Cucurbitacin Research Articles

Transcription Factor CmWRKY13 Regulates Cucurbitacin B Biosynthesis Leading to Bitterness in Oriental Melon Fruit (Cucumis melo var. Makuwa Makino).

Bitterness, caused by cucurbitacin B (CuB), is one of the important traits that affects melon fruit quality and consumer acceptance. Therefore, the detailed mechanism behind the regulation of CuB biosynthesis on melon fruit needs to be further explored. This study investigated CuB content and transcriptomes of "YMR" melon fruit treated by 5 and 20 mg L-1 CPPU. The content of CuB reaches its peak in 5 days and then decreases. WGCNA identified the WRKY transcription factor (TF), CmWRKY13, coexpressed with CuB biosynthetic genes (Cm180, Cm170, Cm160, and CmACT). Yeast one-hybrid, dual-luciferase, and transient gene expression assays were conducted and suggested that the nucleus-localized CmWRKY13 transactivated the promoters of CuB biosynthetic genes and participated in the regulation of CuB biosynthesis. Furthermore, CmWRKY13 could interact with CmBt, the fruit bitterness-specific TF, which synergistically activated CuB biosynthetic gene expression. These findings provide a novel mechanistic insight for CuB biosynthesis and regulation in melon cultivation.

Metabolic fate of the natural anticancer agent cucurbitacin B: an LC-MS/MS-enabled profiling of its major phase I and II conjugates in vivo.

Cucurbitacin B (CuB) is a natural triterpenoid with diverse pharmacological effects including potent anticancer activity. However, its oral bioavailability is hampered by limited metabolism in vivo. We characterized CuB's in vivo metabolism in rats to uncover bioactive metabolites retaining therapeutic potential, using a robust UHPLC-Q-TOF-MS/MS workflow. This workflow combined molecular networking, fragmentation filtering, and mass defect filtering to identify CuB metabolites in rat urine, plasma, and feces following oral administration. Thirteen metabolites were identified and seven were confirmed. Major phase I transformations involved hydrolysis, reduction, epoxidation, and amination. Phase II conjugation included cysteine, glutathione, glucuronide, and gluconic acid conjugates. Notably, one of the main metabolites formed was the cysteine conjugate CuB-Cys. CuB-Cys maintained similar in vitro antiproliferative activity to CuB on HepG2, MCF-7, and PANC-1 cancer cell lines. However, it demonstrated lower cytotoxicity towards non-cancerous L02 cells, highlighting improved therapeutic selectivity. Mechanistically, CuB-Cys induced greater apoptotic signaling in HepG2 cells than CuB via enhanced caspase activation and disrupted BAX-Bcl-2 balance. This represents the first systematic characterization of CuB's in vivo metabolic pathway. The identification and confirmation of CuB-Cys provide insight for drug development efforts aiming to maintain therapeutic efficacy while reducing toxicity, via metabolite-based approaches. Our findings shed light on strategies for improving CuB's clinical potential.

Cucurbitacins mitigate vascular neointimal hyperplasia by suppressing cyclin A2 expression and inhibiting VSMC proliferation.

Restenosis frequently occurs after percutaneous angioplasty in patients with vascular occlusion and seriously threatens their health. Substantial evidence has revealed that preventing vascular smooth muscle cell proliferation using a drug-eluting stent is an effective approach to improve restenosis. Cucurbitacins have been demonstrated to exert an anti-proliferation effect in various tumors and a hypotensive effect. This study aims to investigate the role of cucurbitacins extracted from Cucumis melo L. (CuECs) and cucurbitacin B (CuB) on restenosis. C57BL/6 mice were subjected to left carotid artery ligation and subcutaneously injected with CuECs or CuB for 4 weeks. Hematoxylin-Eosin, immunofluorescence and immunohistochemistry staining were used to evaluate the effect of CuECs and CuB on neointimal hyperplasia. Western blot, real-time PCR, flow cytometry analysis, EdU staining and cellular immunofluorescence assay were employed to measure the effects of CuECs and CuB on cell proliferation and the cell cycle invitro. The potential interactions of CuECs with cyclin A2 were performed by molecular docking. The results demonstrated that both CuECs and CuB exhibited significant inhibitory effects on neointimal hyperplasia and proliferation of vascular smooth muscle cells. Furthermore, CuECs and CuB mediated cell cycle arrest at the S phase. Autodocking analysis demonstrated that CuB, CuD, CuE and CuI had high binding energy for cyclin A2. Our study also showed that CuECs and CuB dramatically inhibited FBS-induced cyclin A2 expression. Moreover, the expression of cyclin A2 in CuEC- and CuB-treated neointima was downregulated. CuECs, especially CuB, exert an anti-proliferation effect in VSMCs and may be potential drugs to prevent restenosis.

