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Cisplatin, which induces DNA damage, is standard chemotherapy for advanced bladder cancer (BCa). However, efficacy is limited due to resistance development. Since artesunate (ART), a derivative of artemisinin originating from Traditional Chinese Medicine, has been shown to exhibit anti-tumor activity, and to inhibit DNA damage repair, the impact of artesunate on cisplatin-resistant BCa was evaluated. Cisplatin-sensitive (parental) and cisplatin-resistant BCa cells, RT4, RT112, T24, and TCCSup, were treated with ART (1–100 µM). Cell growth, proliferation, and cell cycle phases were investigated, as were apoptosis, necrosis, ferroptosis, autophagy, metabolic activity, and protein expression. Exposure to ART induced a time- and dose-dependent significant inhibition of tumor cell growth and proliferation of parental and cisplatin-resistant BCa cells. This inhibition was accompanied by a G0/G1 phase arrest and modulation of cell cycle regulating proteins. ART induced apoptos is by enhancing DNA damage, especially in the resistant cells. ART did not induce ferroptosis, but led to a disturbance of mitochondrial respiration and ATP generation. This impairment correlated with autophagy accompanied by a decrease in LC3B-I and an increase in LC3B-II. Since ART significantly inhibits proliferative and metabolic aspects of cisplatin-sensitive and cisplatin-resistant BCa cells, it may hold potential in treating advanced and therapy-resistant BCa.
Severe acute respiratory syndrome virus 2 (SARS-CoV-2) is the cause of the current coronavirus disease 19 (COVID-19) pandemic. Protease inhibitors are under consideration as virus entry inhibitors that prevent the cleavage of the coronavirus spike (S) protein by cellular proteases. Herein, we showed that the protease inhibitor aprotinin (but not the protease inhibitor SERPINA1/alpha-1 antitrypsin) inhibited SARS-CoV-2 replication in therapeutically achievable concentrations. An analysis of proteomics and translatome data indicated that SARS-CoV-2 replication is associated with a downregulation of host cell protease inhibitors. Hence, aprotinin may compensate for downregulated host cell proteases during later virus replication cycles. Aprotinin displayed anti-SARS-CoV-2 activity in different cell types (Caco2, Calu-3, and primary bronchial epithelial cell air–liquid interface cultures) and against four virus isolates. In conclusion, therapeutic aprotinin concentrations exert anti-SARS-CoV-2 activity. An approved aprotinin aerosol may have potential for the early local control of SARS-CoV-2 replication and the prevention of COVID-19 progression to a severe, systemic disease.
Drug resistance of childhood cancer neuroblastoma is a serious clinical problem. Patients with relapsed disease have a poor prognosis despite intense treatment. In the present study, we aimed to identify chemoresistance gene expression signatures in vincristine resistant neuroblastoma cells. We found that vincristine-resistant neuroblastoma cells formed larger clones and survived under reduced serum conditions as compared with non-resistant parental cells. To identify the possible mechanisms underlying vincristine resistance in neuroblastoma cells, we investigated the expression profiles of genes known to be involved in cancer drug resistance. This specific gene expression patterns could predict the behavior of a tumor in response to chemotherapy and for predicting the prognosis of high-risk neuroblastoma patients. Our signature could help chemoresistant neuroblastoma patients in avoiding useless and harmful chemotherapy cycles.
SARS-CoV-2 is the causative agent of COVID-19. Severe COVID-19 disease has been associated with disseminated intravascular coagulation and thrombosis, but the mechanisms underlying COVID-19-related coagulopathy remain unknown. The risk of severe COVID-19 disease is higher in males than in females and increases with age. To identify gene products that may contribute to COVID-19-related coagulopathy, we analyzed the expression of genes associated with the Gene Ontology (GO) term “blood coagulation” in the Genotype-Tissue Expression (GTEx) database and identified four procoagulants, whose expression is higher in males and increases with age (ADAMTS13, F11, HGFAC, KLKB1), and two anticoagulants, whose expression is higher in females and decreases with age (C1QTNF1, SERPINA5). However, the expression of none of these genes was regulated in a proteomics dataset of SARS-CoV-2-infected cells and none of the proteins have been identified as a binding partner of SARS-CoV-2 proteins. Hence, they may rather generally predispose individuals to thrombosis without directly contributing to COVID-19-related coagulopathy. In contrast, the expression of the procoagulant transferrin (not associated to the GO term “blood coagulation”) was higher in males, increased with age, and was upregulated upon SARS-CoV-2 infection. Hence, transferrin warrants further examination in ongoing clinic-pathological investigations.
