Transcriptional regulation and small compound targeting of ACE2 in lung epithelial cells.

Angiotensin-converting enzyme 2 (ACE2) is the receptor of COVID-19 pathogen SARS-CoV-2, but the transcription factors (TFs) that regulate the expression of the gene encoding ACE2 (ACE2) have not been systematically dissected. In this study we evaluated TFs that control ACE2 expression, and screened for small molecule compounds that could modulate ACE2 expression to block SARS-CoV-2 from entry into lung epithelial cells. By searching the online datasets we found that 24 TFs might be ACE2 regulators with signal transducer and activator of transcription 3 (Stat3) as the most significant one. In human normal lung tissues, the expression of ACE2 was positively correlated with phosphorylated Stat3 (p-Stat3). We demonstrated that Stat3 bound ACE2 promoter, and controlled its expression in 16HBE cells stimulated with interleukin 6 (IL-6). To screen for medicinal compounds that could modulate ACE2 expression, we conducted luciferase assay using HLF cells transfected with ACE2 promoter-luciferase constructs. Among the 64 compounds tested, 6-O-angeloylplenolin (6-OAP), a sesquiterpene lactone in Chinese medicinal herb Centipeda minima (CM), represented the most potent ACE2 repressor. 6-OAP (2.5 µM) inhibited the interaction between Stat3 protein and ACE2 promoter, thus suppressed ACE2 transcription. 6-OAP (1.25-5 µM) and its parental medicinal herb CM (0.125%-0.5%) dose-dependently downregulated ACE2 in 16HBE and Beas-2B cells; similar results were observed in the lung tissues of mice following administration of 6-OAP or CM for one month. In addition, 6-OAP/CM dose-dependently reduced IL-6 production and downregulated chemokines including CXCL13 and CX3CL1 in 16HBE cells. Moreover, we found that 6-OAP/CM inhibited the entry of SARS-CoV-2 S protein pseudovirus into target cells. These results suggest that 6-OAP/CM are ACE2 inhibitors that may potentially protect lung epithelial cells from SARS-CoV-2 infection.

6-O-angeloylplenolin exerts neuroprotection against lipopolysaccharide-induced neuroinflammation in vitro and in vivo.

Neuroinflammation is one of the critical events in neurodegenerative diseases, whereas microglia play an important role in the pathogenesis of neuroinflammation. In this study, we investigated the effects of a natural sesquiterpene lactone, 6-O-angeloylplenolin (6-OAP), isolated from the traditional Chinese medicine Centipeda minima (L.) A.Br., on neuroinflammation and the underlying mechanisms. We showed that treatment with lipopolysaccharide (LPS) caused activation of BV2 and primary microglial cells and development of neuroinflammation in vitro, evidenced by increased production of inflammatory cytokines TNF-α and IL-1ß, the phosphorylation and nuclear translocation of NF-κB, and the transcriptional upregulation of COX-2 and iNOS, leading to increased production of proinflammatory factors NO and PGE2. Moreover, LPS treatment induced oxidative stress through increasing the expression levels of NOX2 and NOX4. Pretreatment with 6-OAP (0.5-4 µM) dose-dependently attenuated LPS-induced NF-κB activation and oxidative stress, thus suppressed neuroinflammation in the cells. In a mouse model of LPS-induced neuroinflammation, 6-OAP (5-20 mg·kg-1·d-1, ip, for 7 days before LPS injection) dose-dependently inhibited the production of inflammatory cytokines, the activation of the NF-κB signaling pathway, and the expression of inflammatory enzymes in brain tissues. 6-OAP pretreatment significantly ameliorated the activation of microglia and astrocytes in the brains. 6-OAP at a high dose caused a much stronger antineuroinflammatory effect than dexamethansone (DEX). Furthermore, we demonstrated that 6-OAP pretreatment could inhibit LPS-induced neurite and synaptic loss in vitro and in vivo. In conclusion, our results demonstrate that 6-OAP exerts antineuroinflammatory effects and can be considered a novel drug candidate for the treatment of neuroinflammatory diseases.

Emerging Anti-Mitotic Activities and Other Bioactivities of Sesquiterpene Compounds upon Human Cells.

