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Senescent cells that accumulate with age have been shown to contribute to age-related diseases and organ dysfunction and have attracted attention as a target for anti-aging therapy. In particular, the use of senescent cell-depleting agents, or senolytics, has been shown to improve the aging phenotype in animal models. Since senescence has been implicated in the skin, particularly in fibroblasts, this study used aged human skin fibroblasts to investigate the effects of resibufogenin. A component of the traditional Chinese medicine toad venom, resibufogenin was investigated for senolytic and/or senomorphic activity. We found that the compound selectively caused senescent cell death without affecting proliferating cells, with a marked effect on the suppression of the senescence-associated secretory phenotype. We also found that resibufogenin causes senescent cell death by inducing a caspase-3-mediated apoptotic program. Administration of resibufogenin to aging mice resulted in an increase in dermal collagen density and subcutaneous fat, improving the phenotype of aging skin. In other words, resibufogenin ameliorates skin aging through selective induction of senescent cell apoptosis without affecting non-aged cells. This traditional compound may have potential therapeutic benefits in skin aging characterized by senescent cell accumulation.

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Toad venom is a traditional Chinese medicine that has a long history in treating infectious and inflammatory diseases, such as carbuncle, pharyngitis. As one of the major active components in toad venom, resibufogenin (RBG) possesses a variety of pharmacological activities, including lowering blood pressure, reducing proteinuria and preventing oxidative stress. But only its antitumor activity attracts widespread attention in these years. This study aimed to explore the nonnegligible anti-inflammatory activity of RBG in vivo and in vitro. In endotoxemia mice, a single intraperitoneal administration of RBG significantly lowered serum TNF-α, IL-6 and MCP-1 levels. In LPS-stimulated macrophages, RBG decreased LPS-induced pro-inflammatory mediators' productions (e.g., iNOS, IL-6, TNF-α and MCP-1) through suppressing their transcriptions. Mechanism study showed that RBG hindered IκBα phosphorylation and prevented nuclear translocation of p65, thus inactivating nuclear factor-κB (NF-κB) signaling. Concurrently, RBG also dampened activator protein-1 (AP-1) signaling through inhibiting the phosphorylation levels of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK). Besides LPS (TLR4 ligand) model, RBG also inhibited Pam3CSK4 (TLR2 ligand)- or poly I:C (TLR3 ligand)-induced inflammatory reactions, suggesting that its target(s) site is(are) not on the cytomembrane. These findings not only support the pharmacological basis for the traditional use of toad venom in inflammatory diseases, but also provide a promising anti-inflammatory candidate.

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OBJECTIVE: To investigate the effects and underlying mechanism of miR-532-3p and resibufogenin (RES) by regulating Wnt/ß-catenin signaling on diffuse Large B-cell lymphoma (DLBCL) cells proliferation. METHODS: DLBCL tissues and adjacent normal tissues were collected from patients had been diagnosed with DLBCL at the First Hospital of Lanzhou University from October 2019 to October 2021. Four groups including mimics-NC, miR-532-3p mimics, RES control and RES treatment in SU-DHL-4 cells were designed. The expression level of miR-532-3p was detected by RT-qPCR. The protein content of ß-catenin was detected by Western blot. MTT assay was used to detect the proliferation activity of SU-DHL-4 cells. RESULTS: miR-532-3p expression was significantly decreased in DLBCL tissues compared with adjacent normal tissues (P<0.001). The miR-532-3p content in lymphoma cells was significantly lower than that in normal lymphocytes (P<0.001). After overexpression of miR-532-3p, the viability of SU-DHL 4 cells was significantly decreased (P<0.001), with a reduced expression of ß-catenin (P<0.05). RES treatment inhibited the proliferation of SU-DHL-4 cells and decreased ß-catenin expression in SU-DHL-4 cells compared with the control group. CONCLUSION: Overexpression of miR-532-3p reduced Wnt/ß-catenin signaling and inhibited the proliferation of lymphoma cells. Moreover, RES treatment inhibited lymphoma cells growth partially through Wnt/ß-catenin signaling suppression.

