Small Natural Compound Research Articles

Hypocrellin A against intrahepatic Cholangiocarcinoma via multi-target inhibition of the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways

BackgroundIntrahepatic cholangiocarcinoma (ICC) is an aggressive and highly lethal cancer with an increasing incidence worldwide that lacks effective treatment regimens. Hypocrellin A (HA), a natural small compound isolated from S. bambusicola, has multiple biomedical activities, including antitumor activity. PurposeWe intended to investigate the therapeutic effects of HA on ICC and its potential mechanisms. MethodsRBE and HuccT1 cell lines were utilized for in vitro experiments. CCK8 assay, colony formation analysis, RTCA, and immunofluorescence staining of ki67 were employed to evaluate the suppression effects of HA on proliferation. The inhibitory effects of HA on cell migration and invasion were evaluate through transwell and wound healing assays, and Hoechst 33,258 staining was performed to evaluate apoptosis. Additionally, we performed transcriptome sequencing and molecular docking for targeting identification, and immunoblotting and immunofluorescence of key molecules for validation. Two in vivo models, HuccT1 xenografts, and the primary ICC model (KRAS/P19/SB) established via hydrodynamic tail-vein injection were implemented. Multiplex immunohistochemistry (mIHC) was used to illustrate the multi-target inhibitory effects of HA. ResultsThe IC50 values of HA against RBE and HuccT1 cells were 4.612 μM and 10.01 μM for 24 h, as determined through the CCK8 assay. Our results confirmed that HA significantly repressed the proliferation, migration, invasion, and promoted the apoptosis of ICC cells at low concentrations. Moreover, HA exerted its anti-cancer effects through multi-target inhibition of the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways. This inhibitory effect was rescued by Recilisib, an activator of the PI3K-AKT-mTOR pathway. Bioinformatics analysis of a multi-center RNA-Seq cohort (n = 90) demonstrated significant associations between these target pathways and the occurrence and poor prognosis of ICC. Animal studies suggested that HA strongly inhibited tumor growth in xenograft ICC models, and repressed the tumor number and size in the liver of primary ICC models by suppressing these three crucial pathways. ConclusionHA, a novel natural small molecule, demonstrated promising therapeutic efficacy against ICC through its multi-target inhibitory effects on the PI3K-AKT-mTOR, MAPK, and STAT3 signaling pathways. Moreover, it exhibited notable therapeutic benefits in a primary ICC model (KRAS/P19/SB), positioning it as a novel therapeutic agent for ICC.

Abstract 1345: NAV-201, an orally bioavailable small molecule NK cell activator enhancing antibody-mediated cancer therapies

Abstract Tumors employ a number of mechanisms to suppress a patient’s immune anti-tumor responses. Recently, we uncovered an immunosuppression mechanism that tumors employ to avoid humoral immune responses that involves tumor-produced proteins, called Humoral Immuno-Oncology (HIO) factors. These HIO factors are able to suppress antibody-mediated tumor cell killing via direct binding to antibodies that alter their Fc domain interactions with the C1q protein to suppress complement dependent cytotoxicity (CDC) and reduce antibody engagement with CD16a Fc receptors on immune-effector cells, including NK cells, to suppress antibody dependent cellular cytotoxicity (ADCC). HIO factor immunosuppression of ADCC appears to involve the reduced binding affinity of antibody to CD16a Fc receptors. In an attempt to identify agents that may enhance antibody-CD16a binding affinity to overcome HIO factor immunosuppression, we sought to identify agents that can restore this interaction by screening natural small compound libraries via an IgG1-CD16a based ELISA assay. One lead compound was identified that was subsequently optimized by structural activity relationship analysis. This compound, named NAV-201, specifically binds CD16a and enhances the affinity of antibody binding to both the low affinity CD16a-158F and high affinity CD16a-158V allotypes. Moreover, we found that NAV-201 can also bind to the mouse CD16a ortholog and enhance its antibody binding affinity, making NAV-201 suitable for efficacy testing in mouse models. In vivo, NAV-201 was found to be well tolerated up to at least 120 mg/kg when administered orally and daily to mice, have a serum half-life of 18 hours, enhance systemic levels of the NK cell produced INF-γ and TNF-α cytokines, and enhance the therapeutic efficacy of antibodies in mouse xenografts. Together, these data support the further development of NAV-201 for human testing to improve and/or enhance the efficacy of antibody-based therapies in HIO factor positive cancers. Citation Format: Nicholas C. Nicolaides, Luigi Grasso, J. Brad Kline. NAV-201, an orally bioavailable small molecule NK cell activator enhancing antibody-mediated cancer therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1345.

