PMID: 

Oncol Res. 2019 Mar 29 ;27(4):475-486. Epub 2018 May 24. PMID: 29793559

Abstract Title: 

Bufalin Induces Apoptosis and Improves the Sensitivity of Human Glioma Stem-Like Cells to Temozolamide.

Abstract: 

Glioma is the most common malignant tumor of the central nervous system, and it is characterized by high relapse and fatality rates and poor prognosis. Bufalin is one of the main ingredients of Chan-su, a traditional Chinese medicine (TCM) extracted from toad venom. Previous studies revealed that bufalin exerted inhibitory effects on a variety of tumor cells. To demonstrate the inhibitory effect of bufalin on glioma cells and glioma stem-like cells (GSCs) and discuss the underlying mechanism, the proliferation of glioma cells was detected by MTT and colony formation assays following treatment with bufalin. In addition, we investigated whether bufalin inhibits or kills GSCs using flow cytometry, Western blotting, and reverse transcription polymerase chain reaction analysis (RT-PCR). Finally, we investigated whether bufalin could improve the therapeutic effect of temozolomide (TMZ) and discussed the underlying mechanism. Taken together, our data demonstrated that bufalin inhibits glioma cell growth and proliferation, inhibits GSC proliferation, and kills GSCs. Bufalin was found to induce the apoptosis of GSCs by upregulating the expression of the apoptotic proteins cleaved caspase 3 and poly(ADP-ribose) polymerase (PARP) and by downregulating the expression of human telomerase reverse transcriptase, which is a marker of telomerase activity. Bufalin also improved the inhibitory effect of TMZ on GSCs by activating the mitochondrial apoptotic pathway. These results suggest that bufalin damages GSCs, induces apoptosis, and enhances the sensitivity of GSCs to TMZ.

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PMID: 

Oncol Lett. 2018 Jun ;15(6):9339-9346. Epub 2018 Apr 18. PMID: 29805658

Abstract Title: 

Bufalin induces apoptosis in human esophageal carcinoma ECA109 cells by inhibiting the activation of the mTOR/p70S6K pathway.

Abstract: 

The present study examined whether bufalin could induce human esophageal carcinoma ECA109 cells apoptosis via inhibiting the activation of mechanistic target of rapamycin (mTOR)/p70 S6 kinase (p70S6K) pathway is discussed in this article. The present study used the esophageal squamous cell carcinoma ECA109 cell line to assess the apoptosis-inducing effects of bufalin via inhibition of the mTOR/p70S6K pathways. A plasmid containing the wild-type mTOR gene (wtmTOR) was transfected into ECA109 cells. The levels of p70S6K, phosphorylated (p)-p70S6K, cellular inhibitor of apoptosis-1 (cIAP-1) and Bcl-2-associated death promoter (BAD) in ECA109 cells were examined by western blot analysis, and apoptosis was detected by flow cytometry analysis and Giemsa staining. The results revealed that the expression of p-p70S6K was increased as the time progressed (at 0, 12 and 24 h), and then decreased at 30, 36, 42 and 48 h after transfection. The expression of cIAP-1 was significantly decreased as time progressed following the addition of bufalin, whereas that of BAD was increased. The levels of p-p70S6K and cIAP-1 were significantly higher in the wtmTOR-transfected group than in the control and empty vector-transfected groups, and then reduced following addition of bufalin; however, BAD expression was significantly lower in the wtmTOR-transfected group. The results of flow cytometry revealed the cell cycle of ECA109 was arrested at G/M phase and the apoptotic rate was significantly lower in the wtmTOR-transfected group than in the control and empty vector-transfected groups, and then increased following addition of bufalin. In conclusion, the findings of the present study demonstrated that bufalin induced apoptosis in esophageal carcinoma cells via the inhibition of the mTOR/p70S6K pathway and indicated that treatment with bufalin could be combined with chemotherapy to overcome the resistance of esophageal carcinoma cells to chemotherapeutic-induced apoptosis.

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PMID: 

Oncotarget. 2018 May 4 ;9(34):23320-23333. Epub 2018 Jan 11. PMID: 29805736

Abstract Title: 

Bufalin inhibits gastric cancer invasion and metastasis by down-regulating Wnt/ASCL2 expression.