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Resibufogenin (RBG) is an active ingredient of toad venom that also has antitumor potential. The present study aimed to investigate the role of RBG in multiple myeloma (MM) and the underlying action mechanism involving the PI3K/Akt signaling pathway. A human MM cell line, RPMI8226, was treated with RBG and/or insulin-like growth factor 1 (IGF-1; an activator of the PI3K/AKT signaling pathway). Cell viability and apoptosis were detected using Cell Counting Kit-8 and flow cytometry, respectively. Cell migration and invasion were detected using a Transwell assay. In addition, the epithelial-mesenchymal transition (EMT)-associated proteins (E-cadherin, N-cadherin and Vimentin) and the PI3K/AKT pathway-associated proteins [AKT, phosphorylated (p)-AKT, PI3K and p-PI3K] were measured using western blotting. RBG inhibited the viability, migration and invasion, and promoted the apoptosis of RPMI8226 cells in a dose-dependent manner. RBG at concentrations of 4 and 8 µM upregulated E-cadherin, and downregulated N-cadherin and Vimentin in RPMI8226 cells. RBG also decreased the protein expression of p-AKT and p-PI3K in a dose-dependent manner. In addition, the intervention of IGF-1 weakened the inhibitory effects of RBG on the malignant characteristics of MM cells. RBG-induced inhibition of EMT and the PI3K/AKT pathway were also weakened by IGF-1 treatment. In conclusion, RBG inhibited viability, migration, invasion and EMT, and promoted the apoptosis of MM cells by blocking the PI3K/AKT signaling pathway.

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Resibufogenin (RB) is a major active ingredient in the traditional Chinese medicine Chansu and has garnered considerable attention for its efficacy in the treatment of cancer. However, the anticancer effects and underlying mechanisms of RB on glioblastoma (GBM) remain unknown. Here, we found that RB induced G2/M phase arrest and inhibited invasion in a primary GBM cell line, P3#GBM, and two GBM cell lines, U251 and A172. Subsequently, we demonstrated that RB-induced G2/M phase arrest occurred through downregulation of CDC25C and upregulation of p21, which was caused by activation of the MAPK/ERK pathway, and that RB inhibited GBM invasion by elevating intercellular Ca2+ to suppress the Src/FAK/Paxillin focal adhesion pathway. Intriguingly, we confirmed that upon RB binding to ATP1A1, Na+-K+-ATPase was activated as a receptor and then triggered the intracellular MAPK/ERK pathway and Ca2+-mediated Src/FAK/Paxillin focal adhesion pathway, which led to G2/M phase arrest and inhibited the invasion of GBM cells. Taken together, our findings reveal the antitumor mechanism of RB by targeting the ATP1A1 signaling cascade and two key signaling pathways and highlight the potential of RB as a new class of promising anticancer agents.

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Resibufogenin (RBG) is a natural medicinal ingredient with promising cardiac protection and antitumor activity. However, poor solubility and severe gastric mucosa irritation restrict its application in the pharmaceutical field. In this study, the inclusion complex of RBG with ß-cyclodextrin (ß-CD) and 2-hydroxypropyl-ß-cyclodextrin (HP-ß-CD) was prepared using the co-evaporation method, and the molar ratio of RBG to CD was determined to be approximately 1:2 by continuous variation plot for both CDs. The formation of inclusion complexes between RBG and each CD (RBG/ß-CD and RBG/HP-ß-CD) was evaluated by phase solubility study, Fourier transform infrared spectroscopy, and thin-layer chromatography. Powder X-ray diffraction and differential scanning calorimetry confirmed drug amorphization and encapsulation in the molecular cage for both CDs. Moreover, the inclusion complexes' morphologies were observed using scanning electron microscopy. The dissolution rate of the inclusion complexes was markedly improved compared to that of RBG, and the complexes retained their antitumor activity, as shown in the in vitro cytotoxicity assay on a human lung adenocarcinoma cancer (A549) cell line. Moreover, less gastric mucosal irritation was observed for the inclusion complex. Thus, the inclusion complex should be considered a promising strategy for the delivery of poorly water-soluble anticancer agents, such as RBG.