Resistance formation after initial therapy response (acquired resistance) is common in high-risk neuroblastoma patients. YM155 is a drug candidate that was introduced as a survivin suppressant. This mechanism was later challenged, and DNA damage induction and Mcl-1 depletion were suggested instead. Here we investigated the efficacy and mechanism of action of YM155 in neuroblastoma cells with acquired drug resistance. The efficacy of YM155 was determined in neuroblastoma cell lines and their sublines with acquired resistance to clinically relevant drugs. Survivin levels, Mcl-1 levels, and DNA damage formation were determined in response to YM155. RNAi-mediated depletion of survivin, Mcl-1, and p53 was performed to investigate their roles during YM155 treatment. Clinical YM155 concentrations affected the viability of drug-resistant neuroblastoma cells through survivin depletion and p53 activation. MDM2 inhibitor-induced p53 activation further enhanced YM155 activity. Loss of p53 function generally affected anti-neuroblastoma approaches targeting survivin. Upregulation of ABCB1 (causes YM155 efflux) and downregulation of SLC35F2 (causes YM155 uptake) mediated YM155-specific resistance. YM155-adapted cells displayed increased ABCB1 levels, decreased SLC35F2 levels, and a p53 mutation. YM155-adapted neuroblastoma cells were also characterized by decreased sensitivity to RNAi-mediated survivin depletion, further confirming survivin as a critical YM155 target in neuroblastoma. In conclusion, YM155 targets survivin in neuroblastoma. Furthermore, survivin is a promising therapeutic target for p53 wild-type neuroblastomas after resistance acquisition (neuroblastomas are rarely p53-mutated), potentially in combination with p53 activators. In addition, we show that the adaptation of cancer cells to molecular-targeted anticancer drugs is an effective strategy to elucidate a drug’s mechanism of action.
The efficacy of cisplatin-based chemotherapy in ovarian cancer is often limited by the development of drug resistance. In most ovarian cancer cells, cisplatin activates extracellular signal-regulated kinase1/2 (ERK1/2) signalling. Phosphoprotein enriched in astrocytes (PEA-15) is a ubiquitously expressed protein, capable of sequestering ERK1/2 in the cytoplasm and inhibiting cell proliferation. This and other functions of PEA-15 are regulated by its phosphorylation status. In this study, the relevance of PEA-15 phosphorylation state for cisplatin sensitivity of ovarian carcinoma cells was examined. The results of MTT-assays indicated that overexpression of PEA-15AA (a non-phosphorylatable variant) sensitised SKOV-3 cells to cisplatin. Phosphomimetic PEA-15DD did not affect cell sensitivity to the drug. While PEA-15DD facilitates nuclear translocation of activated ERK1/2, PEA-15AA acts to sequester the kinase in the cytoplasm as shown by Western blot. Microarray data indicated deregulation of thirteen genes in PEA-15AA-transfected cells compared to non-transfected or PEA-15DD-transfected variants. Data derived from The Cancer Genome Atlas (TCGA) showed that the expression of seven of these genes including EGR1 (early growth response protein 1) and FLNA (filamin A) significantly correlated with the therapy outcome in cisplatin-treated cancer patients. Further analysis indicated the relevance of nuclear factor erythroid 2-related factor 2/antioxidant response element (Nrf2/ARE) signalling for the favourable effect of PEA-15AA on cisplatin sensitivity. The results warrant further evaluation of the PEA-15 phosphorylation status as a potential candidate biomarker of response to cisplatin-based chemotherapy.
Incorporation of doxorubicin in different polymer nanoparticles and their anticancer activity
(2019)
Background: Nanoparticles are under investigation as carrier systems for anticancer drugs. The expression of efflux transporters such as the ATP-binding cassette (ABC) transporter ABCB1 is an important resistance mechanism in therapy-refractory cancer cells. Drug encapsulation into nanoparticles has been shown to bypass efflux-mediated drug resistance, but there are also conflicting results. To investigate whether easy-to-prepare nanoparticles made of well-tolerated polymers may circumvent transporter-mediated drug efflux, we prepared poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), and PEGylated PLGA (PLGA-PEG) nanoparticles loaded with the ABCB1 substrate doxorubicin by solvent displacement and emulsion diffusion approaches and assessed their anticancer efficiency in neuroblastoma cells, including ABCB1-expressing cell lines, in comparison to doxorubicin solution.
Results: The resulting nanoparticles covered a size range between 73 and 246 nm. PLGA-PEG nanoparticle preparation by solvent displacement led to the smallest nanoparticles. In PLGA nanoparticles, the drug load could be optimised using solvent displacement at pH 7 reaching 53 µg doxorubicin/mg nanoparticle. These PLGA nanoparticles displayed sustained doxorubicin release kinetics compared to the more burst-like kinetics of the other preparations. In neuroblastoma cells, doxorubicin-loaded PLGA-PEG nanoparticles (presumably due to their small size) and PLGA nanoparticles prepared by solvent displacement at pH 7 (presumably due to their high drug load and superior drug release kinetics) exerted the strongest anticancer effects. However, nanoparticle-encapsulated doxorubicin did not display increased efficacy in ABCB1-expressing cells relative to doxorubicin solution.
Conclusion: Doxorubicin-loaded nanoparticles made by different methods from different materials displayed substantial discrepancies in their anticancer activity at the cellular level. Optimised preparation methods resulted in PLGA nanoparticles characterised by increased drug load, controlled drug release, and high anticancer efficacy. The design of drug-loaded nanoparticles with optimised anticancer activity at the cellular level is an important step in the development of improved nanoparticle preparations for anticancer therapy. Further research is required to understand under which circumstances nanoparticles can be used to overcome efflux-mediated resistance in cancer cells.