Melatonin facts: Lack of evidence that melatonin is a radical scavenger in living systems.

Melatonin is a small natural compound, so called a neuro-hormone that is synthesized mainly in pineal gland in animals. Its main role is to master the clock of the body, under the surveillance of light. In other words, it transfers the information concerning night and day to the peripheral organs which, without it, could not "know" which part of the circadian rhythm the body is in. Besides its main circadian and circannual rhythms mastering, melatonin is reported to be a radical scavenger and/or an antioxidant. Because radical scavengers are chemical species able to neutralize highly reactive and toxic species such as reactive oxygen species, one would like to transfer this property to living system, despite impossibilities already largely reported in the literature. In the present commentary, we refresh the memory of the readers with this notion of radical scavenger, and review the possible evidence that melatonin could be an in vivo radical scavenger, while we only marginally discuss here the fact that melatonin is a molecular antioxidant, a feature that merits a review on its own. We conclude four things: (i) the evidence that melatonin is a scavenger in acellular systems is overwhelming and could not be doubted; (ii) the transposition of this property in living (animal) systems is (a) theoretically impossible and (b) not proven in any system reported in the literature where most of the time, the delay of the action of melatonin is over several hours, thus signing a probable induction of cellular enzymatic antioxidant defenses; (iii) this last fact needs a confirmation through the discovery of a nuclear factor-a key relay in induction processes-that binds melatonin and is activated by it and (iv) we also gather the very important description of the radical scavenging capacity of melatonin in acellular systems that is now proven and shared by many other double bond-bearing molecules. We finally discussed briefly on the reason-scientific or else-that led this description, and the consequences of this claim, in research, in physiology, in pathology, but most disturbingly in therapeutics where a vast amount of money, hope, and patient bien-être are at stake.

Upregulation of WDR6 drives hepatic de novo lipogenesis in insulin resistance in mice.

Under normal conditions, insulin promotes hepatic de novo lipogenesis (DNL). However, during insulin resistance (IR), when insulin signalling is blunted and accompanied by hyperinsulinaemia, the promotion of hepatic DNL continues unabated and hepatic steatosis increases. Here, we show that WD40 repeat-containing protein 6 (WDR6) promotes hepatic DNL during IR. Mechanistically, WDR6 interacts with the beta-type catalytic subunit of serine/threonine-protein phosphatase 1 (PPP1CB) to facilitate PPP1CB dephosphorylation at Thr316, which subsequently enhances fatty acid synthases transcription through DNA-dependent protein kinase and upstream stimulatory factor 1. Using molecular dynamics simulation analysis, we find a small natural compound, XLIX, that inhibits the interaction of WDR6 with PPP1CB, thus reducing DNL in IR states. Together, these results reveal WDR6 as a promising target for the treatment of hepatic steatosis.

Cheminformatics and machine learning approaches for repurposing anti-viral compounds against monkeypox virus thymidylate kinase.

One of the emerging epidemic concerns is Monkeypox disease which is spreading globally. This disease is caused by the monkeypox virus (MPXV), with an increasing global incidence with an outbreak in 2022. One of the novel targets for monkeypox disease is thymidylate kinase, which is involved in pyrimidine metabolism. In this study, docking-based virtual screening and molecular dynamics techniques were employed in addition to the machine learning (ML) model to investigate the potential anti-viral natural small compounds to inhibit thymidylate kinase of MPXV. Several potential hits were identified through high-throughput virtual screening, and further top three candidates were selected, which ranked using the ML model. These three compounds were then examined under molecular dynamics simulation and MM/GBSA-binding free energy analysis. Among these, Chlorhexidine HCl showed high potential for binding to the thymidylate kinase with stable and consistent conformation with RMSD < 0.3nm. The MM/GBSA analysis also showed the minimum binding free energy (ΔGTOTAL) of -62.41kcal/mol for this compound. Overall, this study used structure-based drug design complemented by machine learning-guided ligand-based drug design to screen potential hit compounds from the anti-viral natural compound database.

