RESUMO
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.
Assuntos
Bufanolídeos , Sistema Enzimático do Citocromo P-450 , Bufanolídeos/química , Bufanolídeos/metabolismo , Bufanolídeos/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Animais , Humanos , Antineoplásicos/farmacologia , Antineoplásicos/química , Antineoplásicos/metabolismo , Hidroxilação , Linhagem Celular Tumoral , Bufonidae/metabolismo , Proliferação de Células/efeitos dos fármacosRESUMO
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.
Assuntos
Bufanolídeos , Neoplasias Colorretais , Proteínas de Choque Térmico HSP90 , Fator de Transcrição STAT3 , Ensaios Antitumorais Modelo de Xenoenxerto , Bufanolídeos/farmacologia , Animais , Neoplasias Colorretais/tratamento farmacológico , Humanos , Camundongos , Fator de Transcrição STAT3/metabolismo , Linfócitos T/efeitos dos fármacos , Linhagem Celular Tumoral , Antígeno B7-H1 , Camundongos Nus , Camundongos Endogâmicos BALB C , Venenos de Anfíbios/farmacologia , FemininoRESUMO
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.
Assuntos
Venenos de Anfíbios , Antineoplásicos , Bufanolídeos , Neoplasias da Próstata , Humanos , Masculino , Animais , Neoplasias da Próstata/tratamento farmacológico , Antineoplásicos/farmacologia , Venenos de Anfíbios/farmacologia , Bufanolídeos/farmacologia , Bufonidae , Lactamas , LactonasRESUMO
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.
Assuntos
Bufanolídeos , Carcinoma Hepatocelular , Proliferação de Células , Colesterol , Neoplasias Hepáticas , Camundongos Endogâmicos C57BL , Pró-Proteína Convertase 9 , Microambiente Tumoral , Macrófagos Associados a Tumor , Animais , Bufanolídeos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Pró-Proteína Convertase 9/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Macrófagos Associados a Tumor/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Camundongos , Colesterol/metabolismo , Proliferação de Células/efeitos dos fármacos , Linhagem Celular Tumoral , Masculino , Movimento Celular/efeitos dos fármacos , Venenos de Anfíbios/farmacologiaRESUMO
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.
Assuntos
Bufanolídeos , Neoplasias Colorretais , Neoplasias Pulmonares , Animais , Camundongos , Humanos , Fator 2 Relacionado a NF-E2/metabolismo , Neoplasias Colorretais/patologia , Linhagem Celular Tumoral , Bufanolídeos/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Transição Epitelial-Mesenquimal , Movimento Celular , Regulação Neoplásica da Expressão Gênica , Proliferação de Células , Metástase NeoplásicaRESUMO
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.
Assuntos
Bufanolídeos , Bufanolídeos/farmacologia , Bufanolídeos/uso terapêutico , Cardiotônicos/farmacologia , Cardiotônicos/uso terapêutico , Coração , Pressão SanguíneaRESUMO
Chinese medicine cinobufacini is an extract from the dried skin of Bufo bufo gargarizans Cantor, with active ingredients of bufadienolides and indole alkaloids. With further research and clinical applications, it is found that cinobufacini alone or in combination with other therapeutic methods can play an anti-tumor role by controlling proliferation of tumor cells, promoting apoptosis, inhibiting formation of tumor neovascularization, reversing multidrug resistance, and regulating immune response; it also has the functions of relieving cancer pain and regulating immune function. In this paper, the chemical composition, pharmacological effects, clinical applications, and adverse reactions of cinobufacini are summarized. However, the extraction of monomer components of cinobufacini, the relationship between different mechanisms, and the causes of adverse reactions need to be further studied. Also, high-quality clinical studies should be conducted.