We review the bio-activities of natural product sesquiterpenes and present the first description of their effects upon mitosis. This type of biological effect upon cells is unexpected because sesquiterpenes are believed to inactivate proteins through Michael-type additions that cause non-specific cytotoxicity. Yet, certain types of sesquiterpenes can arrest cells in mitosis as measured by cell biology, biochemical and imaging techniques. We have listed the sesquiterpenes that arrest cells in mitosis and analyzed the biological data that support those observations. In view of the biochemical complexity of mitosis, we propose that a subset of sesquiterpenes have a unique chemical structure that can target a precise protein(s) required for mitosis. Since the process of mitotic arrest precedes that of cell death, it is possible that some sesquiterpenes that are currently classified as cytotoxic might also induce a mitotic arrest. Our analysis provides a new perspective of sesquiterpene chemical biology.

6-O-angeloylplenolin inhibits osteoclastogenesis in vitro via suppressing c-Src/NF-κB/NFATc1 pathways and ameliorates bone resorption in collagen-induced arthritis mouse model.

One of the effective therapeutic strategies to treat rheumatoid arthritis (RA)-related bone resorption is to target excessive activation of osteoclasts. We discovered that 6-O-angeloylplenolin (6-OAP), a pseudoguaianolide from Euphorbia thymifolia Linn widely used for the treatment of RA in traditional Chinese medicine, could inhibit RANKL-induced osteoclastogenesis and bone resorption in both RAW264.7 cells and BMMs from 1 µM and protect a collagen-induced arthritis (CIA) mouse model from bone destruction in vivo. The severity of arthritis and bone erosion observed in paw joints and the femurs of the CIA model were attenuated by 6-OAP administered at both dosages (1 or 5 mg/kg, i.g.). BMD, Tb.N and BV/TV were also improved by 6-OAP treatment. Histological analysis and TRAP staining of femurs further confirmed the protective effects of 6-OAP on bone erosion, which is mainly due to reduced osteoclasts. Molecular docking indicated that c-Src might be a target of 6-OAP and phosphorylation of c-Src was suppressed by 6-OAP treatment. CETSA and SPR assay further confirmed the potential interaction between 6-OAP and c-Src. Three signaling molecules downstream of c-Src that are vital to the differentiation and function of osteoclasts, NF-κB, c-Fos and NFATc1, were also suppressed by 6-OAP in vitro. In summary, the results demonstrated that the function of c-Src was disrupted by 6-OAP, which led to the suppression of downstream signaling vital to osteoclast differentiation and function. In conclusion, 6-OAP has the potential to be further developed for the treatment of RA-related bone erosion.

Centipeda minima and 6-O-angeloylplenolin enhance the efficacy of immune checkpoint inhibitors in non-small cell lung cancer.

BACKGROUND: Chemotherapeutic agents including cisplatin, gemcitabine, and pemetrexed, significantly enhance the efficacy of immune checkpoint inhibitors (ICIs) in non-small cell lung cancer (NSCLC) by increasing PD-L1 expression and potentiating T cell cytotoxicity. However, the low response rate and adverse effects limit the application of chemotherapy/ICI combinations in patients. METHODS: We screened for medicinal herbs that could perturb PD-L1 expression and enhance T cell cytotoxicity in the presence of anti-PD-L1 antibody, and investigated the underlying mechanisms. RESULTS: We found that the aqueous extracts of Centipeda minima (CM) significantly enhanced the cancer cell-killing activity and granzyme B expression level of CD8+ T cells, in the presence of anti-PD-L1 antibody. Both CM and its active component 6-O-angeloylplenolin (6-OAP) upregulated PD-L1 expression by suppressing GSK-3ß-ß-TRCP-mediated ubiquitination and degradation. CM and 6-OAP significantly enhanced ICI-induced reduction of tumor burden and prolongation of overall survival of mice bearing NSCLC cells, accompanied by upregulation of PD-L1 and increase of CD8+ T cell infiltration. CM also exhibited anti-NSCLC activity in cells and in a patient-derived xenograft mouse model. CONCLUSIONS: These data demonstrated that the induced expression of PD-L1 and enhancement of CD8+ T cell cytotoxicity underlay the beneficial effects of 6-OAP-rich CM in NSCLCs, providing a clinically available and safe medicinal herb for combined use with ICIs to treat this deadly disease.

6-O-angeloylplenolin inhibits anti-IgE-stimulated human mast cell activation via suppressing calcium influx and ERK phosphorylation.