Cucurbitacin B targets STAT3 to induce ferroptosis in non-small cell lung cancer

Cucurbitacin B (CuB) is a compound found in plants like Cucurbitaceae that has shown promise in fighting cancer, particularly in lung cancer. However, the specific impact of CuB on ferroptosis and how it works in lung cancer cells has not been fully understood. Our research has discovered that CuB can effectively slow down the growth of non-small cell lung cancer (NSCLC) cells. Even in small amounts, it was able to inhibit the growth of various NSCLC cell lines. This inhibitory effect was reversed when ferroptosis inhibitors DFO, Lip-1 and Fer-1 were introduced. CuB was found to increase the levels of reactive oxygen species (ROS), lipid ROS, MDA, and ferrous ions within H358 lung cancer cells, leading to a decrease in GSH, mitochondrial membrane potential (MMP) and changes in ferroptosis-related proteins in a dose-dependent manner. These findings were also confirmed in A549 lung cancer cells. In A549 cells, different concentrations of CuB induced the accumulation of intracellular lipid ROS, ferrous ions and changes in ferroptosis-related indicators in a concentration-dependent manner. Meanwhile, the cytotoxic effect induced by CuB in A549 cells was counteracted by ferroptosis inhibitors DFO and Fer-1. Through network pharmacology, we identified potential targets related to ferroptosis in NSCLC cells treated with CuB, with STAT3 targets showing high scores. Further experiments using molecular docking and cell thermal shift assay (CETSA) revealed that CuB interacts with the STAT3 protein. Western blot and immunofluorescence staining demonstrated that CuB inhibits the phosphorylation of STAT3 (P-STAT3) in H358 cells. Silencing STAT3 enhanced CuB-induced accumulation of lipid ROS and iron ions, as well as the expression of ferroptosis-related proteins. On the other hand, overexpression of STAT3 reversed the effects of CuB-induced ferroptosis. The results indicate that CuB has the capability to suppress STAT3 activation, resulting in ferroptosis, and could be a promising treatment choice for NSCLC.

Cucurbitacin B attenuates osteoarthritis development by inhibiting NLRP3 inflammasome activation and pyroptosis through activating Nrf2/HO-1 pathway.

Osteoarthritis (OA) is a complicated joint disorder characterized by inflammation that causes joint destruction. Cucurbitacin B (CuB) is a naturally occurring triterpenoid compound derived from plants in the Cucurbitaceae family. The aim of this study is to investigate the potential role and mechanisms of CuB in a mouse model of OA. This study identified the key targets and potential pathways of CuB through network pharmacology analysis. In vivo and in vitro studies confirmed the potential mechanisms of CuB in OA. Through network pharmacology, 54 potential targets for CuB in treating OA were identified. The therapeutic potential of CuB is associated with the nod-like receptor pyrin domain 3 (NLRP3) inflammasome and pyroptosis. Molecular docking results indicate a strong binding affinity of CuB to nuclear factor erythroid 2-related factor 2 (Nrf2) and p65. In vitro experiments demonstrate that CuB effectively inhibits the expression of pro-inflammatory factors induced by interleukin-1β (IL-1β), including cyclooxygenase-2, inducible nitric oxide synthase, IL-1β, and IL-18. CuB inhibits the degradation of type II collagen and aggrecan in the extracellular matrix (ECM), as well as the expression of matrix metalloproteinase-13 and a disintegrin and metalloproteinase with thrombospondin motifs-5. CuB protects cells by activating the Nrf2/hemeoxygenase-1 (HO-1) pathway and inhibiting nuclear factor-κB (NF-κB)/NLRP3 inflammasome-mediated pyroptosis. Moreover, in vivo experiments show that CuB can slow down cartilage degradation in an OA mouse model. CuB effectively prevents the progression of OA by inhibiting inflammation in chondrocytes and ECM degradation. This action is further mediated through the activation of the Nrf2/HO-1 pathway to inhibit NF-κB/NLRP3 inflammasome activation. Thus, CuB is a potential therapeutic agent for OA.