Assuntos
Venenos de Anfíbios , Bufanolídeos , Neoplasias , Animais , Humanos , Neoplasias/tratamento farmacológico , Bufonidae , Venenos de Anfíbios/farmacologia , Venenos de Anfíbios/uso terapêutico , Venenos de Anfíbios/química , Bufanolídeos/farmacologia , Bufanolídeos/uso terapêuticoRESUMO
Bufonis Venenum, an animal medicinal material, is widely used for treating cardiovascular diseases and pain induced by rheumatics or malignant tumors. In view of the high activity and high toxicity, it is of great significance to pay attention to the quality control of Bufonis Venenum to ensure the safety and effectiveness of its preparations. China's drug standards involve 102 preparations(474 batch numbers) containing Bufonis Venenum approved for sale, including 14 preparations in the Chinese Pharmacopoeia(2020 edition) and 68 preparations in the standards issued by the Ministry of Health Drug Standard of the People's Republic of China. Bufonis Venenum is mostly used in pill and powder preparations in the form of raw powder, with the main functions of clearing heat, removing toxin, relieving swelling and pain, replenishing qi, activating blood, opening orifice, and awakening brain. Except the high level of quality control for Bufonis Venenum in the preparations in the Chinese Pharmacopoeia(2020 edition), the quality control standards of Bufonis Venenum in other preparations are low or even absent. Therefore, it is urgent to conduct research on the improvement of quality standards for the preparations containing Bufonis Venenum. This study retrieved the reports focusing on the quality evaluation and quality control of the preparations containing Bufonis Venenum from CNKI, PubMed, and Web of Science. Qualitative and quantitative analysis methods for 64 preparations containing Bufonis Venenum have been reported, mainly including thin-layer chromatography, HPLC fingerprint, and multi-component content determination. The index components mainly involved bufadienolides, such as gamabufalin, arenobufagin, bufotalin, bufalin, cinobufagin, and resibufogenin. According to the literature information, this paper suggests that attention should be paid to the correlations between the analysis methods and detection indexes of medicinal materials, decoction pieces and preparations, the monitoring of indole alkaloids, and the content uniformity inspection for further improving the quality standards for the preparations containing Bufonis Venenum.
Assuntos
Bufanolídeos , Bufonidae , Animais , Humanos , Pós , Bufanolídeos/farmacologia , Controle de Qualidade , Cromatografia Líquida de Alta Pressão , Dor/tratamento farmacológicoRESUMO
Toad venom is a traditional Chinese medicine with high medicinal value. The existing quality evaluation standards of toad venom have obvious limitations because of the lack of research on proteins. Thus, it is necessary to screen suitable quality markers and establish appropriate quality evaluation methods for toad venom proteins to guarantee their safety and efficacy in clinical applications. SDS-PAGE, HPLC, and cytotoxicity assays were used to analyze differences in protein components of toad venom from different areas. Functional proteins were screened as potential quality markers by proteomic and bioinformatic analyses. The protein components and small molecular components of toad venom were not correlated in content. Additionally, the protein component had strong cytotoxicity. Proteomics analysis showed that 13 antimicrobial proteins, four anti-inflammatory and analgesic proteins, and 20 antitumor proteins were differentially expressed extracellular proteins. A candidate list of functional proteins was coded as potential quality markers. Moreover, Lysozyme C-1, which has antimicrobial activity, and Neuropeptide B (NPB), which has anti-inflammatory and analgesic activity, were identified as potential quality markers for toad venom proteins. Quality markers can be used as the basis of quality studies of toad venom proteins and help to construct and improve safe, scientific, and comprehensive quality evaluation methods.
Assuntos
Venenos de Anfíbios , Bufanolídeos , Animais , Venenos de Anfíbios/química , Proteômica , Bufonidae , Medicina Tradicional Chinesa , Anti-Inflamatórios , Bufanolídeos/farmacologiaRESUMO
Intrinsic or acquired drug resistance of tumor cells is the main cause of tumor chemotherapy failure and tumor-related death. Bufalin (BF) is the main active monomer component extracted from the Traditional Chinese Medicine Toad venom (secretions of glands behind the ears and epidermis of bufo gargarizans and Bufo Melanostictus Schneider). It is a cardiotonic steroid with broad-spectrum anti-cancer effects and has been widely used against various malignant tumors in clinical practice. Pharmacological studies also found that BF has the effect of reversing drug resistance, which provides a new perspective for the application of Traditional Chinese Medicine as a chemosensitizer in cancer therapy. This article provides an extensive search and summary of published research on mitigating drug resistance to BF and reviews its potential mechanisms.