Celastrol impairs tumor growth by modulating the CIP2A-GSK3β-MCL-1 axis in gastric cancer cells.

High Cancerous Inhibitor of PP2A (CIP2A) expression has been reported in solid and hematologic malignancies and is inversely associated with prognosis in Gastric Cancer, the non-small cell lung cancer, et al. CIP2A can be a drug target for the development of novel anti-gastric cancer agent. Our study was designed to explore the anti-cancer effect of celastrol, a small natural compound, and whether it has an anti-proliferative effect through inducing CIP2A degradation against gastric cancer cells. Employing human gastric cancer cells AGS and BCG-823 cells, the effects of celastrol on cell proliferation, apoptosis and cell cycle was specifically investigated via Annexin V-FITC/PI staining and CCK8 assay. The functional association between celastrol and CIP2A was evaluated by using CIP2A knockdown and overexpression technique. The mechanism of underlying celastrol-triggering anti-gastric cancer effect was detected by real-time PCR and western blot analysis. Celastrol concentration- and time-dependently induced CIP2A degradation and led to gastric cancer cell apoptosis. More in depth studies revealed specific activation of Protein phosphatase 2A (PP2A)-GSK3β-MCL-1 signaling pathway was involved in pro-apoptosis effect of celastrol, due to celastrol-triggering degradation of CIP2A, which mainly suppressed PP2A activity. Our findings highlight that celastrol has therapeutic potential via inducing apoptosis of gastric cancer cells.

Diosgenin inhibits prostate cancer progression by inducing UHRF1 protein degradation

Prostate cancer (PCa) represents the second cause of cancer death in adult men. Aberrant overexpression of UHRF1 has been reported in several cancer types, and is regarded as a novel drug target for cancer therapy. Nevertheless, no UHRF1-targeted small molecule inhibitor has been testing in clinical trials. Traditional Chinese medicine (TCM) prescriptions have a long history for the treatment of PCa in China, and Chinese herbal extracts are important resources for new drug discovery. In the present study, we first screened the potentially effective components from the commonly used TCMs for PCa treatment in clinic by using network pharmacology together with molecular docking. We identified diosgenin (DSG) as a small molecule natural compound specifically targeting UHRF1 protein. Furthermore, we validated the results by using the wet lab experiments. DSG, by directly binding UHRF1 protein, induced UHRF1 protein degradation through the ubiquitin-proteasome pathway. Importantly, DSG induced UHRF1 protein degradation by reducing the protein interaction with a deubiquitinase USP7. DSG reduced the level of genomic DNA methylation, and elevated the expression of such tumor suppressor genes as p21, p16 and LXN, thereby resulting in cell cycle arrest, cellular senescence and the inhibition of xenograft tumor growth. We here presented the first report that DSG specifically induced UHRF1 protein degradation, thereby revealing a novel anticancer mechanism of DSG. Altogether, this present study provided a promising strategy to discover new molecule-targeted drugs from small-molecule natural products.

Cirsilineol Treatment Attenuates PM2.5-Induced Lung Injury in Mice.

