly increased anti-CD20/rituximab-mediated CDC. CONCLUSIONS: These results demonstrate that acquired resistance to BTZ in B cells can be overcome by next generation PIs and by anti-CD20/rituximab-induced CDC, thereby paving the way for salvage therapy in BTZ-resistant disease

Reversal of Tetracycline Resistance by Cepharanthine, Cinchonidine, Ellagic Acid and Propyl Gallate in a Multi-drug Resistant Escherichia coli

Bacterial resistance to antibiotics is an increasing threat to global healthcare systems. We therefore sought compounds with potential to reverse antibiotic resistance in a clinically relevant multi-drug resistant isolate of Escherichia coli (NCTC 13400). 200 natural compounds with a history of either safe oral use in man, or as a component of a traditional herb or medicine, were screened. Four compounds; ellagic acid, propyl gallate, cinchonidine and cepharanthine, lowered the minimum inhibitory concentrations (MICs) of tetracycline, chloramphenicol and tobramycin by up to fourfold, and when combined up to eightfold. These compounds had no impact on the MICs of ampicillin, erythromycin or trimethoprim. Mechanistic studies revealed that while cepharanthine potently suppressed efflux of the marker Nile red from bacterial cells, the other hit compounds slowed cellular accumulation of this marker, and/or slowed bacterial growth in the absence of antibiotic. Although cepharanthine showed some toxicity in a cultured HEK-293 mammalian cell-line model, the other hit compounds exhibited no toxicity at concentrations where they are active against E. coli NCTC 13400. The results suggest that phytochemicals with capacity to reverse antibiotic resistance may be more common in traditional medicines than previously appreciated, and may offer useful scaffolds for the development of antibiotic-sensitising drugs

Fourth mRNA COVID-19 vaccination in immunocompromised patients with haematological malignancies (COBRA KAI): a cohort study

Background Patients with haematological malignancies have impaired antibody responses to SARS-CoV-2 vaccination. We aimed to investigate whether a fourth mRNA COVID-19 vaccination improved antibody quantity and quality. Methods In this cohort study, conducted at 5 sites in the Netherlands, we compared antibody concentrations 28 days after 4 mRNA vaccinations (3-dose primary series plus 1 booster vaccination) in SARS-CoV-2 naive, immunocompromised patients with haematological malignancies to those obtained by age-matched, healthy individuals who had received the standard primary 2-dose mRNA vaccination schedule followed by a first booster mRNA vaccination. Prior to and 4 weeks after each vaccination, peripheral blood samples and data on demographic parameters and medical history were collected. Concentrations of antibodies that bind spike 1 (S1) and nucleocapsid (N) protein of SARS-CoV-2 were quantified in binding antibody units (BAU) per mL according to the WHO International Standard for COVID-19 serological tests. Seroconversion was defined as an S1 IgG concentration > 10 BAU/mL and a previous SARS-CoV-2 infection as N IgG > 14.3 BAU/mL. Antibody neutralising activity was tested using lentiviral-based pseudoviruses expressing spike protein of SARS-CoV-2 wildtype (D614G), Omicron BA.1, and Omicron BA.4/5 variants. This study is registered with EudraCT, number 2021-001072-41. Findings Between March 24, 2021 and May 4, 2021, 723 patients with haematological diseases were enrolled, of which 414 fulfilled the inclusion criteria for the current analysis. Although S1 IgG concentrations in patients significantly improved after the fourth dose, they remained significantly lower compared to those obtained by 58 age-matched healthy individuals after their first booster (third) vaccination. The rise in neutralising antibody concentration was most prominent in patients with a recovering B cell compartment, although potent responses were also observed in patients with persistent immunodeficiencies. 19% of patients never seroconverted, despite 4 vaccinations. Patients who received their first 2 vaccinations when they were B cell depleted and the third and fourth vaccination during B cell recovery demonstrated similar antibody induction dynamics as patients with normal B cell numbers during the first 2 vaccinations. However, the neutralising capacity of these antibodies was significantly better than that of patients with normal B cell numbers after two vaccinations. Interpretation A fourth mRNA COVID-19 vaccination improved S1 IgG concentrations in the majority of patients with a haematological malignancy. Vaccination during B cell depletion may pave the way for better quality of antibody responses after B cell reconstitution