Assuntos
Bufanolídeos , Neoplasias , Humanos , Biofarmácia , Bufanolídeos/farmacologia , Bufanolídeos/uso terapêutico , Neoplasias/tratamento farmacológico , Resistência a MedicamentosRESUMO
OBJECTIVE: To explore the active compounds and targets of cinobufotalin (huachansu) compared with the osteosarcoma genes to obtain the potential therapeutic targets and pharmacological mechanisms of action of cinobufotalin on osteosarcoma through network pharmacology. METHODS: The composition of cinobufotalin was searched by literature retrieval, and the target was selected from the CTD and TCMSP databases. The osteosarcoma genes, found from the GeneCards, OMIM, and other databases, were compared with the cinobufotalin targets to obtain potential therapeutic targets. The protein-protein interaction (PPI) network of potential therapeutic targets, constructed through the STRING database, was inputted into Cytoscape software to calculate the hub genes, using the NetworkAnalyzer. The hub genes were inputted into the Kaplan-Meier Plotter online database for exploring the survival curve. Functional enrichment analysis was identified using the DAVID database. RESULTS: 28 main active compounds of cinobufotalin were explored, including bufalin, adenosine, oleic acid, and cinobufagin. 128 potential therapeutic targets on osteosarcoma are confirmed among 184 therapeutic targets form cinobufotalin. The hub genes included TP53, ACTB, AKT1, MYC, CASP3, JUN, TNF, VEGFA, HSP90AA1, and STAT3. Among the hub genes, TP53, ACTB, MYC, TNF, VEGFA, and STAT3 affect the patient survival prognosis of sarcoma. Through function enrichment analysis, it is found that the main mechanisms of cinobufotalin on osteosarcoma include promoting sarcoma apoptosis, regulating the cell cycle, and inhibiting proliferation and differentiation. CONCLUSION: The possible mechanisms of cinobufotalin against osteosarcoma are preliminarily predicted through network pharmacology, and further experiments are needed to prove these predictions.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Bufanolídeos/farmacologia , Osteossarcoma/tratamento farmacológico , Antineoplásicos/química , Biomarcadores Tumorais/genética , Neoplasias Ósseas/genética , Bufanolídeos/química , Biologia Computacional , Bases de Dados de Compostos Químicos , Bases de Dados de Produtos Farmacêuticos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Medicina Tradicional Chinesa , Farmacologia em Rede , Osteossarcoma/genética , Mapas de Interação de Proteínas/efeitos dos fármacos , Mapas de Interação de Proteínas/genéticaRESUMO
Toad venom (Chansu) is used in the treatment of infectious and inflammatory diseases in China and East/Southeast Asian countries. However, the anti-inflammatory components of toad venom have not yet been systematically evaluated and clearly defined. To investigate the anti-inflammatory effects of toad venom and identify new anti-inflammatory ingredients, we used zebrafish, an alternative drug screening model, to evaluate the anti-inflammatory effects of 14 bufadienolides previously isolated from toad venom. Most of the bufadienolides were found to exert significant anti-inflammatory effects on lipopolysaccharide-, CuSO4-, or tail transection-induced zebrafish inflammatory models. Moreover, gammabufotalin ( 6: ) inhibits lipopolysaccharide-induced inflammation by suppressing the myeloid differentiation primary response 88/nuclear factor-kappa B and STAT3 signal pathways. This study confirms the potential of zebrafish in drug screening, clarifies the anti-inflammatory effects of bufadienolides from toad venom, and indicates that gammabufotalin may be developed as a novel therapeutic agent for inflammatory diseases in the future.