Ultrafine particulate matter with less than 2.5 μm diameter (PM2.5) is an air pollutant that causes severe lung damage. Currently, effective treatment and preventive methods for PM2.5-induced lung damage are limited. Cirsilineol (CSL) is a small natural compound isolated from Artemisia vestita. In this study, the efficacy of CSL on PM2.5-induced lung toxicity was tested, and its mechanism was identified. Lung injury was caused by intratracheal administration of PM2.5 suspension in animal models. Two days after PM2.5 pretreatment, CSL was injected via mouse tail vein for two days. The effects of CSL on PM2.5-induced lung damage, autophagy, apoptosis, and pulmonary inflammation in a mouse model and their mechanisms were investigated. CSL significantly suppressed histological lung damage and lung wet/dry weight proportion. CSL also significantly reduced PM2.5-induced autophagy dysfunction, apoptosis, lymphocyte suppression, and inflammatory cytokine levels in bronchoalveolar fluid (BALF). Furthermore, CSL increased mammalian target of rapamycin (mTOR) phosphorylation and significantly inhibited the expression of Toll-like receptors (TLR) 2 and 4, MyD88, and the autophagy proteins, Beclin1 and LC3II. Thus, CSL exerts protective effects on pulmonary damage by regulating mTOR and TLR2,4-myD88 autophagy pathways. Therefore, CSL can be used as an effective treatment for PM2.5-induced lung damage.

Isoprocurcumenol Supports Keratinocyte Growth and Survival through Epidermal Growth Factor Receptor Activation.

Although proliferation of keratinocytes, a major type of skin cells, is a key factor in maintaining the function of skin, their ability to proliferate tends to diminish with age. To solve such a problem, researchers in medical and skin cosmetic fields have tried to utilize epidermal growth factor (EGF), but achieved limited success. Therefore, a small natural compound that can mimic the activity of EGF is highly desired in both medical and cosmetic fields. Here, using the modified biosensor system, we observed that natural small-compound isoprocurcumenol, which is a terpenoid molecule derived from turmeric, can activate EGFR signaling. It increased the phosphorylation of ERK and AKT, and upregulated the expression of genes related to cell growth and proliferation, such as c-myc, c-jun, c-fos, and egr-1. In addition, isoprocurcumenol induced the proliferation of keratinocytes in both physical and UVB-induced cellular damage, indicative of its function in skin regeneration. These findings reveal that EGF-like isoprocurcumenol promotes the proliferation of keratinocytes and further suggest its potential as an ingredient for medical and cosmetics use.

Small molecule natural compound targets the NF-κB signaling and ameliorates the development of osteoarthritis.

Osteoarthritis (OA) is a multifactorial and chronic disease describing the destruction of cartilage that can lead to defects in the elderly. There is currently no practical strategy that can reverse the OA process. Here, we describe nepetin, a small natural compound with extracellular matrix (ECM) and inflammation regulating functions. In this study, we investigated the therapeutic effects of nepetin on interleukin-1β (IL-1β)-induced inflammation in mice chondrocyte and OA model. In chondrocytes, treatment with nepetin inhibited the overexpression of pro-inflammatory cytokines and mediators induced by IL-1β. Moreover, pretreatment or posttreatment with nepetin also reduced the ECM catabolism and enhanced the ECM anabolism. Mechanistically, nepetin suppressed NF-κB signaling pathway in IL-1β stimulated chondrocyte. Meanwhile, our molecular docking studies indicated nepetin had a powerful binding capacity to p65. Furthermore, nepetin showed a protective and therapeutic effect on the mouse OA model. To sum up, this study indicated nepetin had a new potential therapeutic option in OA.

Tripartite Motif-Containing 44 is Involved in the Tumorigenesis of Laryngeal Squamous Cell Carcinoma, and its Expression is Downregulated by Nuciferine.