Cucurbitacin B induces ferroptosis in oral leukoplakia via the SLC7A11/mitochondrial oxidative stress pathway

BackgroundOral leukoplakia (OLK), characterized by abnormal epithelial hyperplasia, is the most common precancerous oral mucosa lesion and is closely related to oxidative stress. Cucurbitacin B (CuB), a tetracyclic triterpenoid molecule derived from plants, has shown promising anti-proliferative and antioxidant effects in preclinical studies. However, whether CuB can play an antiproliferative role in OLK by regulating oxidative stress remains elusive. PurposeTo investigate the role of CuB in inhibiting the malignant progression of oral leukoplakia and to further explore its underlying mechanisms of action. MethodsIn vitro, the effect of CuB on the proliferation, migration, apoptosis, and cell cycle of OLK cells DOK was detected. The core genes and key pathways of OLK and CuB were analyzed in the transcriptome database, by using immunofluorescence, qRT-PCR, and Western blot to evaluate the expression levels of the ferroptosis markers ROS, GSH, MDA, Fe2+, and marker genes SLC7A11, GPX4, and FTH1. Immunohistochemistry of human tissue was performed to investigate the expression of the SLC7A11. In vivo, the model of OLK was established in C57BL/6 mice and the biosafety of CuB treatment for OLK was further evaluated. ResultsCuB substantially suppressed the proliferation of DOK cells. Bioinformatics analysis showed that the core targets of OLK crossing with CuB include SLC7A11 and that the essential pathways involve ROS and ferroptosis. In vitro experiments indicated that CuB might promote ferroptosis by down-regulating the expression of SLC7A11. We observed a gradual increase in SLC7A11 expression levels during the progression from normal oral mucosa to oral leukoplakia with varying degrees of epithelial dysplasia. In vivo experiments demonstrated that CuB inhibited the malignant progression of OLK by promoting ferroptosis in OLK mice and exhibited a certain level of biosafety. ConclusionThis study demonstrated for the first time that CuB could effectively inhibit the malignant progression of OLK by inducing ferroptosis via activating the SLC7A11/ mitochondrial oxidative stress pathway. These findings indicate that CuB could serve as the lead compound for the future development of anti-oral leukoplakia drugs.

Exploring the Cucurbitacin E (CuE) as an Anti-Lung Cancer Lead Compound through Molecular Docking, ADMET, Pass Prediction and Drug Likeness Analysis

Exploring the Cucurbitacin E (CuE) as an Anti-Lung Cancer Lead Compound through Molecular Docking, ADMET, Pass Prediction and Drug Likeness Analysis

Cucurbitacin B modulates M2 macrophage differentiation and attenuates osteosarcoma progression via PI3K/AKT pathway.

Osteosarcoma is a common malignant bone tumour characterised by an aggressive metastatic potential. The tumour microenvironment, particularly the M2-polarised macrophages, is crucial for tumour progression. Cucurbitacin B (CuB), a triterpenoid derivative, is recognised for its anti-inflammatory and antitumour properties. This study investigates CuB and its effect on M2 macrophage differentiation and osteosarcoma progression, aiming to contribute to new treatment strategies. In vitro, THP-1 monocytes were stimulated with PMA, IL-13 and IL-4 to induce differentiation into M2 macrophages. Additionally, the influence of CuB on the proliferation, migration and invasion of osteosarcoma cells in the context of M2 macrophages was scrutinised. Crucial signalling pathways, especially the PI3K/AKT pathway, affected by CuB were identified and validated. In vivo, the osteosarcoma model was employed to gauge the effects of CuB on tumour weight, lung metastasis, angiogenesis, cell proliferation and M2 macrophage markers. The results showed that CuB inhibited M2 macrophage differentiation, leading to reduced proliferation, migration and invasion of osteosarcoma cells. CuB manifested an inhibitory effect on the PI3K/AKT pathway during the differentiation of M2 macrophages. In mouse models, CuB markedly reduced the tumour weight and the number of lung metastases. It also reduced the expression of angiogenesis and cell proliferation markers in tumour tissues, decreased the quantity of M2 macrophages and their associated markers and pathway proteins. In conclusion, CuB impedes osteosarcoma progression by inhibiting M2 macrophage differentiation via the PI3K/AKT pathway, presenting the potential for therapeutic advancements in osteosarcoma treatment.