Assuntos
Venenos de Anfíbios , Bufanolídeos , Animais , Anti-Inflamatórios/farmacologia , Bufanolídeos/farmacologia , Lipopolissacarídeos , Peixe-ZebraRESUMO
Hepatocellular carcinoma (HCC) is one of the deadliest cancers with high mortality and poor prognosis, and the investigation on new approaches and effective drugs for HCC therapy is of great significance. In our study, we demonstrate that treatment with cinobufagin, a natural compound isolated from traditional chinese medicine Chansu, reduces proliferation and the colony formation capacity of the human hepatoma cells in vitro, in addition, cinobufagin induces mitotic arrest in human hepatoma cells. The results of a network pharmacology-based analysis show that EGFR, MAPK1, PTK2, CDK2, MAPK3, ESR1, CDK1, PRKCA, AR, and CSNK2A1 are the key targets involved in the anti-tumor activities of cinobufagin, additionally, several signaling pathways such as proteoglycans in cancer, pathways in cancer, HIF-1 signaling pathway, VEGF signaling pathway, ErbB signaling pathway, and PI3K-AKT signaling pathway are identified as the potential pathways involved in the inhibitory effects of cinobufagin against HCC. Furthermore, at the molecular level, we find that cinobufagin decreases EGFR expression and CDK2 activity in human hepatoma cells. Inhibition of EGFR or CDK2 expression could not only suppress the growth of tumor cells but also enhance the inhibitory effects of cinobufagin on the proliferative potential of human hepatoma cells. We also demonstrate that EGFR positively regulates CDK2 expression. Furthermore, EGFR inhibitor gefitinib or CDK2 inhibitor CVT-313 synergistically enhances anticancer effects of cinobufagin in human hepatoma cells. Taken together, these findings indicate that cinobufagin may exert antitumor effects by suppressing EGFR-CDK2 signaling, and our study suggests that cinobufagin may be a novel, promising anticancer agent for the treatment of HCC.
Assuntos
Antineoplásicos/farmacologia , Bufanolídeos/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Quinase 2 Dependente de Ciclina/metabolismo , Neoplasias Hepáticas/tratamento farmacológico , Farmacologia em Rede , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Quinase 2 Dependente de Ciclina/genética , Regulação para Baixo , Sinergismo Farmacológico , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Receptores ErbB/metabolismo , Gefitinibe/farmacologia , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Células Hep G2 , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Pontos de Checagem da Fase M do Ciclo Celular/efeitos dos fármacos , Mapas de Interação de Proteínas , Inibidores de Proteínas Quinases/farmacologia , Purinas/farmacologia , Transdução de SinaisRESUMO
Bufonis Venenum,the dried secretion of Bufo bufo gargarizans or B. melanostictus,is toxic and hard with the efficacy of removing toxicity for detumescence and relieving pain. The processing of Bufonis Venenum dates back to the Song dynasty. In addition to the wine-processing,milk-processing and talcum powder-processing,there were some other kinds of processing methods in ancient times,such as baking,calcining,water-soaking and vinegar-processing. Modern studies have shown that the Bufonis Venenum has the main chemical components of bufadienolides,indole alkaloids sterols,and other compounds. It has the pharmacological effects of antitumor,cardiac,antibacterial,and analgesic activities,local anesthesia,and so on. This paper reviews the processing evolution,chemical components and pharmacological effects of Bufonis Venenum,providing references for its special processing and modern research as well as the theoretical basis for the research on its processing mechanism and quality control.
Assuntos
Bufanolídeos , Animais , Bufanolídeos/farmacologia , Bufonidae , Controle de QualidadeRESUMO
Peritoneal dissemination threatens the survival of patients with gastric cancer (GC). Bufalin is an extract of traditional Chinese medicine, which has been proved to have anticancer effect. The target of bufalin in suppressing gastric cancer peritoneal dissemination (GCPD) and the underlying mechanism are still unclear. In this research, GC cell line MGC-803 and high-potential peritoneal dissemination cell line MKN-45P were treated with bufalin or L-NAME. Malignant biological behavior and protein level of GC cell lines were detected with MTT, wound healing, transwell, adhesion, and western blotting. Bioinformatics analysis and patient tissues were used to verify the role of endothelial nitric oxide synthase (NOS3) in GC. Mice model was used to assess the effect of bufalin and role of NOS3 in vivo. We found that bufalin inhibited the proliferation, invasion, and migration in GC cell lines. NOS3, which was an independent prognostic factor of GC patients, was predicted to be a potential target of bufalin. Further experiments proved that bufalin reduced the phosphorylation of NOS3, thereby inhibiting the mitogen-activated protein kinase (MAPK) signaling pathway, and ultimately suppressed GCPD by inhibiting EMT process. In conclusion, NOS3 was a potential therapeutic target and prognostic biomarker of GC. Bufalin could suppress GCPD through NOS3-MAPK signaling pathway, which provided more evidence support for intraperitoneal perfusion of bufalin to treat GCPD.