Tripartite motif-containing 44 (TRIM44) was reported to be involved in the tumorigenesis of several tumors, but its function in laryngeal squamous cell carcinoma has not been investigated yet. In the present study, we aimed to elucidate the function of TRIM44 in laryngeal squamous cell carcinoma, and identify the compounds which could inhibit TRIM44 expression. Our results showed that TRIM44 was upregulated in tumor tissues and cell lines of laryngeal squamous cell carcinoma. Knockdown of TRIM44 significantly inhibited cell growth of laryngeal squamous cell carcinoma by suppressing TLR4, phosphorylated AKT and phosphorylated NF-κB p65 expression in vitro. Moreover, TRIM44 knockdown inhibited tumor growth in nude mice, which further suggested that TRIM44 exerted oncogenic activity in laryngeal squamous cell carcinoma. Interestingly, it was found that nuciferine significantly inhibited the mRNA levels of TRIM44 after screening a small natural compound library. Our further studies showed nuciferine markedly downregulated the protein levels of TRIM44 and its substrate TLR4 in a concentration-dependent manner in laryngeal squamous cell carcinoma cells. Moreover, the activation of downstream kinases of TLR4 such as AKT signaling pathway was also inhibited by nuciferine. Additionally, nuciferine markedly inhibited cell survival of laryngeal squamous cell carcinoma in a concentration-dependent manner. In contrast, TRIM44 overexpression significantly reduced the cytotoxicity of nuciferine in laryngeal squamous cell carcinoma cells. In conclusion, this study indicated that inhibiting TRIM44 would be a useful strategy for the treatment of laryngeal squamous cell carcinoma, and nuciferine could be a potential chemical applicated in the therapy of laryngeal squamous cell carcinoma.

Polydatin Alleviates Septic Myocardial Injury by Promoting SIRT6-Mediated Autophagy.

Sepsis is a life-threatening condition. Polydatin (PD), a small natural compound from Polygonum cuspidatum, possesses antioxidant and anti-inflammatory properties. However, the protective mechanism of PD on sepsis-induced acute myocardial damage is still unclear. The aim of this study was to investigate the effect and mechanism of action of PD on lipopolysaccharide (LPS)-induced H9c2 cells and in a rat model of sepsis, and explored the role of PD-upregulated sirtuin (SIRT)6. LPS-induced H9c2 cells were used to simulate sepsis. Cecal ligation and puncture (CLP)-induced sepsis in rats were used to verify the protective effect of PD. ELISA, western blotting, immunofluorescence, immunohistochemistry, and flow cytometry were used to study the protective mechanism of PD against septic myocardial injury. PD pretreatment suppressed LPS-induced H9c2 cell apoptosis by promotion of SIRT6-mediated autophagy. Downregulation of SIRT6 or inhibition of autophagy reversed the protective effect of PD on LPS-induced apoptosis. PD pretreatment also suppressed LPS-induced inflammatory factor expression. CLP-induced sepsis in rats showed that PD pretreatment decreased CLP-induced myocardial apoptosis and serum tumor necrosis factor-α, interleukin (IL)-1β, and IL-6 expression. 3-Methyladenine (autophagy inhibitor) pretreatment prevented the protective effect of PD on septic cardiomyopathy. SIRT6 expression was increased with PD treatment, which confirmed that PD attenuates septic cardiomyopathy by promotion of SIRT6-mediated autophagy. All these results indicate that PD has potential therapeutic effects that alleviate septic myocardial injury by promotion of SIRT6-mediated autophagy.

Abstract P3-10-19: Precise targeting of POLR2A for treating triple negative breast cancer with Chr17p loss

