[Mechanism of Huachansu Injection against colorectal cancer based on network pharmacology and cellular experimental].

This study aimed to elucidate the mechanism of Huachansu Injection(HCSI) against colorectal cancer(CRC) using network pharmacology, molecular docking technology, and cellular experimental. This research group initially used LC-MS/MS to detect the content of 16 bufadienolides in HCSI. Ten bufadienolide components were selected based on a content threshold of greater than 10 ng·mL~(-1). Their potential targets were further predicted using the SwissTargetPrediction database. CRC-related targets were obtained through GeneCards, OMIM, TTD, and PharmGKB databases. The intersection targets of HCSI in the treatment of CRC were obtained through Venny. The "active component-target-disease" network and target protein-protein interaction(PPI) network were constructed via Cytoscape software. Core targets were screened based on the degree values. Gene Ontology(GO) function and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analyses were performed on these key targets. Molecular docking was conducted using AutoDock software on major bufadienolide active components and key targets. Different concentrations of HCSI, psi-bufarenogin(BUF), and bufotalin(BFT) were tested for their effects on cell viability, migration, and apoptosis rates in CRC HCT116 cells. Western blot was conducted to detect the expression of proteins related to the PI3K/Akt/mTOR signaling pathway in HCT116 cells. Eight main active components of HCSI, including arenobufagin, BUF, and BFT, as well as 20 key targets of HCSI in combating CRC, such as EGFR, IL6, and mTOR, were identified. Based on KEGG pathway enrichment and molecular docking results, the PI3K/Akt/mTOR signaling pathway was selected for further verification. Cellular experimental demonstrated that HCSI, BUF, and BFT significantly inhibited the proliferation and migration abilities of HCT116 cells, induced apoptosis in these cells, and downregulated the expression of PI3K/Akt/mTOR pathway-related proteins. This result suggests that HCSI, BUF, and BFT may exert their anti-CRC effects by regulating the PI3K/Akt/mTOR signaling pathway through targets such as mTOR and PIK3CA. This study provides theoretical evidence for exploring the active ingredients and mechanism of HCSI against CRC.

Cinobufagin inhibits M2­like tumor­associated macrophage polarization to attenuate the invasion and migration of lung cancer cells.

Macrophages have crucial roles in immune responses and tumor progression, exhibiting diverse phenotypes based on environmental cues. In the present study, the impact of cinobufagin (CB) on macrophage polarization and the consequences on tumor­associated behaviors were investigated. Morphological transformations of THP­1 cells into M0, M1 and M2 macrophages were observed, including distinct changes in the size, shape and adherence properties of these cells. CB treatment inhibited the viability of A549 and LLC cells in a concentration­dependent manner, with an IC50 of 28.8 and 30.12 ng/ml, respectively. CB at concentrations of <30 ng/ml had no impact on the viability of M0 macrophages and lung epithelial (BEAS­2B) cells. CB influenced the expression of macrophage surface markers, reducing CD206 positivity in M2 macrophages without affecting CD86 expression in M1 macrophages. CB also altered certain expression profiles at the mRNA level, notably downregulating macrophage receptor with collagenous structure (MARCO) expression in M2 macrophages and upregulating tumor necrosis factor­α and interleukin­1ß in both M0 and M1 macrophages. Furthermore, ELISA analyses revealed that CB increased the levels of pro­inflammatory cytokines in M1 macrophages and reduced the levels of anti­inflammatory factors in M2 macrophages. CB treatment also attenuated the migration and invasion capacities of A549 and LLC cells stimulated by M2 macrophage­conditioned medium. Additionally, CB modulated peroxisome proliferator­activated receptor γ (PPARγ) and MARCO expression in M2 macrophages and epithelial­mesenchymal transition in A549 cells, which was partially reversed by rosiglitazone, a PPARγ agonist. Finally, CB and cisplatin treatments hindered tumor growth in vivo, with distinct impacts on animal body weight and macrophage marker expression in tumor tissues. In conclusion, the results of the present study demonstrated that CB exerted complex regulatory effects on macrophage polarization and tumor progression, suggesting its potential as a modulator of the tumor microenvironment and a therapeutic for cancer treatment.

Bufalin-Loaded Multifunctional Photothermal Nanoparticles Inhibit the Anaerobic Glycolysis by Targeting SRC-3/HIF-1α Pathway for Improved Mild Photothermal Therapy in CRC.