Assuntos
Biomarcadores Tumorais/metabolismo , Bufanolídeos/farmacologia , Regulação Neoplásica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Neoplasias Peritoneais/tratamento farmacológico , Neoplasias Gástricas/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Apoptose , Biomarcadores Tumorais/genética , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas Quinases Ativadas por Mitógeno/genética , Óxido Nítrico Sintase Tipo III/genética , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Transdução de Sinais , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Bufalin is an active component of the traditional Chinese medicine "Chan Su" and is reported to play anti-tumor roles in cancer development, but its functional mechanism is largely unclear. This study intends to explore a potential action mode of bufalin in NSCLC. MATERIALS AND METHODS: The malignant properties of NSCLC, including cell viability, proliferation, adhesion capacity, migration and invasion, were monitored by cell counting kit-8 (CCK-8), adhesion assay and transwell assay, respectively. The expression of circ_0046264 and miR-522-3p was detected by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of proliferation- and migration-related markers was examined by western blot. The putative relationship between circ_0046264 and miR-522-3p was verified by dual-luciferase reporter assay, RIP assay and RNA pull-down assay. Animal experiments in nude mice were performed to investigate the role of bufalin in vivo. RESULTS: Bufalin treatment inhibited cell viability, colony formation, cell adhesion capacity, migration and invasion in NSCLC cells. Bufalin facilitated the expression of circ_0046264, and circ_0046264 overexpression also inhibited NSCLC cell viability, colony formation, cell adhesion capacity, migration and invasion. Besides, circ_0046264 knockdown partially counteracted the effects of bufalin. Further, miR-522-3p was identified as a target of circ_0046264, and its deficiency reversed the effects of circ_0046264 knockdown to suppress malignant activities of NSCLC cells. In addition, bufalin restrained the tumor growth and development in vivo via enhancing the expression of circ_0046264. CONCLUSION: Bufalin played an anti-tumor role in NSCLC by modulating the circ_0046264/miR-522-3p pathway, which might be a potential functional mechanism of bufalin in NSCLC.
Assuntos
Antineoplásicos/administração & dosagem , Bufanolídeos/administração & dosagem , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , MicroRNAs/genética , RNA Circular/genética , Células A549 , Animais , Antineoplásicos/farmacologia , Bufanolídeos/farmacologia , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Pulmonares/genética , Masculino , Camundongos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
CONTEXT: Bufadienolide compounds occur in many plants and animal species and have strong cardiac and anti-inflammatory properties. The compounds have been recently investigated for cytotoxic and antitumor activity. OBJECTIVE: The cytotoxic effect of bersaldegenin-1,3,5-orthoacetate - a bufadienolide steroid occuring in plants from Kalanchoe genus (Crassulaceae), was evaluated with cervical cancer HeLa cells in vitro. MATERIALS AND METHODS: The cytotoxic activity of the compound (at 0.1-20.0 µg/mL) on the cells was determined by Real-Time Cell Analysis (RTCA) system for 24 h. The estimation of cell cycle arrest, reactive oxygen species (ROS) production, reduction of mitochondrial membrane potential (MMP), and caspases-3/7/9 activity in the HeLa cells treated with the compound was done by flow cytometry and luminometric technique. DNA damage in the cells was estimated by immunofluorescence staining and the comet assay with etoposide as a positive control. RESULTS: The compound had strong effect on the cells (IC50 = 0.55 µg/mL) by the suppression of HeLa cells proliferation in G2/M phase of cell cycle and induction of cell death through double-stranded DNA damage and reactive oxygen species overproduction. Furthermore, we did not observe an increase in the activity of caspase-3/7/9 in the treated cells as well as a decrease in cellular mitochondrial membrane potential. Gene expression analysis revealed the overexpression of NF-Kappa-B inhibitors genes (>2-fold higher than control) in the treated cells. CONCLUSIONS: Bersaldegenin-1,3,5-orthoacetate induces cell cycle arrest and caspase-independent cell death through double-stranded DNA damage. These results are an important step in further studies on cell death signalling pathways induced by bufadienolides.