Incorporation of doxorubicin in different polymer nanoparticles and their anti-cancer activity
(2018)
Nanoparticles are under investigation as carrier systems for anti-cancer drugs. They have been shown to accumulate in cancer tissues through the enhanced permeability and retention (EPR) effect, to reduce toxicity to non-target tissues, and to protect drugs from preliminary inactivation. However, nanoparticle preparations are not commonly compared for their anti-cancer effects at the cellular level. Here, we prepared doxorubicin-loaded nanoparticles based on poly(lactic-co-glycolic acid) (PLGA), polylactic acid (PLA), and PEGylated PLGA (PLGA-PEG) by solvent displacement and emulsion diffusion approaches. The resulting nanoparticles covered a size range between 73 and 246 nm. PLGA-PEG nanoparticle preparation by solvent displacement resulted in the smallest nanoparticles. In PLGA nanoparticles, the drug load could be optimised using solvent displacement at pH7 reaching 53 µg doxorubicin/mg nanoparticle. In addition, these PLGA nanoparticles displayed sustained doxorubicin release kinetics compared to the more burst-like kinetics of the other preparations. In neuroblastoma cells, doxorubicin-loaded PLGA-PEG nanoparticles (presumably due to their small size) and PLGA nanoparticles prepared by solvent displacement at pH7 (presumably due to their high drug load and superior drug release kinetics) exerted the strongest anti-cancer effects. In conclusion, doxorubicin-loaded nanoparticles made by different methods from different materials displayed substantial discrepancies in their anti-cancer activity at the cellular level. Optimised preparation methods resulted in PLGA nanoparticles characterised by increased drug load, controlled drug release, and high anti-cancer efficacy. The design of drug-loaded nanoparticles with optimised anti-cancer activity at the cellular level is an important step in the development of improved nanoparticle preparations for anti-cancer therapy.
Omeprazole was shown to improve the anti-cancer effects of the nucleoside analogue 5-fluorouracil. Here, we combined omeprazole with the antiviral nucleoside analogues ribavirin and acyclovir. Omeprazole did not affect the antiviral effects of ribavirin in non-toxic concentrations up to 80 μg/mL but increased the acyclovir-mediated effects on herpes simplex virus 1 and 2 (HSV-1 and -2) replication in a dose-dependent manner. Omeprazole alone reduced HSV-1 and -2 titers [but not HSV-induced formation of cytopathogenic effects (CPE)] at concentrations ≥40 μg/mL. However, it exerted substantially stronger effects on acyclovir activity and also increased acyclovir activity at lower concentrations that did not directly interfere with HSV replication. Omeprazole 80 μg/mL caused a 10.8-fold (Vero cells) and 47.7-fold (HaCaT cells) decrease of the acyclovir concentrations that reduced HSV-1-induced CPE formation by 50% (IC50). In HSV-2-infected cells, omeprazole 80 μg/mL reduced the acyclovir IC50 by 7.3- (Vero cells) and 12.9-fold (HaCaT cells). In HaCaT cells, omeprazole 80 μg/mL reduced the HSV-1 titer in the presence of acyclovir 1 μg/mL by 1.6 × 105-fold and the HSV-2 titer in the presence of acyclovir 2 μg/mL by 9.2 × 103-fold. The proton pump inhibitors pantoprazole, rabeprazole, lansoprazole, and dexlansoprazole increased the antiviral effects of acyclovir in a similar fashion as omeprazole, indicating this to be a drug class effect. In conclusion, proton pump inhibitors increase the anti-HSV activity of acyclovir and are candidates for antiviral therapies in combination with acyclovir, in particular for topical preparations for the treatment of immunocompromised individuals who are more likely to suffer from severe complications.
The nucleoside analogue nelarabine, the prodrug of arabinosylguanine (AraG), is effective against T-cell acute lymphoblastic leukaemia (T-ALL) but not against B-cell ALL (B-ALL). The underlying mechanisms have remained elusive. Here, data from pharmacogenomics studies and a panel of ALL cell lines reveal an inverse correlation between nelarabine sensitivity and the expression of SAMHD1, which can hydrolyse and inactivate triphosphorylated nucleoside analogues. Lower SAMHD1 abundance is detected in T-ALL than in B-ALL in cell lines and patient-derived leukaemic blasts. Mechanistically, T-ALL cells display increased SAMHD1 promoter methylation without increased global DNA methylation. SAMHD1 depletion sensitises B-ALL cells to AraG, while ectopic SAMHD1 expression in SAMHD1-null T-ALL cells induces AraG resistance. SAMHD1 has a larger impact on nelarabine/AraG than on cytarabine in ALL cells. Opposite effects are observed in acute myeloid leukaemia cells, indicating entity-specific differences. In conclusion, SAMHD1 promoter methylation and, in turn, SAMHD1 expression levels determine ALL cell response to nelarabine.