Purpose: Compared with traditional photothermal therapy (PTT, >50°C), mild PTT (≤45°C) is a promising strategy for tumor therapy with fewer adverse effects. Unfortunately, its anti-tumor efficacy is hampered by thermoresistance induced by overexpression of heat shock proteins (HSPs). In our previous study, we found bufalin (BU) is a glycolysis inhibitor that depletes HSPs, which is expected to overcome thermotolerance of tumor cells. In this study, BU-loaded multifunctional nanoparticles (NPs) were developed for enhancing the mild PTT of colorectal cancer (CRC). Methods: Fe3O4 NPs coated with the polydopamine (PDA) shell modified with polyethylene glycol (PEG) and cyclic arginine-glycyl-aspartic peptide (cRGD) for loading BU (Fe3O4@PDA-PEG-cRGD/BU NPs) were developed. The thermal variations in Fe3O4@PDA-PEG-cRGD/BU NPs solution under different conditions were measured. Glycolysis inhibition was evaluated by measuring the glucose uptake, extracellular lactate, and intracellular adenosine triphosphate (ATP) levels. The cellular cytotoxicity of Fe3O4@PDA-PEG-cRGD/BU NPs was analyzed using a cell counting kit-8 assay, Calcein-AM/PI double staining, and flow cytometry in HCT116 cells. The magnetic resonance imaging (MRI) performance and anti-tumor therapeutic efficacy of Fe3O4@PDA-PEG-cRGD/BU NPs were evaluated in HCT116-tumor bearing mice. Results: Fe3O4@PDA-PEG-cRGD/BU NPs had an average diameter of 260.4±3.5 nm, the zeta potential of -23.8±1.6 mV, the drug loading rate of 1.1%, which had good thermal stability, photothermal conversion efficiencies and MRI performance. In addition, the released BU not only killed tumor cells but also interfered with glycolysis by targeting the steroid receptor coactivator 3 (SRC-3)/HIF-1α pathway, preventing intracellular ATP synthesis, and combating HSP-dependent tumor thermoresistance, ultimately strengthening the thermal sensitivity toward mild PTT both in vitro and in vivo. Conclusion: This study provides a highly effective strategy for enhancing the therapeutic effects of mild PTT toward tumors.

Multi-omics reveals bufadienolide Q-markers of Bufonis Venenum based on antitumor activity and cardiovascular toxicity in zebrafish.

BACKGROUND: Bufonis Venenum (BV) is a traditional animal-based Chinese medicine with therapeutic effects against cancer. However, its clinical use is significantly restricted due to associated cardiovascular risks. BV's value in China's market is typically assessed based on "content priority," focusing on indicator components. However, these components of BV possess both antitumor activity and toxicity, and the correlation between the antitumor activity and toxicity of BV has not yet been elucidated. PURPOSE: This study employs an integrated multi-omics approach to identify bufadienolide Q-markers and explore the correlation between BV's antitumor activity and toxicity. The aim is to establish a more comprehensive method for BV's quality. METHODS: Normal zebrafish and HepG2 xenograft zebrafish were chosen as activity and toxicity evaluation models. Ultra-high performance liquid chromatography (UHPLC) coupled with a linear ion trap orbitrap (LTQ-Orbitrap) mass spectrometry was used to quantify eight batches of BV and key "toxic and effective" components were screened out. Transcriptomic and metabolomic analyses were performed to elucidate the regulatory mechanisms underlying the antitumor activity and cardiovascular toxicity of the key components in BV. RESULTS: Eight key "toxic and effective" compounds were identified: resibufogenin, cinobufagin, arenobufagin, bufotalin, bufalin, gamabufotalin, desacetylcinobufagin, and telocinobufagin. The findings showed that bufalin and cinobufagin interfered with calcium homeostasis through CaV and CaSR, induced cardiotoxicity, and upregulated CASP9 to activate myocardial cell apoptosis. However, desacetylcinobufagin exhibited greater potential in terms of anti-tumor effects. Combining the results of untargeted and targeted metabolomics revealed that desacetylcinobufagin could have a callback effect on differential lipids and correct abnormal energy and amino acid metabolism caused by cancer, similar to cinobufagin and bufalin. Microscale thermophoresis (MST) ligand binding measurements also showed that the binding of desacetylcinobufagin to GPX4 has a more potent ability to induce ferroptosis in tumor cells compared to cinobufagin. CONCLUSION: An innovative evaluation method based on the zebrafish was developed to investigate the relationship between the toxicity and efficacy of BV. This study identified toxicity and activity Q-markers and explored the mechanism between the two effects of BV. The research data could offer valuable insights into the efficacy of BV. Additionally, desacetylcinobufagin, an active ingredient with low toxicity, was found to enhance the quality of BV.