Cucurbitacin E elicits apoptosis in laryngeal squamous cell carcinoma by enhancing reactive oxygen species-regulated mitochondrial dysfunction and endoplasmic reticulum stress.

Laryngeal squamous cell carcinoma (LSCC) is a prevalent head and neck neoplasm with escalating global morbidity and mortality rates. Despite the increasing burden of LSCC, the drugs currently approved for its treatment are limited. Therefore, it is necessary to identify novel and promising drugs that target LSCC. Cucurbitacin E (CuE) is a naturally oxygenated tetracyclic triterpenoid that suppresses several cancers. However, its anti-LSCC activity and the molecular mechanisms of action remain unclear. This study explored its impact on LSCC, revealing cell viability attenuation and apoptosis enhancement in vitro. Further investigations indicated that CuE significantly decreased mitochondrial membrane potential, thereby promoting cytochrome c release, increasing cleaved-Caspase 3 and cleaved-PARP levels, and triggering mitochondria-dependent apoptosis. Concurrently, exposure of LSCC cells to CuE enhanced endoplasmic reticulum (ER) stress, mobilized the protein kinase RNA-like endoplasmic reticulum kinase/initiation factor 2a/ATF4/C-EBP homologous protein pathway, and induced LSCC cell apoptosis. Finally, CuE markedly elevated intracellular reactive oxygen species (ROS) levels. When ROS were eliminated with N-acetylcysteine, CuE-mediated mitochondrial dysfunction, ER stress, and cell apoptosis were nearly abolished. Similar outcomes were observed in murine LSCC models. Together, these results highlight that CuE suppresses proliferation while triggering apoptosis in LSCC cells via ROS-regulated mitochondrial dysfunction and the ER stress pathway. Hence, CuE may serve as a promising candidate for LCSS treatment.

Cucurbitacin B Inhibits the Malignancy of Esophageal Carcinoma through the KIF20A/JAK/STAT3 Signaling Pathway.

This study intends to explore the effects of Cucurbitacin B (CuB) and KIF20A on esophageal carcinoma (ESCA). Data were downloaded from the Cancer Genome Atlas (TCGA) database. The expression properties of KIF20A have been confirmed by GEPIA and ualcan from TCGA. The expression of KIF20A was determined using western blotting in ECA109 and KYSE150 cells after transfection with KIF20A, KIF20A siRNA, or numerical control siRNA (si-NC). Then, different concentrations of CuB were used to treat ECA109 and KYSE150 cells. CCK-8 and colony formation assays were used to measure cell viability, and a Transwell assay was utilized to assess cell migration and invasion ability. N-cadherin, E-cadherin, snail, p-Janus kinase 2 (JAK2), JAK2, p-signal transducer and activator of transcription 3 (STAT3), and STAT3 expression levels were evaluated using western blot. KIF20A was higher expressed in ESCA than in normal cells, and its overexpression was associated with squamous cell carcinoma, TNM stage, and lymph nodal metastasis of ESCA patients. In ECA109 and KYSE150 cells, increased KIF20A facilitated cell proliferation, migration, and invasion, whereas the knockdown of KIF20A can reverse these effects with N-cadherin. Snail expression diminished and E-cadherin increased. Similarly, CuB treatment could inhibit cell proliferation, migration, and invasion concentration dependently. Furthermore, KIF20A accelerated the expression of p-JAK2 and p-STAT3, while the application of CuB inhibited KIF20A expression and attenuated the activation of the JAK/STAT3 pathway. These findings revealed that CuB could inhibit the growth, migration, and invasion of ESCA through downregulating the KIF20A/JAK/STAT3 signaling pathway, and CuB could serve as an essential medicine for therapeutic intervention.

Genetic mutation of Tas2r104/Tas2r105/Tas2r114 cluster leads to a loss of taste perception to denatonium benzoate and cucurbitacin B.