Objectives: Mast cells are important immune cells that primarily localize in the interface between the host and external environment, and protect us from pathogen infection. However, they are also involved in the pathology of allergic diseases such as asthma and atopic dermatitis. A novel S phase kinase-associated protein 1 (SKP1) inhibitor 6-O-angeloylplenolin (6-OAP), was studied with its potential ability to alleviate the anti-IgE-induced inflammatory responses of primary human cultured mast cells (HCMCs) and LAD2 cell line. Materials and Methods: We isolated the HCMCs from the buffy coat of voluntary blood donors. The effects of 6-OAP on mast cell activation were evaluated by measuring degranulation, cytokine release, migration, calcium influx, and ERK phosphorylation using spectro-fluorescence assay, multiplex cytometric bead assay/ELISA, migration assay, Fluo-4 calcium flux assay, and western blot, respectively. Results: It was found that 6-OAP exerted anti-inflammatory effects on human mast cells by dose-dependently suppressing the anti-IgE-mediated degranulation and release of cytokines such as proinflammatory cytokines (IL-8 and TNF-α), growth factors (GM-CSF, VEGF, and FGF), and chemokines (CCL2 and CCL3) in HCMC and LAD2 cells. It also suppressed the migration of immature HCMCs induced by CXCL12. Moreover, the process of calcium influx and ERK phosphorylation in activated HCMC cells were inhibited by 6-OAP administration. Conclusion: Our results showed that 6-OAP inhibited anti-IgE-induced inflammatory responses of human mast cells via suppressing calcium influx and ERK phosphorylation.

Promising anticancer activities and mechanisms of action of active compounds from the medicinal herb Centipeda minima (L.) A. Braun & Asch.

BACKGROUND: Centipeda minima (L.) A. Braun & Asch (C. minima) has been used as a traditional Chinese herbal medicine to treat multiple diseases, including sinusitis, rhinitis, headache, and allergy. To date, the anticancer properties of C. minima have drawn considerable attention owing to the anticancer potential of C. minima extracts, the identification of active components, and the elucidation of underlying molecular mechanisms. However, the anticancer properties and significance of active components in C. minima have rarely been summarized. PURPOSE: This review presents a comprehensive summary of the anticancer properties exhibited by active components of C. minima. METHODS: An extensive search for published articles on the anticancer activities and active components of C. minima was performed using Web of Science, PubMed, Science Direct, and Google Scholar. RESULTS: C. minima extracts exhibited both anticancer and chemosensitizing effects. Phytochemical studies have identified the active anticancer components of C. minima extracts. Sesquiterpene lactones, such as 6-O-angeloylplenolin (6-OAP, or brevilin A) and arnicolide D, have similar structures and anticancer mechanisms. As the most abundant sesquiterpene lactone in C. minima, 6-OAP exhibits anticancer activities mainly by targeting Skp1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase and signal transducers and activators of transcription 3 (STAT3). Clinical trials have assessed the potential of 6-OAP in patients with vertex balding and alopecia areata, given its effect on JAK-STATs signaling. Chlorogenic acid, a representative organic acid in C. minima, reportedly possesses anticancer potential and inhibits tumor growth by affecting tumor microenvironment and has been approved for phase II clinical trials in patients with glioma in China. CONCLUSION: In the present review, we highlight intriguing anticancer properties mediated by active compounds isolated from C. minima extracts, particularly sesquiterpene lactones, which might provide clues for developing novel anticancer drugs. Relevant clinical trials on chlorogenic acid and 6-OAP can promote anticancer clinical applications. Therefore, it is worth comprehensively elucidating underlying anticancer mechanisms and conducting clinical trials on C. minima and its active components.

Sesquiterpene lactone 6-O-angeloylplenolin reverses vincristine resistance by inhibiting YB-1 nuclear translocation in colon carcinoma cells.

Multidrug resistance (MDR) is a major obstacle to cancer chemotherapy efficacy. In the present study, 6-O-angeloylplenolin repressed the overexpression of ATP binding cassette subfamily B member 1 (MDR1) and increasing the intracellular concentration of anticancer drugs. A reduction in P-glycoprotein expression (encoded by MDR1) was observed in parallel with a decline in mRNA expression in vincristine-resistant HCT (HCT-8/VCR) cells treated with 6-O-angeloylplenolin. In addition, 6-O-angeloylplenolin suppressed the activity of the MDR1 gene promoter. Treatment with 6-O-angeloylplenolin also decreased the amount of the specific protein complex that interacted with the MDR1 gene promoter in HCT-8/VCR cells, potentially leading to the suppression of MDR1 expression. Treatment with 6-O-angeloylplenolin inhibited the nuclear translocation of Y-box binding protein-1 in HCT-8/VCR cells treated with 6-O-angeloylplenolin, contributing to the negative regulation of MDR1. Finally, 6-O-angeloylplenolin reversed VCR resistance in an HCT/VCR xenograft model. In conclusion, 6-O-angeloylplenolin exhibited a MDR-reversing effect by downregulating MDR1 expression and could represent a novel adjuvant agent for chemotherapy.