Antibody to Endogenous Cardiotonic Steroid Reverses Vascular Fibrosis and Restores Vasorelaxation in Chronic Kidney Disease.

Marinobufagenin (MBG) is implicated in chronic kidney disease, where it removes Fli1-induced inhibition of the collagen-1. We hypothesized that (i) in nephrectomized rats, aortic fibrosis develops due to elevated plasma MBG and inhibited Fli1, and (ii) that the antibody to MBG reduces collagen-1 and improves vasodilatation. A partial nephrectomy was performed in male Sprague-Dawley rats. Sham-operated animals comprised the control group. At 5 weeks following nephrectomy, rats were administered the vehicle (n = 8), or the anti-MBG antibody (n = 8). Isolated aortic rings were tested for their responsiveness to sodium nitroprusside following endothelin-1-induced constriction. In nephrectomized rats, there was an increase in the intensity of collagen staining in the aortic wall vs. the controls. In antibody-treated rats, the structure of bundles of collagen fibers had ordered organization. Western blots of the aorta had lower levels of Fli1 (arbitrary units, 1 ± 0.05 vs. 0.2 ± 0.01; p < 0.001) and greater collagen-1 (arbitrary units, 1 ± 0.01 vs. 9 ± 0.4; p < 0.001) vs. the control group. Administration of the MBG antibody to rats reversed the effect of the nephrectomy on Fli1 and collagen-1 proteins. Aortic rings pretreated with endothelin-1 exhibited 50% relaxation following the addition of sodium nitroprusside (EC50 = 0.28 µmol/L). The responsiveness of the aortic rings obtained from nephrectomized rats was markedly reduced (EC50 = 3.5 mol/L) compared to the control rings. Treatment of rats with the antibody restored vasorelaxation. Thus, the anti-MBG antibody counteracts the Fli1-collagen-1 system and reduces aortic fibrosis.

Advances on Delivery System of Active Ingredients of Dried Toad Skin and Toad Venom.

Dried toad skin (TS) and toad venom (TV) are the dried skin of the Bufo bufo gargarizans Cantor and the Bufo melanostictus Schneider, which remove the internal organs and the white secretions of the skin and retroauricular glands. Since 2005, cinobufacini preparations have been approved by the State Food and Drug Administration for use as adjuvant therapies in the treatment of various advanced cancers. Meanwhile, bufalenolides has been identified as the main component of TS/TV, exhibiting antitumor activity, inducing apoptosis of cancer cells and inhibiting cancer cell proliferation or metastasis through a variety of signaling pathways. However, clinical agents frequently face limitations such as inherent toxicity at high concentrations and insufficient tumor targeting. Additionally, the development and utilization of these active ingredients are hindered by poor water solubility, low bioavailability, and rapid clearance from the bloodstream. To address these challenges, the design of a targeted drug delivery system (TDDS) aims to enhance drug bioavailability, improve targeting within the body, increase drug efficacy, and reduce adverse reactions. This article reviews the TDDS for TS/TV, and their active components, including passive, active, and stimuli-responsive TDDS, to provide a reference for advancing their clinical development and use.

Pharmacological insights and role of bufalin (bufadienolides) in inflammation modulation: a narrative review.

Bufadienolides, specifically bufalin, have garnered attention for their potential therapeutic application in modulating inflammatory pathways. Bufalin is derived from toad venom and exhibits promising anti-inflammatory properties. Its anti-inflammatory effects have been demonstrated by influencing crucial signaling pathways like NF-B, MAPK, and JAK-STAT, resulting in the inhibition of pro-inflammatory substances like cytokines, chemokines, and adhesion molecules. Bufalin blocks inflammasome activation and reduces oxidative stress, hence increasing its anti-inflammatory properties. Bufalin has shown effectiveness in reducing inflammation-related diseases such as cancer, cardiovascular problems, and autoimmune ailments in preclinical investigations. Furthermore, producing new approaches of medication delivery and combining therapies with bufalin shows potential for improving its effectiveness and reducing adverse effects. This review explores the pharmacological effects and mechanistic approaches of bufalin as an anti-inflammatory agent, which further highlights its potential for therapy and offers the basis for further study on its therapeutic application in inflammation-related disorders.

Bufadienolide-Fatty Acid Conjugates from the Fertilized Eggs of Toad Bufo gargarizans: Isolation, Characterization, Toxicity, and Antiproliferative Evaluation.