Abstract Triple-negative breast cancer (TNBC) is a molecularly diverse and clinically heterogeneous disease. The challenges for developing novel treatment approaches for TNBC are the paucity of actionable targets, lack of targeted therapies, and poor prognosis of patients. Breast cancer genomics revealed that heterozygous deletion of chromosome 17p (Chr17p) is the most prevalent (53%) event in TNBC. Within the 17p deletion region is the tumor suppressor TP53 (encoding p53), whose deletion or mutation has been long known as a primary tumorigenic driver. However, it remains unclear whether the deletion event, which often includes as many as 200 genes, impacts tumorigenesis beyond TP53 loss alone. While p53 aberrancy promotes proliferation, metabolism and metastatic potential of the TNBC cells, we found that heterozygous deletion of Chr17p is tightly correlated with poor cytotoxicity of tumor infiltrating lymphocytes (TILs) and poor clinical outcomes in patients with TNBC. This result suggests that selective advantage due to Chr17p loss reflects the combined impact of TP53 loss and the reduced dosage of co-deleted genes. Our recent study also identified POLR2A in the TP53-neighboring region as a collateral vulnerability target in the TNBC tumors with Chr17p loss, suggesting that inhibition of POLR2A may be a precision therapy approach for TNBC. As a proof-of-concept, we designed pH-activated nanoparticles (termed as nanobomb) for delivery of POLR2A siRNA, which enhances bioavailability and improves endo/lysosomal escape of the siRNA. In this study (Nature Nanotechnology, 2019)1, we demonstrated that suppressing POLR2A expression with the siRNA-laden nanoparticles led to marked growth reduction of TNBC tumors with 17p loss. Despite recent success of the first FDA-approved RNAi-based therapy for polyneuropathy, the issues of RNAi-based drugs, such as non-specific and incomplete knockdown, can make RNAi unpredictable, slow, and risky, in particular in drug discovery, where speed and reliability of results are crucial factors. To accelerate the translational development of our important finding, we will use α-amanitin, a natural small compound isolated from Amanita phalloides, to specifically inhibit POLR2A (Kd ∼10−9 M). However, free form of α-amanitin causes liver toxicity via the interaction with the hepatocyte-specific OATP1B3 transporter, limiting its clinical applications. To overcome the drug toxicity of α-amanitin, we have been developing α-amanitin-based antibody-drug conjugates (ADC). This approach inhibits the specific uptake of α-amanitin into hepatocytes and increases tumor-specific targeting using tumor-specific monoclonal antibodies. This type of ADC showed significant efficacy in inhibiting the growth of colorectal tumors with heterozygous deletion of TP53 and POLR2A (Nature, 2015)2. Here, we propose that heterozygous deletion of Chr17p not only contributes to TNBC tumorigenesis, but also confers therapeutic vulnerabilities, which can be utilized to develop novel targeted cancer therapy. Refernces 1. Xu,J. et al. Precise targeting of POLR2A as a therapeutic strategy for human triple negative breast cancer. Nat. Nanotechnol. 14, 388-397 (2019). 2. Liu,Y. et al. TP53 loss creates therapeutic vulnerability in colorectal cancer. Nature 520, 697-701 (2015). Citation Format: Xiongbin Lu, Yujing Li, Yifan Sun, Kevin Van der Jeught, Michael Kulke, Pahl Andreas, Xinna Zhang, Xiaoming He, Chi Zhang. Precise targeting of POLR2A for treating triple negative breast cancer with Chr17p loss [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-10-19.

Dual therapeutic strategy targeting tumor cells and tumor microenvironment in triple-negative breast cancer

Objective: Triple-negative breast cancer (TNBC) is characterized by a lack of estrogen receptors (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2/neu). Only 30% of TNBC patients show a pathologic complete response, and the other 70% of patients exhibit a less pronounced response followed by relapse and metastasis to distant organs after neoadjuvant chemotherapy. Achievements of immunotherapy targeting programmed cell death 1 ligand 1 (PD-L1) in clinical trials for treating melanoma, nonsmall-cell lung cancer, renal cell carcinoma, and TNBC suggest that targeting the interaction of tumor cells with tumor microenvironment is highly beneficial for cancer treatment. Finding a novel dual-targeting therapy against tumor cells and the tumor microenvironment (TME) may provide options for improved responses in TNBC patients. Data Sources: We searched the potential targeted therapy candidates that regulate tumor cells as well as the TME of cancer diseases, including TNBC, based on our previous and recent other publications. Study Selection: We selected the potential targeted therapies supported by relevance clinical data, in vitro and in vivo studies. Results: In this review, we found the KDM5B, Cadherin 11, β-catenin, CDK2, signal peptide CUB-EGF domain-containing protein 2, and PDL1 regulate the tumor cells and TME of TNBC cells. In addition, we also highlighted the Antrocin, Ovatodiolide, and Pterostilbene as natural small compound possess anti-cancer through the disruption of tumor cell–TME interactions. Conclusion: The new therapy approach targeting tumor cells-TME interaction may improve the response and survival rate of TNBC patients. Later, natural small compounds could provide alternative therapy options for TNBC patients.