Proteomic identification of the oncoprotein STAT3 as a target of a novel Skp1 inhibitor.

The S phase kinase-associated protein 1 (Skp1), an adaptor protein of the Skp1-Cul1-F-box protein complex, binds the ubiquitin E3 ligase Skp2 and is critical to its biological functions. Targeting of Skp1 by a small compound 6-O-angeloylplenolin (6-OAP) results in dissociation and degradation of Skp2 and mitotic arrest of lung cancer cells. Here, by using a proteome microarray containing 16,368 proteins and a biotinylated 6-OAP, we identified 99 proteins that could bind 6-OAP, with Skp1 and STAT3 sitting at the central position of the 6-OAP interactome. 6-OAP formed hydrogen bonds with Ser611/Ser613/Arg609 at the SH2 domain of STAT3 and inhibited the constitutive and interleukin-6-induced phosphorylated STAT3 (pSTAT3), leading to inhibitory effects on lung cancer cells and suppression of Skp2 transcription. STAT3 was overexpressed in tumor samples compared to counterpart normal lung tissues and was inversely associated with prognosis of the patients. 6-OAP inhibited tumor growth in SCID mice intravenously injected with lung cancer cells, and downregulated both STAT3 and Skp2 in tumor samples. Given that 6-OAP is a Skp1 inhibitor, our data suggest that this compound may target Skp1 and STAT3 to suppress Skp2, augmenting its anti-lung cancer activity.

Skp1 in lung cancer: clinical significance and therapeutic efficacy of its small molecule inhibitors.

Skp1 is an essential adaptor protein of the Skp1-Cul1-F-box protein complex and is able to stabilize the conformation of some ubiquitin E3 ligases. However, the role Skp1 plays during tumorigenesis remains unclear and Skp1-targeting agent is lacking. Here we showed that Skp1 was overexpressed in 36/64 (56.3%) of non-small cell lung cancers, and elevated Skp1 was associated with poor prognosis. By structure-based high-throughput virtual screening, we found some Skp1-targeting molecules including a natural compound 6-O-angeloylplenolin (6-OAP). 6-OAP bound Skp1 at sites critical to Skp1-Skp2 interaction, leading to dissociation and proteolysis of oncogenic E3 ligases NIPA, Skp2, and ß-TRCP, and accumulation of their substrates Cyclin B1, P27 and E-Cadherin. 6-OAP induced prometaphase arrest and exerted potent anti-lung cancer activity in two murine models and showed low adverse effect. These results indicate that Skp1 is critical to lung cancer pathogenesis, and Skp1 inhibitor inactivates crucial oncogenic E3 ligases and exhibits significant therapeutic potentials.

Small compound 6-O-angeloylplenolin induces caspase-dependent apoptosis in human multiple myeloma cells.

6-O-angeloylplenolin (6-OAP) is a sesquiterpene lactone agent that has been previously demonstrated to inhibit the growth of multiple myeloma (MM) cells through mitotic arrest with accumulated cyclin B1. In the present study, the levels of apoptosis were analyzed in dexamethasone-sensitive (MM.1S), dexamethasone-resistant (U266) and chemotherapy-sensitive (RPMI 8226) myeloma cell lines. Enhanced apoptosis was identified following a 48-h incubation with 6-OAP (0-10 µM) that induced a dose-dependent decrease in pro-casp-3 and the cleavage of its substrate, anti-poly (ADP-ribose) polymerase (PARP). In addition, time-dependent cleavage of PARP was also detected in U266 and MM.1S cells. The mechanism of 6-OAP cytotoxicity in all cell lines was associated with the induction of apoptosis with the presence of cleaved caspase-3 and PARP. In conclusion, 6-OAP-induced apoptosis is caspase-dependent. These observations are likely to provide a framework for future studies of 6-OAP therapy in MM.