Abstract: 

Achaete-scute-like 2 (ASCL2) is a transcription factor containing a basic helix-loop-helix (bHLH) domain and is a downstream target of Wnt signaling in intestinal stem cells. Bufalin is the primary active ingredient in Chan Su, a traditional Chinese medicine obtained from the skin and parotid venom glands of toads. The purpose of this study was to research the anti-invasion and anti-metastasis activity of bufalin in gastric cancer and to identify the potential mechanism. Bufalin inhibited gastric cancer cell invasion and metastasis, suppressed cancer cell colony formation, and inhibited the growth of subcutaneous xenografted tumors in nude mice. Furthermore, bufalin inhibited ASCL2 expression and down-regulated the expression of invasion-related genes such as MMP2, MMP9, and vimentin, thereby suppressing epithelial-mesenchymal transition (EMT) in gastric cancer. A Wnt signaling inhibitor (XAV939) down-regulated invasion and the expression of ASCL2,β-catenin, and vimentin but up-regulated E-cadherin expression. In nude mice, bufalin inhibited the tumorigenic behavior of gastric cancer cells, induced cancer cell apoptosis, and regulated invasion-related gene expression. Together, our results suggest that bufalin arrests invasion and metastasisand that its mechanism of action may involve down-regulating Wnt/ASCL2 expression.

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PMID: 

Oncol Lett. 2018 May ;15(5):8019-8026. Epub 2018 Mar 23. PMID: 29849804

Abstract Title: 

Bufalin and 5-fluorouracil synergistically induce apoptosis in colorectal cancer cells.

Abstract: 

5-fluorouracil (5-FU) has been used in the treatment of colorectal cancer for>50 years. However, drug resistance remains an obstacle in the application of 5-FU-based chemotherapy. Bufalin, a type of steroid with anti-tumor activity, may be purified from the skin and parotid venom glands of toads. In order to improve the anti-tumor effect of 5-FU, the present study examined the combined effects of bufalin with 5-FU on human colorectal cancer HCT116 cells. Following treatment, cell proliferation was quantified using MTT assay and apoptotic cell percentage was assessed by flow cytometry. The apoptosis-associated protein expression was evaluated by western blotting. It was observed that bufalin enhanced the cytotoxicity of 5-FU in HCT116 cells via the induction of the mitochondrial apoptotic pathway. Additionally, bufalin combined with 5-FU reduced the expression levels of anti-apoptotic proteins, such as Mcl-1, XIAP and Bcl-2 and upregulated the levels of the pro-apoptotic proteins, Bax and Bad. To verify the role of Bax, RNA interference was used to knock-down Bax. It was determined that the synergistic effect between 5-FU and bufalin was diminished following the silencing of Bax. In summary, bufalin in combination with 5-FU may induce a higher level of apoptosis compared with monotherapy, and the combination mat be a potential therapeutic strategy for the treatment of colorectal cancer.

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PMID: 

Am J Cancer Res. 2018 ;8(7):1288-1296. Epub 2018 Jul 1. PMID: 30094101

Abstract Title: 

Bufalin inhibits glycolysis-induced cell growth and proliferation through the suppression of Integrinβ2/FAK signaling pathway in ovarian cancer.

Abstract: 

Bufalin is the major digoxin-like component of the traditional Chinese medicine Chansu and has obvious anti-tumor effect in major malignancies, but the role of bufalin in glucose metabolism in ovarian cancer remains illustrated. Here, we sought to elucidate the regulatory function of bufalin on cell glucose metabolism in ovarian cancer. The treatment of bufalin on ovarian cancer cells effectively inhibited glucose uptake and lactate production in ovarian cancer cells. The expression levels of glycolysis-related proteins, including GLUT4, LDHB and HK2, were decreased by the treatment of bufalin detected by qRT-PCR and immunoblotting. Mechanistically, bufalin exerted its anti-tumor effect by targeting ITGB2/FAK signaling pathwayand, which could be rescued by the introduction of ITGB2 cDNA in ovarian cancer cells. These findings provide evidence that bufalin inhibited cellular glycolysis-induced cell growth and proliferation through repression of the ITGB2/FAK pathway, indicating that bufalin may be developed as a chemotherapeutic agent to treat ovarian cancer.