Assuntos
Bufanolídeos/farmacologia , Caspases/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Extratos Vegetais/farmacologia , Neoplasias do Colo do Útero/metabolismo , Animais , Bufanolídeos/isolamento & purificação , Bufanolídeos/uso terapêutico , Bufonidae , Pontos de Checagem do Ciclo Celular/fisiologia , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Dano ao DNA/fisiologia , Feminino , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Potencial da Membrana Mitocondrial/fisiologia , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/uso terapêutico , Espécies Reativas de Oxigênio/antagonistas & inibidores , Espécies Reativas de Oxigênio/metabolismo , Neoplasias do Colo do Útero/tratamento farmacológicoRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Cinobufacini is extracted from the skins and parotid venom glands of the toad for treating symptoms like swelling and pain in ancient times. Nowadays, cinobifucini injection has also achieved satisfactory therapeutic effects on hepatocellular carcinoma (HCC) in China. AIM OF THE STUDY: Our previous work found that bufothionine, an alkaloid abundant in cinobufacini injection, induced mitochondria-mediated apoptosis. In this work, the underlying effects of bufothionine on autophagy in HCC and its possible dependent pathway were investigated. METHODS: CCK-8 and Hoechst staining assays were performed to verify effects of drugs on proliferation and apoptosis of SMMC7721 cell. H22-tumor-bearing mice model was established by inoculating ascites fluid. HE staining was used to observe pathological changes in liver and tumor tissues. ELISA and Western blot experiments were conducted to investigate IL-6/JAK2/STAT3 signaling pathway. The effects of drugs on expressions of autophagic relative proteins were investigated by Western blot in vitro and in vivo. RESULTS: In vitro, CCK-8 and Hoechst staining assays showed that bufothionine inhibited SMMC7721 cell proliferation and promoted apoptosis at 100 µM. In vivo, bufothionine relieved symptoms of H22-tumor-bearing mice and exerted anti-inflammation activity. ELISA and Western blot demonstrated that bufothionine significantly reduced serum IL-6 concentration, suppressed p-Stat3tyr705, p-Stat3ser727 and Jak2 expressions in tumor tissues and upregulated Atg5, Atg7 and LC3â ¡ expressions in SMMC7721 cell and H22 tumor. CONCLUSION: This is the first report showing that bufothionine might induce autophagy in HCC by inhibiting JAK2/STAT3 pathway, presenting a possible anti-cancer mechanism of bufothionine in cinobufacini injection.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Autofagia/efeitos dos fármacos , Bufanolídeos/farmacologia , Alcaloides Indólicos/farmacologia , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias/patologia , Compostos de Quinolínio/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Bufanolídeos/química , Bufanolídeos/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Alcaloides Indólicos/uso terapêutico , Interleucina-6/antagonistas & inibidores , Janus Quinase 2/antagonistas & inibidores , Janus Quinase 2/metabolismo , Neoplasias Hepáticas Experimentais/metabolismo , Neoplasias Hepáticas Experimentais/patologia , Masculino , Camundongos , Neoplasias/metabolismo , Compostos de Quinolínio/uso terapêutico , Fator de Transcrição STAT3/antagonistas & inibidores , Fator de Transcrição STAT3/metabolismoRESUMO