Bufadienolides (BDs) are a class of naturally occurring toxins present in amphibian toads. Serving as the chemical weapons, they exist not only in the adult toads but also in toad eggs. Guided by mass spectrometry (MS)-based component analysis and feature-based molecular networking (FBMN), 30 bufadienolide-fatty acid conjugates (BDFs) were isolated from the fertilized eggs of toad Bufo gargrizans, including 25 previously undescribed compounds (1-25). Their chemical structures were elucidated by extensive spectroscopic analysis, chemical methods, and GC-MS. The toxicities of all BDFs and their corresponding free BDs were assessed using the zebrafish model. The structure-toxicity relationship analysis showed that the modification of BDs by hydroxy fatty acids can cause a significant increase of the toxicity. Furthermore, all the isolated compounds were evaluated for their antiproliferative activities in pancreatic cancer cell lines ASPC-1 and PANC10.05. The structure-activity relationship (SAR) analysis revealed that BDFs with hellebrigenin as the bufogenin moiety (6 and 7) exhibited the most potent antiproliferative effect. Further investigation into their functional mechanism demonstrated that 6 and 7 induced apoptosis in pancreatic cancer cells PANC10.05 and significantly suppressed the expression of the apoptosis-related gene c-MYC. In addition, 6 and 7 effectively inhibited the expression of the PI3K/Akt/mTOR pathway in PANC10.05. Moreover, we assessed the efficacy of 6 and 7 on cancer cells from various tissues and observed their broad-spectrum antiproliferative activity.

Bufotalin enhances apoptosis and TMZ chemosensitivity of glioblastoma cells by promoting mitochondrial dysfunction via AKT signaling pathway.

Glioblastoma multiforme (GBM) is the most prevalent and lethal primary intracranial neoplasm in the adult population, with treatments of limited efficacy. Recently, bufotalin has been shown to have anti-cancer activity in a variety of cancers. This investigation aims to investigate the effect of bufotalin on GBM and elucidate its potential underlying mechanism. Our results show that bufotalin not only inhibits the proliferation and epithelial-mesenchymal transition (EMT) but also triggers apoptosis in GBM cells. The result of RNA-seq indicated that bufotalin could induce mitochondrial dysfunction. Moreover, our observations indicate that bufotalin induces an excessive accumulation of intracellular reactive oxygen species (ROS) in GBM cells, leading to mitochondrial dysfunction and the dephosphorylation of AKT. Moreover, bufotalin improved TMZ sensitivity of GBM cells in vitro and in vivo. In conclusion, bufotalin enhances apoptosis and TMZ chemosensitivity of glioblastoma cells by promoting mitochondrial dysfunction via AKT signaling pathway.

Cytochrome P450 Mining for Bufadienolide Diversification.

Bufadienolides are a class of steroids with a distinctive α-pyrone ring at C17, mostly produced by toads and consisting of over 100 orthologues. They exhibit potent cardiotonic and antitumor activities and are active ingredients of the traditional Chinese medicine Chansu and Cinobufacini. Direct extraction from toads is costly, and chemical synthesis is difficult, limiting the accessibility of active bufadienolides with diverse modifications and trace content. In this work, based on the transcriptome and genome analyses, using a yeast-based screening platform, we obtained eight cytochrome P450 (CYP) enzymes from toads, which catalyze the hydroxylation of bufalin and resibufogenin at different sites. Moreover, a reported fungal CYP enzyme Sth10 was found functioning in the modification of bufalin and resibufogenin at multiple sites. A total of 15 bufadienolides were produced and structurally identified, of which six were first discovered. All of the compounds were effective in inhibiting the proliferation of tumor cells, especially 19-hydroxy-bufalin (2) and 1ß-hydroxy-bufalin (3), which were generated from bufalin hydroxylation catalyzed by CYP46A35. The catalytic efficiency of CYP46A35 was improved about six times and its substrate diversity was expanded to progesterone and testosterone, the common precursors for steroid drugs, achieving their efficient and site-specific hydroxylation. These findings elucidate the key modification process in the synthesis of bufadienolides by toads and provide an effective way for the synthesis of unavailable bufadienolides with site-specific modification and active potentials.

Arenobufagin induces cell apoptosis by modulating the cell cycle regulator claspin and the JNK pathway in nasopharyngeal carcinoma cells.