Bitter taste receptors (Tas2rs) are generally considered to sense various bitter compounds to escape the intake of toxic substances. Bitter taste receptors have been found to widely express in extraoral tissues and have important physiological functions outside the gustatory system invivo. To investigate the physiological functions of the bitter taste receptor cluster Tas2r106/Tas2r104/Tas2r105/Tas2r114 in lingual and extraoral tissues, multiple Tas2rs mutant mice and Gnat3 were produced using CRISPR/Cas9 gene-editing technique. A mixture containing Cas9 and sgRNA mRNAs for Tas2rs and Gnat3 gene was microinjected into the cytoplasm of the zygotes. Then, T7EN1 assays and sequencing were used to screen genetic mutation at the target sites in founder mice. Quantitative real-time polymerase chain reaction (qRT-PCR) and immunostaining were used to study the expression level of taste signaling cascade and bitter taste receptor in taste buds. Perception to taste substance was also studied using two-bottle preference tests. We successfully produced several Tas2rs and Gnat3 mutant mice using the CRISPR/Cas9 technique. Immunostaining results showed that the expression of GNAT3 and PLCB2 was not altered in Tas2rs mutant mice. But qRT-PCR results revealed the changed expression profile of mTas2rs gene in taste buds of these mutant mice. With two-bottle preference tests, these mutant mice eliminate responses to cycloheximide due to genetic mutation of Tas2r105. In addition, these mutant mice showed a loss of taste perception to quinine dihydrochloride, denatonium benzoate, and cucurbitacin B (CuB). Gnat3-mediated taste receptor and its signal pathway contribute to CuB perception. These findings implied that these mutant mice would be a valuable means to understand the biological functions of TAS2Rs in extraoral tissues and investigate bitter compound-induced responses mediated by these TAS2Rs in many extraoral tissues.

Exploring the effect and mechanism of cucurbitacin B on cholestatic liver injury based on network pharmacology and experimental verification

Ethnopharmacological relevanceCholestatic liver injury (CLI) is a pathologic process with the impairment of liver and bile secretion and excretion, resulting in an excessive accumulation of bile acids within the liver, which leads to damage to both bile ducts and hepatocytes. This process is often accompanied by inflammation. Cucumis melo L is a folk traditional herb for the treatment of cholestasis. Cucurbitacin B (CuB), an important active ingredient in Cucumis melo L, has significant anti-inflamamatory effects and plays an important role in diseases such as neuroinflammation, skin inflammation, and chronic hepatitis. Though numerous studies have confirmed the significant therapeutic effect of CuB on liver diseases, the impact of CuB on CLI remains uncertain. Consequently, the objective of this investigation is to elucidate the therapeutic properties and potential molecular mechanisms underlying the effects of CuB on CLI. Aim of the studyThe aim of this paper was to investigate the potential protective mechanism of CuB against CLI. MethodsFirst, the corresponding targets of CuB were obtained through the SwissTargetPrediction and SuperPre online platforms. Second, the DisGeNET database, GeneCards database, and OMIM database were utilized to screen therapeutic targets for CLI. Then, protein-protein interaction (PPI) was determined using the STRING 11.5 data platform. Next, the OmicShare platform was employed for the purpose of visualizing the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses. The molecular docking technique was then utilized to evaluate the binding affinity existing between potential targets and CuB. Subsequently, the impacts of CuB on the LO2 cell injury model induced by Lithocholic acid (LCA) and the CLI model induced by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) were determined by evaluating inflammation in both in vivo and in vitro settings. The potential molecular mechanism was explored by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot (WB) techniques. ResultsA total of 122 CuB targets were collected and high affinity targets were identified through the PPI network, namely TLR4, STAT3, HIF1A, and NFKB1. GO and KEGG analyses indicated that the treatment of CLI with CuB chiefly involved the inflammatory pathway. In vitro study results showed that CuB alleviated LCA-induced LO2 cell damage. Meanwhile, CuB reduced elevated AST and ALT levels and the release of inflammatory factors in LO2 cells induced by LCA. In vivo study results showed that CuB could alleviate DDC-induced pathological changes in mouse liver, inhibit the activity of serum transaminase, and suppress the liver and systemic inflammatory reaction of mice. Mechanically, CuB downregulated the IL-6, STAT3, and HIF-1α expression and inhibited STAT3 phosphorylation. ConclusionBy combining network pharmacology with in vivo and in vitro experiments, the results of this study suggested that CuB prevented the inflammatory response by inhibiting the IL-6/STAT3/HIF-1α signaling pathway, thereby demonstrating potential protective and therapeutic effects on CLI. These results establish a scientific foundation for the exploration and utilization of natural medicines for CLI.

