Blocking the terminal complement pathway with the C5 inhibitor eculizumab has revolutionized the clinical management of several complement-mediated diseases and has boosted the clinical development of new inhibitors. Data on the C3 inhibitor Compstatin and the C5 inhibitors eculizumab and Coversin reported here demonstrate that C3/C5 convertases function differently from prevailing concepts. Stoichiometric C3 inhibition failed to inhibit C5 activation and lytic activity during strong classical pathway activation, demonstrating a “C3 bypass” activation of C5. We show that, instead of C3b, surface-deposited C4b alone can also recruit and prime C5 for consecutive proteolytic activation. Surface-bound C3b and C4b possess similar affinities for C5. By demonstrating that the fluid phase convertase C3bBb is sufficient to cleave C5 as long as C5 is bound on C3b/C4b-decorated surfaces, we show that surface fixation is necessary only for the C3b/C4b opsonins that prime C5 but not for the catalytic convertase unit C3bBb. Of note, at very high C3b densities, we observed membrane attack complex formation in absence of C5-activating enzymes. This is explained by a conformational activation in which C5 adopts a C5b-like conformation when bound to densely C3b-opsonized surfaces. Stoichiometric C5 inhibitors failed to prevent conformational C5 activation, which explains the clinical phenomenon of residual C5 activity documented for different inhibitors of C5. The new insights into the mechanism of C3/C5 convertases provided here have important implications for the development and therapeutic use of complement inhibitors as well as the interpretation of former clinical and preclinical data.

Ranibizumab reverses proliferation and cell migration stimulated by VEGF and delocalisation of tight junction proteins induced by VEGF(165) in iBREC.

No abstract

Pentacyclic triterpenic acids from oleogum resins of Boswellia species are of considerable therapeutic interest. Yet, their pharmaceutical development is hampered by uncertainties regarding botanical identification and the complexity of triterpenic acid mixtures. Here, a highly sensitive, selective, and accurate method for the simultaneous quantification of eight boswellic and lupeolic acids by high-performance liquid chromatography with tandem mass spectrometry detection (HPLC-MS/MS) was developed. The method was applied to the comparative analysis of 41 oleogum resins of the species B. sacra, B. dalzielli, B. papyrifera, B. serrata, B. carterii, B. neglecta, B. rivae, B. frereana, and B. occulta. Multivariate statistical analysis of the data revealed differences in the triterpenic acid composition that could be assigned to distinct Boswellia species and to their geographic growth location. Extracts of the oleogum resins exhibited cytotoxicity against the human, treatment-resistant, metastatic breast cancer cell line MDA-MB-231. Extracts from B. sacra were the most potent ones with an average IC50 of 8.3 ± 0.6 µg/mL. The oleogum resin of the B. sacra was further fractionated to enrich different groups of substances. The cytotoxic efficacy against the cancer cells correlates positively with the contents of pentacyclic triterpenic acids in Boswellia extracts.

For centuries, frankincense extracts have been commonly used in traditional medicine, and more recently, in complementary medicine. Therefore, frankincense constituents such as boswellic and lupeolic acids are of considerable therapeutic interest. Sixteen frankincense nutraceuticals were characterized by high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS), revealing major differences in boswellic and lupeolic acid compositions and total contents, which varied from 0.4% to 35.7%. Frankincense nutraceuticals significantly inhibited the release of proinflammatory cytokines, such as TNF-α, IL-6, and IL-8, by LPS-stimulated peripheral blood mononuclear cells (PBMC) and whole blood. Moreover, boswellic and lupeolic acid contents correlated with TNF-α, IL-1β, IL-6, IL-8, and IL-10 inhibition. The nutraceuticals also exhibited toxicity against the human triple-negative breast cancer cell lines MDA-MB-231, MDA-MB-453, and CAL-51 in vitro. Nutraceuticals with total contents of boswellic and lupeolic acids >30% were the most active ones against MDA-MB-231 with a half maximal inhibitory concentration (IC50) ≤ 7.0 µg/mL. Moreover, a frankincense nutraceutical inhibited tumor growth and induced apoptosis in vivo in breast cancer xenografts grown on the chick chorioallantoic membrane (CAM). Among eight different boswellic and lupeolic acids tested, β-ABA exhibited the highest cytotoxicity against MDA-MB-231 with an IC50 = 5.9 µM, inhibited growth of cancer xenografts in vivo, and released proinflammatory cytokines. Its content in nutraceuticals correlated strongly with TNF-α, IL-6, and IL-8 release inhibition.