The effect of honokiol on pulmonary artery endothelium cell autophagy mediated by cyclophilin A in hypoxic pulmonary arterial hypertension

Abnormal autophagy plays critical roles in the structure and function of the pulmonary vasculature. Cyclophilin A (CyPA) can be secreted from cells in response to hypoxia and oxidative stress, which are involved in inducing autophagy and regulating the function of endothelial cells in pulmonary arterial hypertension. Honokiol is a small molecule natural compound; it has many bioactivities, such as antitumor, anti-inflammatory, antioxidant and antiangiogenic properties, but how honokiol mediates autophagy in pulmonary arterial hypertension is unclear. Rat' lungs gavaged with honokiol were examined for autophagy via western blot and fluorescence microscopy. In addition, western blot, quantitative RT-PCR and immunofluorescence were employed to test the expression of CyPA and autophagy markers in pulmonary artery endothelial cells (PAECs). Small interfering RNA targeting CyPA (si-CyPA) was used to knockdown the expression of CyPA, and then autophagy was tested with mRFP-GFP-LC3 fluorescence microscopy and western blot. We found that honokiol could reduce the expression of CyPA and autophagy markers in vivo and in vitro. Furthermore, autophagy was also down-regulated by si-CyPA. Taken together, we revealed a novel mechanism by which honokiol regulates autophagy. The results revealed that honokiol can alleviate autophagy and pulmonary arterial hypertension regulated by CyPA in PAECs.

Small molecule natural compound agonist of SIRT3 as a therapeutic target for the treatment of intervertebral disc degeneration

Oxidative stress-induced mitochondrial dysfunction is implicated in the pathogenesis of intervertebral disc degeneration (IVDD). Sirtuin 3 (SIRT3), a sirtuin family protein located in mitochondria, is essential for mitochondrial homeostasis; however, the role of SIRT3 in the process of IVDD has remained elusive. Here, we explored the expression of SIRT3 in IVDD in vivo and in vitro; we also explored the role of SIRT3 in senescence, apoptosis, and mitochondrial homeostasis under oxidative stress. We subsequently activated SIRT3 using honokiol to evaluate its therapeutic potential for IVDD. We assessed SIRT3 expression in degenerative nucleus pulposus (NP) tissues and oxidative stress-induced nucleus pulposus cells (NPCs). SIRT3 was knocked down by lentivirus and activated by honokiol to determine its role in oxidative stress-induced NPCs. The mechanism by which honokiol affected SIRT3 regulation was investigated in vitro, and the therapeutic potential of honokiol was assessed in vitro and in vivo. We found that the expression of SIRT3 decreased with IVDD, and SIRT3 knockdown reduced the tolerance of NPCs to oxidative stress. Honokiol (10 μM) improved the viability of NPCs under oxidative stress and promoted their properties of anti-oxidation, mitochondrial dynamics and mitophagy in a SIRT3-dependent manner. Furthermore, honokiol activated SIRT3 through the AMPK-PGC-1α signaling pathway. Moreover, honokiol treatment ameliorated IVDD in rats. Our study indicated that SIRT3 is involved in IVDD and showed the potential of the SIRT3 agonist honokiol for the treatment of IVDD.

Uncovering the action mechanism of polydatin via network pharmacological target prediction.

Polydatin (PD), a small natural compound originally extracted from Polygonum cuspidatum, exerts distinct biological functions in a variety of diseases. However, the action mechanism of PD has yet to be systematically explored. In this study, we firstly corroborated the druggability of PD by evaluating the medicinal properties of PD using a TCMSP server. We then conducted in silico target-prediction for PD using PharmMapper and ChemMapper, which led to the identification of 15 potential targets overlapping in both approaches. These 15 targets were subsequently evaluated by GeneMANIA, GO biological process and KEGG pathway analysis, which finally contribute to the construction of a drug-target-pathway network for PD. The network analysis revealed that these targets were mainly associated with cancer, cell growth and apoptosis, hormones and other physiological processes, outlining the pharmacological influences of PD on multiple integrated pathways involved in a particular network.