Cucurbitacin B and erastin co-treatment synergistically induced ferroptosis in breast cancer cells via altered iron-regulating proteins and lipid peroxidation

Ferroptosis is a unique type of cell death which co-exists with elevated iron, suppressed antioxidative function and increased lipid peroxidation. Recent studies have shown that cancer cells are particularly susceptible to the compounds with ferroptotic activities. Cucurbitacin B (CuB) is a triterpenoid with potent biological properties. It has been demonstrated to induce apoptosis and inhibit metastasis in cancer cells. However, the underlying mechanism of the compound is still not fully understood. In the present study, we investigated the ferroptotic effect of CuB in breast cancer cells and evaluated the impact of its combination with erastin, a ferroptosis inducer. In this regard, MTT assay was performed to analyze cell viability. Lipid peroxidation, oxidative stress and the cellular antioxidant capacity were determined with relevant kits. The expression of ferroptotic proteins were analyzed by western blotting. The results indicated that the combined treatment of CuB and erastin activated the ferroptotic pathways significantly in MCF-7 and MDA-MB-231 breast cancer cells. More importantly, the combination treatment altered the expression of iron-related proteins IREB2 and FPN1. In conclusion, this study demonstrated the ferroptotic potential of CuB in breast cancer cells for the first time, and revealed its impact on the expression of iron-regulating proteins.

Depolymerization of actin filaments by Cucurbitacin I through binding G-actin.

Cucurbitacins have high economic value as they are a major source of food and have pharmacological properties. Cucurbitacin I (CuI) is a plant-derived natural tetracyclic triterpenoid compound that shows an anticancer effect via inhibiting the JAK2-STAT3 signaling pathway. The actin cytoskeleton is the most abundant protein in cells and regulates critical events through reorganization in cells. In this study, it is aimed at determining the direct effect of CuI on actin dynamics. The fluorescence profile of G-actin in the presence of CuI (1-200 nM) shifted to a higher temperature, suggesting that G-actin binds CuI and that G-actin-CuI is more thermally stable than the ligand-free form. CuI dose-dependently inhibited the polymerization of F-actin invitro and disrupted actin filaments in endothelial cells. Docking and MD simulations suggested that CuI binds to the binding site formed by residues I136, I175, D154, and A138 that are at the interface of monomers in F-actin. The migration ability of cells treated with CuI for 24 h was significantly lower than the control group (p < .001). This study reveals the molecular mechanisms of CuI in the regulation of actin dynamics by binding G-actin. More importantly, this study indicates a novel role of CuI as an actin-targeting drug by binding directly to G-actin and may contribute to the mode of action of CuI on anticancer activities.

Cucurbitacin B suppresses hepatocellular carcinoma progression through inducing DNA damage-dependent cell cycle arrest

BackgroundThe mortality rate of liver cancer ranks third in the world, and hepatocellular carcinoma (HCC) is a malignant tumor of the digestive tract. Cucurbitacin B (CuB), a natural compound extracted from Cucurbitaceae spp., is the main active component of Chinese patent medicine the Cucurbitacin Tablet, which has been widely used in the treatment of various malignant tumors in clinics, especially HCC. PurposeThis study explored the role and mechanism of CuB in the suppression of liver cancer progression. MethodsCell Counting Kit-8 (CCK-8) and colony formation assays were used to detect the inhibitory function of CuB in Huh7, Hep3B, and Hepa1/6 hepatoma cells. Calcein-AM/propidium iodide (PI) staining and lactate dehydrogenase (LDH) measurement assays were performed to determine cell death. Mitochondrial membrane potential (Δψm) was measured, and flow cytometry was performed to evaluate cell apoptosis and cell cycle. Several techniques, such as proteomics, Western blotting (WB), and ribonucleic acid (RNA) interference, were utilized to explore the potential mechanism. The animal experiment was performed to verify the results of in vitro experiments. ResultsCuB significantly inhibited the growth of Huh7, Hep3B, and Hepa1/6 cells and triggered the cell cycle arrest in G2/M phage without leading to cell death, especially apoptosis. Knockdown of insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1), a target of CuB, did not reverse CuB elicited cell cycle arrest. CuB enhanced phosphorylated ataxia telangiectasia mutated (p-ATM) and phosphorylated H2A histone family member X (γ-H2AX) levels. Moreover, CuB increased p53 and p21 levels and decreased cyclin-dependent kinase 1 (CDK1) expression, accompanied by improving phosphorylated checkpoint kinase 1 (p-CHK1) level and suppressing cell division cycle 25C (CDC25C) protein level. Interestingly, these phenomena were partly abolished by a deoxyribonucleic acid (DNA) protector methylproamine (MPA). Animal studies showed that CuB also significantly suppressed tumor growth in BALB/c mice bearing Hepa1/6 cells. In tumor tissues, CuB reduced the expression levels of proliferating cell nuclear antigen (PCNA) and γ-H2AX but did not change the terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) level. ConclusionThis study demonstrated for the first time that CuB could effectively impede HCC progression by inducing DNA damage-dependent cell cycle arrest without directly triggering cell death, such as necrosis and apoptosis. The effect was achieved through ataxia telangiectasia mutated (ATM)-dependent p53-p21-CDK1 and checkpoint kinase 1 (CHK1)-CDC25C signaling pathways. These findings indicate that CuB may be used as an anti-HCC drug, when the current findings are confirmed by independent studies and after many more clinical phase 1, 2, 3, and 4 testings have been done.