Anticancer potential of Thevetia peruviana fruit methanolic extract

Abstract Background: Thevetia peruviana (Pers.) K. Schum or Cascabela peruviana (L.) Lippold (commonly known as ayoyote, codo de fraile, lucky nut, or yellow oleander), native to Mexico and Central America, is a medicinal plant used traditionally to cure diseases like ulcers, scabies, hemorrhoids and dissolve tumors. The purpose of this study was to evaluate the cytotoxic, antiproliferative and apoptotic activity of methanolic extract of T. peruviana fruits on human cancer cell lines. Methods: The cytotoxic activity of T. peruviana methanolic extract was carried out on human breast, colorectal, prostate and lung cancer cell lines and non-tumorigenic control cells (fibroblast and Vero), using the MTT assay. For proliferation and motility, clonogenic and wound-healing assays were performed. Morphological alterations were monitored by trypan blue exclusion, as well as DNA fragmentation and AO/EB double staining was performed to evaluate apoptosis. The extract was separated using flash chromatography, and the resulting fractions were evaluated on colorectal cancer cells for their cytotoxic activity. The active fractions were further analyzed through mass spectrometry. Results: The T. peruviana methanolic extract exhibited cytotoxic activity on four human cancer cell lines: prostate, breast, colorectal and lung, with values of IC50 1.91 ± 0.76, 5.78 ± 2.12, 6.30 ± 4.45 and 12.04 ± 3.43 μg/mL, respectively. The extract caused a significant reduction of cell motility and colony formation on all evaluated cancer cell lines. In addition, morphological examination displayed cell size reduction, membrane blebbing and detachment of cells, compared to non-treated cancer cell lines. The T. peruviana extract induced apoptotic cell death, which was confirmed by DNA fragmentation and AO/EB double staining. Fractions 4 and 5 showed the most effective cytotoxic activity and their MS analysis revealed the presence of the secondary metabolites: thevetiaflavone and cardiac glycosides. Conclusion: T. peruviana extract has potential as natural anti-cancer product with critical effects in the proliferation, motility, and adhesion of human breast and colorectal cancer cells, and apoptosis induction in human prostate and lung cancer cell lines, with minimal effects on non-tumorigenic cell lines. Keywords: Cytotoxic activity, Anti-proliferative activity, Motility, Apoptosis, Human cancer cells, Flavonoid, Cardiac glycoside

Inhibitors of trypanosoma cruzi Sir2 related protein 1 as potential drugs against Chagas disease.

Chagas disease remains one of the most neglected diseases in the world despite being the most important parasitic disease in Latin America. The characteristic chronic manifestation of chagasic cardiomyopathy is the region's leading cause of heart-related illness, causing significant mortality and morbidity. Due to the limited available therapeutic options, new drugs are urgently needed to control the disease. Sirtuins, also called Silent information regulator 2 (Sir2) proteins have long been suggested as interesting targets to treat different diseases, including parasitic infections. Recent studies on Trypanosoma cruzi sirtuins have hinted at the possibility to exploit these enzymes as a possible drug targets. In the present work, the T. cruzi Sir2 related protein 1 (TcSir2rp1) is genetically validated as a drug target and biochemically characterized for its NAD+-dependent deacetylase activity and its inhibition by the classic sirtuin inhibitor nicotinamide, as well as by bisnaphthalimidopropyl (BNIP) derivatives, a class of parasite sirtuin inhibitors. BNIPs ability to inhibit TcSir2rp1, and anti-parasitic activity against T. cruzi amastigotes in vitro were investigated. The compound BNIP Spermidine (BNIPSpd) (9), was found to be the most potent inhibitor of TcSir2rp1. Moreover, this compound showed altered trypanocidal activity against TcSir2rp1 overexpressing epimastigotes and anti-parasitic activity similar to the reference drug benznidazole against the medically important amastigotes, while having the highest selectivity index amongst the compounds tested. Unfortunately, BNIPSpd failed to treat a mouse model of Chagas disease, possibly due to its pharmacokinetic profile. Medicinal chemistry modifications of the compound, as well as alternative formulations may improve activity and pharmacokinetics in the future. Additionally, an initial TcSIR2rp1 model in complex with p53 peptide substrate was obtained from low resolution X-ray data (3.5 Å) to gain insight into the potential specificity of the interaction with the BNIP compounds. In conclusion, the search for TcSir2rp1 specific inhibitors may represent a valuable strategy for drug discovery against T. cruzi

Reactive oxygen species are involved in BMP-induced dendritic growth in cultured rat sympathetic neurons

Previous studies have shown that bone morphogenetic proteins (BMPs) promote dendritic growth in sympathetic neurons; however, the downstream signaling molecules that mediate the dendrite promoting activity of BMPs are not well characterized. Here we test the hypothesis that reactive oxygen species (ROS)-mediated signaling links BMP receptor activation to dendritic growth. In cultured rat sympathetic neurons, exposure to any of the three mechanistically distinct antioxidants, diphenylene iodinium (DPI), nordihydroguaiaretic acid (NGA) or desferroxamine (DFO), blocked de novo BMP-induced dendritic growth. Addition of DPI to cultures previously induced with BMP to extend dendrites caused dendritic retraction while DFO and NGA prevented further growth of dendrites. The inhibition of the dendrite promoting activity of BMPs by antioxidants was concentration-dependent and occurred without altering axonal growth or neuronal cell survival. Antioxidant treatment did not block BMP activation of SMAD 1,5 as determined by nuclear localization of these SMADs. While BMP treatment did not cause a detectable increase in intracellular ROS in cultured sympathetic neurons as assessed using fluorescent indicator dyes, BMP treatment increased the oxygen consumption rate in cultured sympathetic neurons as determined using the Seahorse XF24 Analyzer, suggesting increased mitochondrial activity. In addition, BMPs upregulated expression of NADPH oxidase 2 (NOX2) and either pharmacological inhibition or siRNA knockdown of NOX2 significantly decreased BMP-7 induced dendritic growth. Collectively, these data support the hypothesis that ROS are involved in the downstream signaling events that mediate BMP7-induced dendritic growth in sympathetic neurons, and suggest that ROS-mediated signaling positively modulates dendritic complexity in peripheral neurons