BACKGROUND: The high recurrence rate and incidence of distant metastasis of nasopharyngeal carcinoma (NPC) result in poor prognosis. It is necessary to identify natural compounds that can complement combination radiation therapy. Arenobufagin is commonly used for heart diseases and liver cancer, but its effectiveness in NPC is unclear. STUDY DESIGN AND METHODS: The effect of arenobufagin-induced apoptosis was measured by a cell viability assay, tumorigenic assay, fluorescence assay, and Western blot assay through NPC-039 and NPC-BM cell lines. The protease array, Western blot assay, and transient transfection were used to investigate the underlying mechanism of arenobufagin-induced apoptosis. An NPC xenograft model was established to explore the antitumor activity of arenobufagin in vivo. RESULTS: Our findings indicated that arenobufagin exerted cytotoxic effects on NPC cells, inhibiting proliferation through apoptosis activation. Downregulation of claspin was confirmed in arenobufagin-induced apoptosis. Combined treatment with arenobufagin and mitogen-activated protein kinase inhibitors demonstrated that arenobufagin induced NPC apoptosis through the c-Jun N-terminal kinases (JNK) pathway inhibition. Furthermore, arenobufagin suppressed NPC tumor proliferation in vivo. CONCLUSION: Our results revealed the antitumor effect of arenobufagin in vitro and in vivo. Arenobufagin may have clinical utility in treating NPC due to its suppression of claspin and inhibition of the JNK pathway.

Arenobufagin enhances T-cell anti-tumor immunity in colorectal cancer by modulating HSP90ß accessibility.

BACKGROUND: Colorectal cancer (CRC) is a significant public health issue, ranking as one of the predominant cancer types globally in terms of incidence. Intriguingly, Arenobufagin (Are), a compound extracted from toad venom, has demonstrated the potential to inhibit tumor growth effectively. PURPOSE: This study aimed to explore Are's molecular targets and unravel its antitumor mechanism in CRC. Specifically, we were interested in its impact on immune checkpoint modulation and correlations with HSP90ß-STAT3-PD-L1 axis activity. METHODS: We investigated the in vivo antitumor effects of Are by constructing a colorectalcancer subcutaneous xenograft mouse model. Subsequently, we employed single-cell multi-omics technology to study the potential mechanism by which Are inhibits CRC. Utilizing target-responsive accessibility profiling (TRAP) technology, we identified heatshock protein 90ß (HSP90ß) as the direct target of Are, and confirmed this through a microscale thermophoresis experiment (MST). Further downstream mechanisms were explored through techniques such as co-immunoprecipitation, Western blotting, qPCR, and immunofluorescence. Concurrently, we arrived at the same research conclusion at the organoid level by co-cultivating with immune cells. RESULTS: We observed that Are inhibits PD-Ll expression in CRC tumor xenografts at low concentrations. Moreover, TRAP revealed that HSP90ß's accessibility significantly decreased upon Are binding. We demonstrated a decrease in the activity of the HSP90ß-STAT3-PD-Ll axis following low-concentration Are treatment in vivo. The PDO analysis showed improved enrichment of lymphocytes, particularly T cells, on the PDOs following Are treatment. CONCLUSION: Contrary to previous research focusing on the direct cytotoxicity of Are towards tumor cells, our findings indicate that it can also inhibit tumor growth at lower concentrations through the modulation of immune checkpoints. This study unveils a novel anti-tumor mechanism of Are and stimulates contemplation on the dose-response relationship of natural products, which is beneficial for the clinical translational application of Are.

Isolation, Identification and Chemical Modification of Bufadienolides from Bufo melanostictus Schneider and Their Cytotoxic Activities against Prostate Cancer Cells.

The traditional Chinese medicine toad venom (Venenum bufonis) has been extensively used to treat various diseases, including cancers, in China and other Southeast Asian countries. The major constituents of toad venom, e.g., bufadienolides and alkaloids, exhibit broad-spectrum pharmacological effects in cancers. Herein, two new bufadienolides (1 and 2), along with eleven known compounds (3-13) were successfully isolated from Bufo melanostictus Schneider. Their structures were elucidated by extensive spectroscopic data and X-ray diffraction analysis. Furthermore, four lactam derivatives were synthesized through the transformation of bufadienolides lactones. The inhibitory effects of these compounds against human prostate cancer cell lines PC-3 and DU145 were evaluated. The outcomes indicated a notable trend, with a substantial subset displaying nanomolar range IC50 values against PC-3 and DU145 cells, underscoring their pronounced cytotoxicity. Moreover, a noteworthy distinction surfaces, wherein lactones consistently outperformed their lactam counterparts, further validating their heightened potency for the treatment of prostate cancer. This study contributes significant preclinical evidence substantiating the therapeutic viability of bufadienolides and toad venom as intervention strategies for prostate cancer.