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This manuscript aims to investigate the effects of resibufogenin on the proliferation, migration and invasion of human hepatocellular carcinoma cells and its related mechanisms. MTT assay was used to determine the inhibitory effect of resibufogenin on the growth of four hepatocellular carcinoma cells in vitro. Wound-healing assay and Transwell assay were used to evaluate the migration and invasion ability of resibufogenin on MHCC-97H cells. Western blot assay was used to detect the expression of migration and invasion related proteins in MHCC-97H cells treated with different concentrations of resibufogenin. The results showed that resibufogenin significantly inhibited the proliferation of hepatocellular carcinoma cells in vitro. The half maximal inhibitory concentration (IC50) values on MHCC-97H, HepG2, SK-Hep-1 and Huh-7 cells were 0.55 ± 0.06, 2.83 ± 0.24, 5.25 ± 0.49, 14.89 ± 2.28 μmol·L-1, respectively. Resibufogenin also suppressed the migration and invasion of MHCC-97H cells in a concentration-dependent manner. The protein expression of integrin α2, integrin α6, integrin β1, N-cadherin, matrix metalloproteinase 2 (MMP2) and transcription factor Twist in MHCC-97H cells were decreased significantly with the increase of the concentration of resibufogenin, while the protein expression of E-cadherin increased. In addition, we found that p-PI3K/PI3K and p-AKT/AKT ratios were significantly reduced after treatment with resibufogenin. In conclusion, resibufogenin can inhibit the proliferation, migration and invasion of hepatocellular carcinoma MHCC-97H cells in vitro, which is related to the regulation of intracellular migration and invasion protein expression and PI3K/AKT signaling pathway.

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ObjectiveTo develop a quantitative analysis of multi-components by single marker （QAMS） for determination of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills， and to provide a method for improving the national standard of the pills. MethodHigh performance liquid chromatography （HPLC） was developed for simultaneous determination of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills and the methodology validation was carried out. The chromatographic separation was performed on a Nucleosil 100-5 C18 column （4.6 mm×250 mm， 5 μm） with the mobile phase of acetonitrile -0.1% potassium dihydrogen phosphate aqueous solution （pH adjusted to 3.2 with phosphoric acid） （48∶52）， and the flow rate was 0.6 mL·min-1， the detection wavelength was set at 296 nm and the column temperature was 35 ℃. Taking cinobufagin as the internal standard， the relative correction factors （RCFs） of bufalin and resibufogenin were calculated， and the key influencing factors of RCFs were investigated. Relative retention time was used for the chromatographic peak location of the analyte， combining with the on-line ultraviolet spectroscopy and accurate relative molecular weight obtained by ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry （UPLC-QTOF/MS）. The external standard method was used to verify the contents of three components obtained by QAMS. ResultQAMS was established for the determination of bufalin， cinobufagin and resibufogenin in the samples， and RCFs of cinobufagin to bufalin and resibufogenin were 0.922 and 1.01， respectively. The total content of the three marker compounds in 11 batches of Shexiang Baoxin pills was 33.7-36.0 µg per pill. There was no significant difference between the quantitative results of QAMS and external standard method. ConclusionThe established method can be used for the quality control of bufalin， cinobufagin and resibufogenin in Shexiang Baoxin pills. It is suggested that bufalin should be considered as one of three marker compounds， and the sum of bufalin， cinobufagin and resibufogenin should be used for the content limit of this preparation.