Resibufogenin (RB) has been used for cancer treatment, but the underlying mechanisms are still unclear. This study aimed to investigate the effects of RB treatment on colorectal cancer (CRC) cells, and to determine the underlying mechanisms. The cell counting kit-8 assay was used to determine cell viability. Cell morphology was observed under light microscopy, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay was employed to detect cell apoptosis. Intracellular ferrous iron (Fe2+ ), malondialdehyde (MDA), glutathione (GSH), and reactive oxygen species levels were detected by using commercial iron assay kit, MDA assay kit, GSH assay kit, and 2,7-dichlorodihydrofluorescein diacetate probes, respectively. The protein expressions were determined by Western blot and immunohistochemistry. RB inhibited cell viability in the CRC cell lines (HT29 and SW480) in a dose- and time-dependent manner, and caused cytotoxicity to the normal colonic epithelial cell line (NCM460) at high dose. Similarly, RB induced morphological changes in CRC cells from normal to round shape, and promoted cell death. Of note, RB triggered oxidative stress and ferroptotic cell death in CRC cells, and only ferroptosis inhibitors (deferoxamine and ferrostatin-1), instead of inhibitors for other types of cell death (apoptosis, autophagy, and necroptosis), reversed the inhibitory effects of RB on CRC cell proliferation. Furthermore, glutathione peroxidase 4 (GPX4) was inactivated by RB treatment, and overexpression of GPX4 alleviated RB-induced oxidative cell death in CRC cells. Consistently, the in vivo experiments validated that RB also triggered oxidative stress, and inhibited CRC cells growth and tumorigenicity in mice models. RB can inhibit CRC cells growth and tumorigenesis by triggering ferroptotic cell death in a GPX4 inactivation-dependent manner.
Assuntos
Bufanolídeos/farmacologia , Carcinogênese/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/patologia , Ferroptose/efeitos dos fármacos , Fosfolipídeo Hidroperóxido Glutationa Peroxidase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Forma Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/metabolismo , Humanos , Estresse Oxidativo/efeitos dos fármacosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Chansu, dried secretions from Bufonidae, has long been used for cancer treatment as a traditional Chinese medicine. In searching for effective anti-hepatoma agents from Chansu, our preliminary drug screening found that a bufadienolide, namely 1ß-hydroxyl-arenobufagin (1ß-OH-ABF), displays anti-hepatoma activities. However, the anti-hepatoma effects and molecular mechanisms of 1ß-OH-ABF have not been defined. AIM OF THE STUDY: To evaluate the anti-hepatoma activity of 1ß-OH-ABF against liver cancer Hep3B and HepG2 cells in vitro and in vivo, as well as explore the underlying mechanisms. MATERIALS AND METHODS: The anti-proliferative effects of 1ß-OH-ABF on liver cancer Hep3B, HepG2, HuH7, SK-HEP-1 and normal hepatocyte LO2 cells were examined by MTT assay and colony formation assay. Hoechst 33258 staining and Annexin V-FITC/PI staining assay were used to analyze apoptosis induced by 1ß-OH-ABF. The collapse of the mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining assay. Western blotting was used to examine the expression levels of targeted proteins. The role of mTOR in 1ß-OH-ABF-induced apoptosis was investigated using small interfering RNA (siRNA) transfection. Zebrafish xenograft model was established to evaluate the anti-hepatoma effects of 1ß-OH-ABF in vivo. RESULTS: We found that 1ß-OH-ABF inhibits the proliferation of Hep3B, HepG2, HuH7, SK-HEP-1 cells but has little cytotoxicity towards LO2 cells. 1ß-OH-ABF induces mitochondria dysfunction and triggers mitochondria apoptotic pathway, which is accompanied by the loss of ΔΨm, upregulation and translocation of Bax, as well as cleavages of caspase-9, caspase-3 and PARP. Mechanistically, 1ß-OH-ABF markedly decreases the expression level of p-AKT/AKT and p-mTOR (Ser2248 and Ser2481)/mTOR in a time-dependent manner. Inhibition of mTOR by siRNA strengthens 1ß-OH-ABF-mediated apoptosis. Critically, 1ß-OH-ABF shows a marked in vivo anti-hepatoma effect on human Hep3B cell xenografts in zebrafish model. CONCLUSION: 1ß-OH-ABF induces mitochondrial apoptosis through the suppression of mTOR signaling in vitro and in vivo, indicating that 1ß-OH-ABF may serve as a potential agent for the treatment of liver cancer.