Validation of Core Ingredients and Molecular Mechanism of Cinobufotalin Injection Against Liver Cancer.

Purpose: Cinobufotalin injection has obvious curative effects on liver cancer patients with less toxicity and fewer side effects than other therapeutic approaches. However, the core ingredients and mechanism underlying these anti-liver cancer effects have not been fully clarified due to its complex composition. Methods: Multidimensional network analysis was used to screen the core ingredients, key targets and pathways underlying the therapeutic effects of cinobufotalin injection on liver cancer, and in vitro and in vivo experiments were performed to confirm the findings. Results: By construction of ingredient networks and integrated analysis, eight core ingredients and ten key targets were finally identified in cinobufotalin injection, and all of the core ingredients are tightly linked with the key targets, and these key targets are highly associated with the cell cycle-related pathways, supporting that both cinobufotalin injection and its core ingredients exert anti-liver cancer roles by blocking cell cycle-related pathways. Moreover, in vitro and in vivo experiments confirmed that either cinobufotalin injection or one of its core ingredients, cinobufagin, significantly inhibited cell proliferation, colony formation, cell cycle progression and xenograft tumor growth, and the key target molecules involved in the cell cycle pathway such as CDK1, CDK4, CCNB1, CHEK1 and CCNE1, exhibit consistent changes in expression after treatment with cinobufotalin injection or cinobufagin. Interestingly, some key targets CDK1, CDK4, PLK1, CHEK1, TTK were predicted to bind with multiple of core ingredients of cinobufotalin injection, and the affinity between one of the critical ingredients cinobufagin and key target CDK1 was further confirmed by SPR assay. Conclusion: Cinobufotalin injection was confirmed to includes eight core ingredients, and they play therapeutic effects in liver cancer by blocking cell cycle-related pathways, which provides important insights for the mechanism of cinobufotalin injection antagonizing liver cancer and the development of novel small molecule anti-cancer drugs.

MiR-2110 induced by chemically synthesized cinobufagin functions as a tumor-metastatic suppressor via targeting FGFR1 to reduce PTEN ubiquitination degradation in nasopharyngeal carcinoma.

Tumor cell metastasis is the key cause of death in patients with nasopharyngeal carcinoma (NPC). MiR-2110 was cloned and identified in Epstein-Barr virus (EBV)-positive NPC, but its role is unclear in NPC. In this study, we investigated the effect of miR-2110 on NPC metastasis and its related molecular basis. In addition, we also explored whether miR-2110 can be regulated by cinobufotalin (CB) and participate in the inhibition of CB on NPC metastasis. Bioinformatics, RT-PCR, and in situ hybridization were used to observe the expression of miR-2110 in NPC tissues and cells. Scratch, Boyden, and tail vein metastasis model of nude mouse were used to detect the effect of miR-2110 on NPC metastasis. Western blot, Co-IP, luciferase activity, colocalization of micro confocal and ubiquitination assays were used to identify the molecular mechanism of miR-2110 affecting NPC metastasis. Finally, miR-2110 induced by CB participates in CB-stimulated inhibition of NPC metastasis was explored. The data showed that increased miR-2110 significantly suppresses NPC cell migration, invasion, and metastasis. Suppressing miR-2110 markedly restored NPC cell migration and invasion. Mechanistically, miR-2110 directly targeted FGFR1 and reduced its protein expression. Decreased FGFR1 attenuated its recruitment of NEDD4, which downregulated NEDD4-induced phosphatase and tensin homolog (PTEN) ubiquitination and degradation and further increased PTEN protein stability, thereby inactivating PI3K/AKT-stimulated epithelial-mesenchymal transition signaling and ultimately suppressing NPC metastasis. Interestingly, CB, a potential new inhibitory drug for NPC metastasis, significantly induced miR-2110 expression by suppressing PI3K/AKT/c-Jun-mediated transcription inhibition. Suppression of miR-2110 significantly restored cell migration and invasion in CB-treated NPC cells. Finally, a clinical sample assay indicated that reduced miR-2110 was negatively correlated with NPC lymph node metastasis and positively related to NPC patient survival prognosis. In summary, miR-2110 is a metastatic suppressor involving in CB-induced suppression of NPC metastasis.

Arenobufagin modulation of PCSK9-mediated cholesterol metabolism induces tumor-associated macrophages polarisation to inhibit hepatocellular carcinoma progression.