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As a new strategy capable of uncovering the characteristics of traditional Chinese medicines, the quantitative analysis of multi-components by single-marker(QAMS) has been widely employed for the quality evaluation of Chinese medicinal materials, slices, and extracts. However, its application in the assessment of Chinese patent medicines is yet to be explored. By referring to the determination of three bufogenins in Bufonis Venenum by QAMS described in Chinese Pharmacopoeia(2020 Edition), this paper selected seven representative preparations containing Bufonis Venenum and explored whether the relative correction factors(RCFs) of cinobufagin(CB) to bufalin(BF) and resibufogenin(RB) could be directly used for the quality control of Bufonis Venenum-contained preparations. Based on the qualitative analyses under the same chromatographic conditions as used for toad venom, combing specificity test, five preparations such as Yatong Yili Pills, Houzheng Pills, Xiongdan Jiuxin Pills, Liushen Pills and Niuhuang Xiaoyan Pills, were expected to use validated RCFs for the direct determination of three components. Taking Houzheng Pills as an example, the methodological validation of bufalin, cinobufagin and resibufogenin was carried out, and the recoveries of bufalin, cinobufagin and resibufogenin were 90.64%-106.1%. The obvious difference was not observed between the contents of bufalin and resibufogenin in 24 batches of preparation samples by QAMS and external reference method. In the tested samples, the content of bufalin, cinobufagin and resibufogenin were 1.27-2.61, 2.44-5.66 and 0.988-3.16 mg·g~(-1) in 10 batches of Liushen Pills samples. The contents of bufalin, cinobufagin and resibufogenin were 0.760-1.32, 1.35-2.39 and 0.600-1.55 mg·g~(-1) in 10 batches of Houzheng Pills samples from three manufacturers. The obtained data contribute to improving the quality standard of Bufonis Venenum-contained preparations, and they also provide some ideas for the application of QAMS in the quality evaluation and control of Chinese patent medicines.

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Metabolic reprogramming, characterized by alterations of cellular metabolic patterns, is fundamentally important in supporting the malignant behaviors of cancer cells. It is considered as a promising therapeutic target against cancer. Traditional Chinese medicine (TCM) and its bioactive components have been used in cancer therapy for an extended period, and they are well-known for their multi-target pharmacological functions and fewer side effects. However, the detailed and advanced mechanisms underlying the anticancer activities of TCM remain obscure. In this review, we summarized the critical processes of cancer cell metabolic reprogramming, including glycolysis, mitochondrial oxidative phosphorylation, glutaminolysis, and fatty acid biosynthesis. Moreover, we systemically reviewed the regulatory effects of TCM and its bioactive ingredients on metabolic enzymes and/or signal pathways that may impede cancer progress. A total of 46 kinds of TCMs was reported to exert antitumor effects and/or act as chemosensitizers via regulating metabolic processes of cancer cells, and multiple targets and signaling pathways were revealed to contribute to the metabolic-modulating functions of TCM. In conclusion, TCM has its advantages in ameliorating cancer cell metabolic reprogramming by its poly-pharmacological actions. This review may shed some new light on the explicit recognition of the mechanisms of anticancer actions of TCM, leading to the development of natural antitumor drugs based on reshaping cancer cell metabolism.

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Resibufogenin (RBF), an active compound from Bufo bufonis, has been used for the treatment of multiple malignant cancers, including pancreatic cancer, colorectal cancer, and breast cancer. However, whether RBF could exert its antitumor effect by inhibiting angiogenesis remains unknown. Here, we aimed to explore the antiangiogenic activity of RBF and its underlying mechanism on human umbilical vein endothelial cell (HUVEC), and the therapeutic efficacy with regard to antiangiogenesis in vivo using two triple-negative breast cancer (TNBC) models. Our results demonstrated that RBF can inhibit the proliferation, migration, and tube formation of HUVECs in a dose-dependent manner. Spheroid sprouts were thinner and shorter after RBF treatment in vitro 3D spheroid sprouting assay. RBF also significantly suppressed VEGF-mediated vascular network formation in vivo Matrigel plug assay. In addition, Western blot analysis was used to reveal that RBF inhibited the phosphorylation of VEGFR2 and its downstream protein kinases FAK and Src in endothelial cells (ECs). Molecular docking simulations showed that RBF affected the phosphorylation of VEGFR2 by competitively binding to the ATP-bound VEGFR2 kinase domain, thus preventing ATP from providing phosphate groups. Finally, we found that RBF exhibited promising antitumor effect through antiangiogenesis in vivo without obvious toxicity. The present study first revealed the high antiangiogenic activity and the underlying molecular basis of RBF, suggesting that RBF could be a potential antiangiogenic agent for angiogenesis-related diseases.