Molecular interactions of cucurbitacins A and B with anaplastic lymphoma kinase for lung cancer treatment

Lung cancer is a major global public health issue and the leading cause of cancer-related deaths. Several medications are commonly used to treat lung cancer, either alone or in combination with other treatments. The anaplastic lymphoma kinase (ALK) protein is one of several target proteins that are thought to be potential therapeutic targets in the context of lung cancer. Several ALK inhibitors have been identified, but many of these have been associated with side effects and toxicity concerns. In this study, we intend to computationally predict the binding potential of cucurbitacins (CBNs), A and B to the active pockets of ALK, in order to estimate their potential ALK inhibitors. Compared to CBN-A, which has a binding energy of −7.9 kcal/mol, CBN B exhibits significantly better binding efficacy with a binding energy of −8.1 kcal/mol. This is closely comparable to the binding energy of Crizotinib, which is −8.2 kcal/mol. The results of the molecular dynamics simulation indicated that the docked complexes remained stable for the duration of the 100 ns simulation period. CBN inhibited the proliferation of both non-small cell lung cancer cell lines, H1299 and A549, in a dose-dependent manner. CBN-B inhibited the proliferation of lung cancer cells, showing IC50 values of 0.08 µM for H1299 cells and 0.10 µM for A549 cells. The computational analyses provide strong evidence that CBN-B has the potential to act as a potent natural inhibitor against ALK, and could prove to be a valuable treatment option for lung cancer. Communicated by Ramaswamy H. Sarma

Diselenium-linked dimeric prodrug nanomedicine breaking the intracellular redox balance for triple-negative breast cancer targeted therapy

Triple-negative breast cancer (TNBC) has been regarded as the strongest malignancy in cases of breast cancer with a poor prognosis. The development of effective treatment strategies for TNBC has always been an urgent and unmet need. The intracellular redox balance is essential for maintaining TNBC cell malignancy. Disrupting intracellular redox balance by enlarging reactive oxygen species (ROS) generation and facilitating glutathione (GSH) depletion to amplify intracellular oxidative stress may be an alternative strategy to eliminate TNBC cells. However, inducing ROS generation and GSH depletion concurrently may be challenging. Herein, a diselenium linked-dimeric prodrug nanomedicine FA-SeSe-NPs was developed to break the intracellular redox homeostasis for TNBC targeted therapy. The dimeric prodrug was synthesized by conjugating two cucurbitacin B (CuB) molecules via one diselenium bond, which was subsequently assembled with FA-PEG-DSPE to form the final nanomedicine FA-SeSe-NPs. Using the active targeting potential of folic acid (FA), FA-SeSe-NPs could accumulate in tumor tissue with elevated levels and then be specifically internalized by cancer cells. In the high ROS and GSH conditions of TNBC cells, the diselenium bond can specifically respond to ROS to produce selenium free radicals to increase ROS and react with GSH to generate S-Se bond to deplete GSH. The released CuB further induced ROS production in TNBC cells. The diselenium bond and CuB functioned synergistically to amplify oxidative stress to kill the TNBC cells. Here, we provide a promising strategy to disrupt the intracellular redox balance of cancer cells for effective TNBC therapy.