Boswellic acids, and particularly 11-keto-boswellic acids, triterpenoids derived from the genus Boswellia (Burseraceae), are known for their anti-inflammatory and potential antitumor efficacy. Although boswellic acids generally occur as α-isomers (oleanane type) and β-isomers (ursane type), 11-keto-boswellic acid (KBA) was found only as the β-isomer, β-KBA. Here, the existence and natural occurrence of the respective α-isomer, 11-keto-α-boswellic acid (α-KBA), is demonstrated for the first time. Initially, α-KBA was synthesized and characterized by high-resolution mass spectrometry (HR-MS) and nuclear magnetic resonance (NMR) spectroscopy, and a highly selective, sensitive, and accurate high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS) method was developed by Design of Experiments (DoE) using a pentafluorophenyl stationary phase. This method allowed the selective quantification of individual 11-keto-boswellic acids and provided evidence for α-KBA in Boswellia spp. oleogum resins. The contents of α-KBA as well as further boswellic acids and the composition of essential oils were used to chemotaxonomically classify 41 Boswellia oleogum resins from 9 different species. Moreover, α-KBA exhibited cytotoxicity against three treatment-resistant triple-negative breast cancer (TNBC) cell lines in vitro and also induced apoptosis in MDA-MB-231 xenografts in vivo. The respective β-isomer and the acetylated form demonstrate higher cytotoxic efficacies against TNBC cells. This provides further insights into the structure-activity relationship of boswellic acids and could support future developments of potential anti-inflammatory and antitumor drugs.

We investigated the cytotoxic potential of the cardenolide glycoside acovenoside A against non-small-cell lung cancer cells. Lung cancer is the leading cause of cancer-related mortality and the second most common cancer diagnosed. Epidemiological studies revealed a direct correlation between the regular administration of cardiac glycosides and a lower incidence of various cancers. Acovenoside A, isolated from the pericarps of Acokanthera oppositifolia, potently inhibited proliferation and induced cytotoxicity in A549 non-small-cell lung cancer cells with an IC of 68 ± 3 nM after 48 h of exposure. Compared to the antineoplastic agent doxorubicin, acovenoside A was more potent in inhibiting the viability of A549 cancer cells. Moreover, acovenoside A exhibited selectivity against cancer cells, being significantly less toxic to lung fibroblasts and nontoxic for peripheral blood mononuclear cells. Analysis of the cell cycle profile in acovenoside A-treated A549 cells revealed mitotic arrest, due to accumulation of the G/M regulators cyclin B and CDK1, and cytokinesis failure. Furthermore, acovenoside A affected the mitochondrial membrane integrity and induced production of radical oxygen species, which resulted in induction of canonical apoptosis, manifested by caspase 3 activation and DNA fragmentation. Based on our results, acovenoside A warrants further exploration as a potential anticancer lead.

Triple negative human breast cancer (TNBC) is an aggressive cancer subtype with poor prognosis. Besides the better-known artemisinin, Artemisia annua L. contains numerous active compounds not well-studied yet. High-performance liquid chromatography coupled with diode-array and mass spectrometric detection (HPLC-DAD-MS) was used for the analysis of the most abundant compounds of an Artemisia annua extract exhibiting toxicity to MDA-MB-231 TNBC cells. Artemisinin, 6,7-dimethoxycoumarin, arteannuic acid were not toxic to any of the cancer cell lines tested. The flavonols chrysosplenol d and casticin selectively inhibited the viability of the TNBC cell lines, MDA-MB-231, CAL-51, CAL-148, as well as MCF7, A549, MIA PaCa-2, and PC-3. PC-3 prostate cancer cells exhibiting high basal protein kinase B (AKT) and no ERK1/2 activation were relatively resistant, whereas MDA-MB-231 cells with high basal ERK1/2 and low AKT activity were more sensitive to chrysosplenol d treatment. In vivo, chrysosplenol d and casticin inhibited MDA-MB-231 tumor growth on chick chorioallantoic membranes. Both compounds induced mitochondrial membrane potential loss and apoptosis. Chrysosplenol d activated ERK1/2, but not other kinases tested, increased cytosolic reactive oxygen species (ROS) and induced autophagy in MDA-MB-231 cells. Lysosomal aberrations and toxicity could be antagonized by ERK1/2 inhibition. The Artemisia annua flavonols chrysosplenol d and casticin merit exploration as potential anticancer therapeutics.