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Resibufogenin (RBG) is a chemical ingredient of Chan Su. In our research, we found RBG affected cardiac rhythm in a negative chronotropic way in vivo. The cardiac Mapping system ex vivo and the patch clamp in vitro were used to explore how RBG influenced the cardiac electrophysiological properties. The negative chronotropic action of RBG at 100 µM might be attribute to prolongation in the atrioventricular conduction time and reduction in the ventricular conduction velocity. Using whole-cell patch clamp in ventricular myocytes of adult rats, we found that RBG prolonged the action potential duration (APD) in APD20, APD50, and APD90 at 100 µM and inhibited calcium currents (ICa), total outward potassium currents (IK), and transient outward potassium current (Ito) in a concentration-dependent manner, but not on the inward rectifying potassium current (IK1). Notably, RBG had a potent proarrhythmic action ex vivo in the isolated perfused guinea pig hearts at 10 µM, but not in rats. To avoid the potential cardiotoxicity derived from the distributional differences of ion channels among species, the effect of RGB on IKr in hERG-HEK293 cells was detected. The IC50 of RGB on IKr was more than 100 µM. In summary, all these results indicated that the negative chronotropic action of RBG relied on the blocking activities on multiple ion channels, and the species-difference of proarrhythmic effects might result from lack of the Ito on the myocardial membrane of guinea pigs. Anyhow, the cardiotoxicity observed in guinea pigs required further detailed studies to mitigate the potential risks in the clinical application of Chan Su.

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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.

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The purpose of this study is to explore the antitumor properties of resibufogenin (RB) in non-small cell lung cancer (NSCLC) and elucidate its underlying mechanism. A549 and H520 cells were treated with various concentrations of RB with or without NLRP3 inhibitor (MCC950), caspase-1 inhibitor (VX765), or N-acetyl-l-cysteine (an ROS scavenger). Cell counting kit-8 and colony formation assays were conducted to determine cell viability. Cell invasion was detected by using the transwell assay. The release of lactate dehydrogenase (LDH) was determined by the LDH detection assay. The protein expression levels of related genes were measured by western blotting and immunohistochemistry. The reactive oxygen species (ROS) level was detected by using a 2,7-dichlorodihydrofluorescein diacetate ROS Assay Kit. The in vivo effects of RB were evaluated in a xenograft mouse model. RB treatment reduced cell viability and invasion in a dose-dependent manner. Furthermore, RB also enhanced pyroptosis levels in A549 and H520 cells, as indicated by the increased release of LDH and pyroptosis-related proteins. Interestingly, we also found that the antiproliferative and antimetastatic effects of RB were alleviated by the blockade of pyroptosis using NLRP3 inhibitor MCC950. Further study demonstrated that RB induced pyroptosis in a caspase-1-dependent manner, as evidenced by the finding that VX765 effectively reversed the effects of RB on A549 and H520 cells. We also found that RB could trigger caspase-1-dependent pyroptosis through ROS-mediated NF-κB suppression. In summary, our findings provide a potential antitumor agent and a novel insight into the mechanism of RB treatment of NSCLC.

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As a new strategy capable of uncovering the characteristics of traditional Chinese medicines, the quantitative analysis of multi-components by single-marker(QAMS) has been widely employed for the quality evaluation of Chinese medicinal materials, slices, and extracts. However, its application in the assessment of Chinese patent medicines is yet to be explored. By referring to the determination of three bufogenins in Bufonis Venenum by QAMS described in Chinese Pharmacopoeia(2020 Edition), this paper selected seven representative preparations containing Bufonis Venenum and explored whether the relative correction factors(RCFs) of cinobufagin(CB) to bufalin(BF) and resibufogenin(RB) could be directly used for the quality control of Bufonis Venenum-contained preparations. Based on the qualitative analyses under the same chromatographic conditions as used for toad venom, combing specificity test, five preparations such as Yatong Yili Pills, Houzheng Pills, Xiongdan Jiuxin Pills, Liushen Pills and Niuhuang Xiaoyan Pills, were expected to use validated RCFs for the direct determination of three components. Taking Houzheng Pills as an example, the methodological validation of bufalin, cinobufagin and resibufogenin was carried out, and the recoveries of bufalin, cinobufagin and resibufogenin were 90.64%-106.1%. The obvious difference was not observed between the contents of bufalin and resibufogenin in 24 batches of preparation samples by QAMS and external reference method. In the tested samples, the content of bufalin, cinobufagin and resibufogenin were 1.27-2.61, 2.44-5.66 and 0.988-3.16 mg·g~(-1) in 10 batches of Liushen Pills samples. The contents of bufalin, cinobufagin and resibufogenin were 0.760-1.32, 1.35-2.39 and 0.600-1.55 mg·g~(-1) in 10 batches of Houzheng Pills samples from three manufacturers. The obtained data contribute to improving the quality standard of Bufonis Venenum-contained preparations, and they also provide some ideas for the application of QAMS in the quality evaluation and control of Chinese patent medicines.