Discovery of Triazolyl Derivatives of Cucurbitacin B Targeting IGF2BP1 against Non-Small Cell Lung Cancer.

Cucurbitacin B (CuB) is a potent but toxic anticancer natural product. Herein, we designed and synthesized 2-OH- and 16-OH-modified CuB derivatives to improve their antitumor efficacy and reduce toxicity. Among them, derivative A11 had the most potent antiproliferative activity against A549 lung cancer cells (IC50 = 0.009 μM) and was approximately 10-fold more potent than CuB, while the cytotoxicity of A11 toward normal L02 cells was about 10-fold less potent, indicating a much wider therapeutic window than CuB. Derivative A11 directly binds to the insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) protein with a KD value of 2.88 nM, which is about 23-fold more potent than CuB, leading to the decreased expression of downstream apoptosis- and cell cycle-related proteins. More importantly, A11 exhibited much more potent anticancer efficacy in an A549 xenograft mouse model with a TGI rate of 80% and a superior in vivo safety profile than that of CuB.

Cucurbitacin B induces apoptosis in colorectal cells through reactive oxygen species generation and endoplasmic reticulum stress pathways.

Cucurbitacin B (CuB) is a member of the cucurbitacin family, which has shown potent anticancer pharmacological activity. Prolonged or severe endoplasmic reticulum stress (ERS) induces apoptosis; therefore, the present study investigated whether CuB may activate the ERS pathway to induce apoptosis. HT-29 and SW620 colorectal cancer (CRC) cells were treated with a range of concentrations of CuB for 48 h, and the viability and proliferation of cells were determined using Cell Counting Kit 8 (CCK8) and colony formation assays. Subsequently, the appropriate CuB concentration (5 µM) was selected for treatment of CRC cells for 48 h. Western blot analysis was used to measure the expression levels of ERS-related proteins, flow cytometry was used to evaluate apoptosis, the dichlorodihydrofluorescein diacetate fluorescent probe was used to detect reactive oxygen species (ROS) production, and the relationship between ROS and ERS was determined by western blot analysis. Furthermore, flow cytometry was used to evaluate apoptosis after treatment with the ERS inhibitor 4-phenylbutyric acid, the ROS inhibitor N-acetylcysteine and following knockdown of CHOP expression. In addition, western blot analysis was performed to measure Bax and Bcl2 protein expression levels, and a CCK8 assay was performed to evaluate the viability of cells following knockdown of CHOP. Notably, CuB treatment increased apoptosis and inhibited cell proliferation in CRC cell lines, and these effects were mediated by ROS and ROS-regulated activation of the PERK and XBP1 ERS pathways. In conclusion, CuB may induce apoptosis in HT-29 and SW620 CRC cells via ROS and ERS.

Network pharmacological analysis and experimental study of cucurbitacin B in oral squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is a malignant tumor with a high incidence and poor prognosis. Cucurbitacin B (CuB) is a tetracyclic triterpenoid small-molecule compound extracted from plants, such as Cucurbitaceae and Brassicaceae, which has powerful anticancer effects. However, the effect and mechanism of CuB on OSCC remain unclear. Within the framework of the current study, network pharmacology was used to analyze the relationship between CuB and OSCC. The network pharmacology analysis showed that CuB and OSCC share 134 common targets; among them, PIK3R1, SRC, STAT3, AKT1, and MAPK1 are the key targets. The molecular docking analysis showed that CuB binds five target proteins. The results of the enrichment analysis showed that CuB exerted effects on OSCC through various pathways; of these pathways, PI3K-AKT was the most important pathway. The results of the in vitro cell experiments showed that CuB could inhibit the proliferation and migration of SCC25 and CAL27 cells, block the cell cycle in the G2 phase, induce cell apoptosis, and regulate the protein expression of the PI3K-AKT signaling pathway. The results of the in vivo animal experiments showed that CuB could inhibit 4NQO-induced oral cancer in mice. Therefore, network pharmacology, molecular docking, cell experiments, and animal experiments showed that CuB could play a role in OSCC by regulating multiple targets and pathways.