BACKGROUND: The tumor microenvironment (TME) of hepatocellular carcinoma is heterogeneous enough to be prone to drug resistance and multidrug resistance during treatment, and reprogramming of cholesterol metabolism in TME mediates tumor-associated macrophages (TAMs) polarization, which has an impact on the regulation of malignant tumor progression. Arenobufagin (ARBU) was extracted and isolated from toad venom (purity ≥98 %), which is the main active ingredient of the traditional Chinese medicine Chan'su with good anti-tumor effects. PURPOSE: To investigate the regulatory effect of ARBU on lipid metabolism in tumor microenvironment, interfere with macrophage polarization, and determine its mechanism of action on liver cancer progression. METHODS: In this study, the inhibitory effect of ARBU on the proliferation of Hepa1-6 in C57 mice and the safety of administration were evaluated by establishing a transplanted tumor model of Hepa1-6 hepatocellular carcinoma mice and using 5-FU as a positive control drug. In addition, we constructed a co-culture system of Hepa1-6 cells and primary mouse macrophages to study the effects of ARBU on the polarization phenotypic transformation of macrophages and the proliferation and migration of hepatoma cells. The influence of ARBU on the metabolism of lipids in the hepatocellular carcinoma mouse model was investigated by combining it with lipidomics technology. The influence of ARBU on the PCSK9/LDL-R signaling pathway and macrophage polarization, which regulate cholesterol metabolism, was tested by using qRT-PCR, gene editing, IF, and WB. CONCLUSION: ARBU significantly inhibited the proliferation of Hepa1-6 in vivo and in vitro, regulated cholesterol metabolism, and promoted the M1-type polarization of macrophages in the tumor microenvironment. ARBU inhibits cholesterol synthesis in the TME through the PCSK9/LDL-R signaling pathway, thereby blocking macrophage M2 polarization, promoting apoptosis of the tumor cells, and inhibiting their proliferation and migration.

Arenobufagin inhibits lung metastasis of colorectal cancer by targeting c-MYC/Nrf2 axis.

BACKGROUND: Colorectal cancer (CRC) is one of the commonest cancers worldwide. Metastasis is the most common cause of death in patients with CRC. Arenobufagin is an active component of bufadienolides, extracted from toad skin and parotid venom. Arenobufagin reportedly inhibits epithelial-to-mesenchymal transition (EMT) and metastasis in various cancers. However, the mechanism through which arenobufagin inhibits CRC metastasis remains unclear. PURPOSE: This study aimed to elucidate the molecular mechanisms by which arenobufagin inhibits CRC metastasis. METHODS: Wound-healing and transwell assays were used to assess the migration and invasion of CRC cells. The expression of nuclear factor erythroid-2-related factor 2 (Nrf2) in the CRC tissues was assessed using immunohistochemistry. The protein expression levels of c-MYC and Nrf2 were detected by immunoblotting. A mouse model of lung metastasis was used to study the effects of arenobufagin on CRC lung metastasis in vivo. RESULTS: Arenobufagin observably inhibited the migration and invasion of CRC cells by downregulating c-MYC and inactivating the Nrf2 signaling pathway. Pretreatment with the Nrf2 inhibitor brusatol markedly enhanced arenobufagin-mediated inhibition of migration and invasion, whereas pretreatment with the Nrf2 agonist tert­butylhydroquinone significantly attenuated arenobufagin-mediated inhibition of migration and invasion of CRC cells. Furthermore, Nrf2 knockdown with short hairpin RNA enhanced the arenobufagin-induced inhibition of the migration and invasion of CRC cells. Importantly, c-MYC acts as an upstream modulator of Nrf2 in CRC cells. c-MYC knockdown markedly enhanced arenobufagin-mediated inhibition of the Nrf2 signaling pathway, cell migration, and invasion. Arenobufagin inhibited CRC lung metastasis in vivo. Together, these findings provide evidence that interruption of the c-MYC/Nrf2 signaling pathway is crucial for arenobufagin-inhibited cell metastasis in CRC. CONCLUSIONS: Collectively, our findings show that arenobufagin could be used as a potential anticancer agent against CRC metastasis. The arenobufagin-targeted c-MYC/Nrf2 signaling pathway may be a novel chemotherapeutic strategy for treating CRC.

Inhibitory Impact Of Cinobufagin In Triple-Negative Breast Cancer Metastasis: Involvements Of Macrophage Reprogramming Through Upregulated MME and Inactivated FAK/STAT3 Signaling.