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Increasing evidence confirmed that the Warburg effect plays an important role involved in the progression of malignant tumors. Resibufogenin (RES) has been proved to have a therapeutic effect in multiple malignant tumors. However, the mechanism of whether RES exerted an antitumor effect on breast cancer through regulating the Warburg effect is largely unknown. The effect of RES on glycolysis was determined by glucose consumption, lactate production, ATP generation, extracellular acidification rate and oxygen consumption rate in breast cancer cells. The total RNA and protein levels were respectively measured by RT-qPCR and western blot. Cell proliferation and apoptosis were examined using the CCK-8 assay, colony formation assay, and flow cytometry, respectively. The interaction between miR-143-3p and HK2 was verified by dual-luciferase reporter gene assay. We also evaluated the influence of RES on the tumor growth and Warburg effect in vivo. RES treatment significantly decreased glycolysis, cell proliferation and induced apoptosis of both MDA-MB-453 and MCF-7 cells. Simultaneously, the expression of HK2 was decreased in breast cancer cells treated with RES, which was positively associated with tumor size and glycolysis. Moreover, HK2 was a direct target gene of miR-143-3p. Mechanistically, upregulation of miR-143-3p by RES treatment inhibited tumor growth by downregulating HK2-mediated Warburg effect in breast cancer. Our findings suggested that RES exerted anti-tumorigenesis and anti-glycolysis activities in breast cancer through upregulating the inhibitory effect of miR-143-3p on HK2 expression, which provided a new potential strategy for breast cancer clinical treatment.

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Patients diagnosed with ovarian clear cell carcinoma (OCCC), a rare histologic subtype of ovarian cancer, often experience poor prognosis owing to the chemoresistance of their disease. Thus, there is an urgent need to identify new therapeutic options for these patients. A drug screen of 172 traditional Chinese herbs identified resibufogenin as a compound that inhibited the growth of cultured OCCC cells. Resibufogenin, a bioactive compound originally isolated from toad venom, is used in traditional Chinese medicine to treat several malignancies. The current study examined the impact of resibufogenin treatment on proliferation, migration, and invasion of ES-2 and TOV-21G OCCC cells in vitro. Flow cytometric analyses were employed to determine if resibufogenin affects apoptosis in OCCC cells. Additionally, the ability of resibufogenin to inhibit tumor growth in vivo was evaluated in murine xenograft models. RNA sequencing, quantitative polymerase chain reactions (qPCR), immunohistochemical assays, and western blotting were used to identify and verify cellular pathways potentially targeted by resibufogenin. Resibufogenin inhibited proliferation, migration, and invasion of OCCC cells, and induced apoptosis in them. Resibufogenin also suppressed the growth of xenograft tumors, which consequently showed lower Ki-67 and higher terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) expression. We observed down-regulation of (a) PI3K and AKT in the PI3K/AKT signaling pathway, and (b) MDM2 and myosin in the actin cytoskeleton pathway upon resibufogenin treatment. Thus, resibufogenin inhibits growth and migration of OCCC cells in vitro and suppresses OCCC growth in vivo through the PI3K/AKT and actin cytoskeleton signaling pathways.