Harmine promotes molar root development via SMAD1/5/8 phosphorylation

Tooth and bone are major tissues involved in physiological calcification in the body, and they use similar molecular pathways for development, homeostasis, and regeneration. Harmine (HMN) is a natural small compound that stimulates osteoblast differentiation in vitro and in vivo. Here we examined the biological effect of HMN on the postnatal development of molar tooth roots and periodontal tissues. HMN supported the formation of tooth roots and periodontal tissues in developing tooth germs. In tooth germ organ culture, HMN promoted the elongation of Hertwig's epithelial root sheath (HERS) and stimulated cell proliferation in HERS and dental follicle-derived tissues, including dental papillae and dental follicles. HMN stimulated cell proliferation and cell movement of HERS-derived cells without mesenchymal cells in vitro and directly induced the phosphorylation of SMAD1/5/8 protein in HERS-derived cells. Our results indicated that HMN was the first natural small compound to stimulate postnatal development of tooth germs.

Chalcone Attenuates Staphylococcus aureus Virulence by Targeting Sortase A and Alpha-Hemolysin.

Staphylococcus aureus (S.aureus) resistance, considered a dilemma for the clinical treatment of this bacterial infection, is becoming increasingly intractable. Novel anti-virulence strategies will undoubtedly provide a path forward in combating these resistant bacterial infections. Sortase A (SrtA), an enzyme responsible for anchoring virulence-related surface proteins, and alpha-hemolysin (Hla), a pore-forming cytotoxin, have aroused great scientific interest, as they have been regarded as targets for promising agents against S. aureus infection. In this study, we discovered that chalcone, a natural small compound with little anti-S. aureus activity, could significantly inhibit SrtA activity with an IC50 of 53.15 μM and Hla hemolysis activity with an IC50 of 17.63 μM using a fluorescence resonance energy transfer (FRET) assay and a hemolysis assay, respectively. In addition, chalcone was proven to reduce protein A (SpA) display in intact bacteria, binding to fibronectin, formation of biofilm and S. aureus invasion. Chalcone could down-regulate the transcriptional levels of the hla gene and the agrA gene, thus leading to a reduction in the expression of Hla and significant protection against Hla-mediated A549 cell injury; more importantly, chalcone could also reduce mortality in infected mice. Additionally, molecular dynamics simulations and mutagenesis assays were used to identify the mechanism of chalcone against SrtA, which implied that the inhibitory activity lies in the bond between chalcone and SrtA residues Val168, Ile182, and Arg197. Taken together, the in vivo and in vitro experiments suggest that chalcone is a potential novel therapeutic compound for S. aureus infection via targeting SrtA and Hla.

Honokiol Induces Apoptosis, G1 Arrest, and Autophagy in KRAS Mutant Lung Cancer Cells.

Aberrant signaling transduction induced by mutant KRAS proteins occurs in 20∼30% of non-small cell lung cancer (NSCLC), however, a direct and effective pharmacological inhibitor targeting KRAS has not yet reached the clinic to date. Honokiol, a small molecular polyphenol natural biophenolic compound derived from the bark of magnolia trees, exerts anticancer activity, however, its mechanism remains unknown. In this study, we sought to investigate the in vitro effects of honokiol on NSCLC cell lines harboring KRAS mutations. Honokiol was shown to induce G1 arrest and apoptosis to inhibit the growth of KRAS mutant lung cancer cells, which was weakened by an autophagy inhibitor 3-methyladenine (3-MA), suggesting a pro-apoptotic role of honokiol-induced autophagy that was dependent on AMPK-mTOR signaling pathway. In addition, we also discovered that Sirt3 was significantly up-regulated in honokiol treated KRAS mutant lung cancer cells, leading to destabilization of its target gene Hif-1α, which indicated that the anticancer property of honokiol maybe regulated via a novel mechanism associated with the Sirt3/Hif-1α. Taken together, these results broaden our understanding of the mechanisms on honokiol effects in lung cancer, and reinforce the possibility of its potential anticancer benefit as a popular Chinese herbal medicine (CHM).