BACKGROUND: Cinobufagin (CBG), a key bioactive component in cinobufacini, exhibits antitumor properties. This study explores CBG's impact on triple-negative breast cancer (TNBC) metastasis and elucidates the underpinning mechanism. METHODS: Murine xenograft and orthotopic metastatic TNBC models were generated and treated with CBG. The burden of metastatic tumor in the mouse lung, the epithelial to mesenchymal transition (EMT) markers, and macrophage polarization markers within the tumors were examined. The phenotype of tumor-associated macrophages (TAMs) and mobility of TNBCs in vitro in a macrophage-TNBC cell coculture system were analyzed. Physiological targets of CBG were identified by bioinformatics analyses. RESULTS: CBG treatment significantly alleviated lung tumor burden and EMT activity. It triggered an M2-to-M1 shift in TAMs, resulting in decreased TNBC cell migration, invasion, and EMT in vitro. CBG upregulated membrane metalloendopeptidase (MME) expression, suppressing FAK and STAT3 phosphorylation. Silencing of MME, either in mice or TAMs, counteracted CBG effects, reinstating M2 TAM predominance and enhancing TNBC cell metastasis. Cotreatment with Defactinib, a FAK antagonist, reversed M2 TAM polarization and TNBC cell metastasis. Notably, MME silencing in TNBC cells had no impact on CBG-suppressed malignant properties, indicating MME's indirect involvement in TNBC cell behavior through TAM mediation. CONCLUSION: This study unveils CBG's ability to enhance MME expression, deactivate FAK/STAT3 signaling, and inhibit TNBC metastasis by suppressing M2-skewed macrophages.

Mechanism of bufalin inhibition of colon cancer liver metastasis by regulating M2-type polarization of Kupffer cells induced by highly metastatic colon cancer cells.

Patients with metastatic colorectal cancer often have poor outcomes, primarily due to hepatic metastasis. Colorectal cancer (CRC) cells have the ability to secrete cytokines and other molecules that can remodel the tumor microenvironment, facilitating the spread of cancer to the liver. Kupffer cells (KCs), which are macrophages in the liver, can be polarized to M2 type, thereby promoting the expression of adhesion molecules that aid in tumor metastasis. Our research has shown that huachanshu (with bufalin as the main active monomer) can effectively inhibit CRC metastasis. However, the underlying mechanism still needs to be thoroughly investigated. We have observed that highly metastatic CRC cells have a greater ability to induce M2-type polarization of Kupffer cells, leading to enhanced metastasis. Interestingly, we have found that inhibiting the expression of IL-6, which is highly expressed in the serum, can reverse this phenomenon. Notably, bufalin has been shown to attenuate the M2-type polarization of Kupffer cells induced by highly metastatic Colorectal cancer (mCRC) cells and down-regulate IL-6 expression, ultimately inhibiting tumor metastasis. In this project, our aim is to study how high mCRC cells induce M2-type polarization and how bufalin, via the SRC-3/IL-6 pathway, can inhibit CRC metastasis. This research will provide a theoretical foundation for understanding the anti-CRC effect of bufalin.

Resibufogenin: An Emerging Therapeutic Compound with Multifaceted Pharmacological Effects - A Comprehensive Review.

Resibufogenin (RBG), a significant bufadienolide compound found in the traditional Chinese medicine Chansu, has garnered increasing attention in recent years for its wide range of pharmacological effects. This compound has shown promising potential in various therapeutic areas, including oncology, cardiology, and respiratory medicine. Among its notable properties, the anticancer effects of RBG are particularly striking, positioning it as a potential candidate for innovative cancer treatments. The mechanism of action of RBG is diverse, impacting various cellular processes. Its anticancer efficacy has been observed in different types of cancer cells, where it induces apoptosis and inhibits cell proliferation. Beyond its oncological applications, RBG also demonstrates substantial anti-inflammatory and antiviral activities. These properties suggest its utility in managing chronic inflammatory disorders and viral infections, respectively. The compound's cardiotonic effects are also noteworthy, providing potential benefits in cardiovascular health, particularly in heart failure management. Additionally, RBG has shown effectiveness in blood pressure regulation and respiratory function improvement, making it a versatile agent in the treatment of hypertension and respiratory disorders. However, despite these promising aspects, systematic reviews focusing specifically on RBG are limited. This article aims to address this gap by comprehensively reviewing RBG's origin, physiological, and pharmacological effects. The review will serve as a crucial reference for clinicians and researchers interested in the therapeutic applications of RBG, highlighting its potential in various medical domains. By synthesizing current research findings, this review will facilitate a deeper understanding of RBG's role in medicine and encourage further investigation into its clinical uses.