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Ovarian cancer is a common human malignancy of the female reproductive system. However, chemotherapy has been proven to have limited effectiveness in a majority of patients. Resibufogenin (RB) is a major active ingredient in cinobufacini, which has been used in the treatment of human malignancies as adjunct agents. This study was designed to examine the anti-cancer effect of RB and the underlying mechanisms in ovarian cancer. Our results showed that RB treatment resulted in cell death, cell cycle arrest, and apoptosis in ovarian cancer cells. The anti-growth and pro-apoptotic effects of RB were also validated in xenograft mice models. Proteomics analysis indicated that RB was able to alter the expressions of several genes, which were involved in the regulation of glycolysis. The suppression effect of RB in the glycolysis pathway of ovarian cancer cells was validated by decreased glucose consumption, lactate production, and extracellular acidification rate (ECAR). We proposed that PIM1 functioned as the key target that mediated the anti-cancer effect of RB against ovarian cancer cells. Our results have revealed that RB downregulated PIM1 in ovarian cancer cells and its downstream genes involved in glycolysis. Moreover, our results indicated that the anti-growth activities and suppressing effect of RB on glycolysis were enhanced significantly by PIM1 knockdown but was attenuated by ectopic PIM1 expression. This provided evidence to support the role of PIM1 in the anti-cancer activities of RB.

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BACKGROUND: Resibufogenin is one of the main active compounds of Venenum Bufonis and exhibits diverse pharmacological activities. It is brought into focus for its potency in heart failure and cancer therapy. PURPOSE: The purpose of this study was to establish a convenient and effective method which was used to simultaneously determine the resibufogenin and its metabolites in rat plasma for further understanding the metabolic profiles of resibufogenin in vivo and pharmacokinetic study by LC-MS/MS. METHODS: The analytes were separated on a BEH C18 column with a mobile phase of water containing 0.05% formic acid and acetonitrile under gradient elution at a flow rate of 0.4 ml/min. Resibufogenin and its eight metabolites were quantified in positive electrospray ionization and MRM mode with transitions of m/z 385.5→349.2 for resibufogenin; m/z 513.7→145.3 for IS (internal standard); m/z 401.23→365.21, m/z 417.23→285.21 and m/z 385.24→349.21 for three main metabolites (hydroxylated-resibufogenin; dihydroxylated-resibufogenin and 3-epi-resibufogenin, respectively). RESULTS: This method was successfully validated with a good linearity over the concentration ranges of 1-200 ng/ml for resibufogenin and the correlation coefficients was more than 0.990. The lower limit of quantification was 1 ng/ml and the precision and accuracy values were less than 15%. The method was applied to study the metabolic profiles of resibufogenin in rat plasma after oral administration of 20 mg/kg. The results indicated that the metabolic reactions of resibufogenin were mainly hydroxylation, dihydroxylation, dehydrogenation and isomerization. Totally eleven metabolites were identified, among which eight were successfully quantified. CONCLUSION: The results could provide further research foundation for the mechanisms study of activity and toxicity in vivo and facilitate the appropriate clinical application of resibufogenin.

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Objective: To develop a rapid, simple, sensitive, and accurate high performance liquid chromatography-tandem mass spectrometry (HPLC-MS) method for determining the contents of cinobufagin and resibufogenin from Bufo Corium. Methods: The contetns were determined by Zorbax SB-C18 column and tested in positive electrospray ionization with multiple reaction monitoring of HPLC-MS. The mobile phase was conswasted with the mixture of acetonitril-0.1 mol/L formic acid water solution (41:59), flow rate 200 μL/min, column temperature 30 oC. Mass spectrometry conditions: nitrogen temperature 350 oC, nitrogen flow rate 12 L/min, nebulizer pressure 101.316 kPa (35 psi). The shredded voltage of cinobufagin was 160 V, the collwasion energy was 15, the parent ion and the daughter ion were 443.2 and 364.8 respectively; The fragmentation voltage of resibufogenin was 130 V, the collwasion energy was 15, and the parent ion and the daughter ion were 385.2 and 366.9 respectively. Results: There was good linearity in the range of 0.99-7.92 μg/mL and 1.04-8.32 μg/mL for the cinobufagin and resibufogenin. The detection limit (S/N ≥ 3) was 0.3 ng/g. The recoveries of cinobufagin and resibufogenin ranged from 97.96% to 103.7% and 96.86% to 102.4%, respectively. The intraday and daytime precwasion were both less than 3%. Conclusion: The results showed that the method was sensitive and reliable, which can meet the needs of analyse of toxic substances in B. Corium.