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PMID: 

Biochem Biophys Res Commun. 2018 09 10 ;503(3):2124-2131. Epub 2018 Aug 6. PMID: 30100060

Abstract Title: 

Bufalin inhibits cell proliferation and migration of hepatocellular carcinoma cells via APOBEC3F induced intestinal immune network for IgA production signaling pathway.

Abstract: 

OBJECTIVE: This study aimed to evaluate functions of APOBEC3F gene in biological process of hepatocellular carcinoma (HCC) and anti-tumor mechanisms of bufalin.METHODS: Effect of APOBEC3F and bufalin on cell proliferation and migration abilities were evaluated by CCK-8, wounding healing tests and transwell assays in SK-Hep1 and Bel-7404 cells. Bioinformatic analysis were also used to compare APOBEC3F expression levels, detect coexpressed genes and enrichment of pathways.RESULTS: APOBEC3F was overexpressed in tumor tissues compared to adjacent tissues in HCC patients. And, APOBEC3F promotes cell proliferation and migration in SK-Hep1 and Bel-7404 cells. Bufalin inhibits cell proliferation and migration and reduces APOBEC3F expression. GO and KEGG enrichment of APOBEC3F-coexpressed genes revealed that APOBEC3F might active intestinal immune network for IgA production signaling pathway, leading to malignant biological behaviors of HCC cells. Additionally, siAPOBEC3F could decrease pIgR, CCR9, CCR10 and CXCR4 protein levels. And, bufalin inhibits the pIgR, CCR9, CCR10 and CXCR4 protein expressions.CONCLUSIONS: Bufalin inhibits cell proliferation and migration of HCC cells via APOBEC3F induced intestinal immune network for IgA production signaling pathway.

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PMID: 

Int J Mol Sci. 2019 Oct 6 ;20(19). Epub 2019 Oct 6. PMID: 31590417

Abstract Title: 

The Effect of Polyphenols on Hypercholesterolemia through Inhibiting the Transport and Expression of Niemann-Pick C1-Like 1.

Abstract: 

The Niemann-Pick C1-like 1 (NPC1L1) protein is a cholesterol transporter that is expressed in the small intestine. This report describes the discovery of NPC1L1, its transport properties, and the inhibitory effects of polyphenols on NPC1L1. NPC1L1 was identified in 2004 while searching for ezetimibe molecular targets. Excessive synthesis of cholesterol results in hyperlipidemia, which increases the amount of bile cholesterol excreted into the duodenum. The inhibition of NPC1L1 decreases blood cholesterol because food and bile cholesterol are also absorbed from NPC1L1 in the intestine. Some polyphenols, particularly luteolin, have been reported as NPC1L1-mediated anti-dyslipidemia constituents. Luteolin affects NPC1L1 through two mechanisms. Luteolin directly inhibits NPC1L1 by binding to it, which occurs in a short timeframe similar to that for ezetimibe. The other mechanism is the inhibition of NPC1L1 expression. Luteolin reduced the binding of Sterol-regulatory element-binding protein 2 (SREBP2) in the promoter region of thegene and decreased mRNA levels ofand. These data suggest that luteolin decreases the expression of NPC1L1 through regulation of transcription factors. This review also explores the effect of other polyphenols on NPC1L1 and hypercholesterolemia.

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PMID: 

Drug Des Devel Ther. 2019 ;13:3899-3911. Epub 2019 Nov 18. PMID: 31819365

Abstract Title: 

Luteolin Attenuates Atherosclerosis Via Modulating Signal Transducer And Activator Of Transcription 3-Mediated Inflammatory Response.

Abstract: 

Background: Inflammatory factors play a crucial role throughout the development and progression of atherosclerosis, which has been considered as a chronic vascular inflammatory disease. Luteolin, a natural flavonoid which exists in many natural medicinal materials, has anti-inflammatory, anti-fibrotic and other pharmacological effects. Recently, the protective effects of luteolin on the cardiovascular disease have been reported. However, there is a paucity of studies on anti-atherosclerosis. Therefore, the anti-atherosclerosis potential of luteolin remains to be elucidated.Method: ApoEmice were fed with a high-fat diet to induce atherosclerosis in an animal model, where they were treated with oral administration of luteolin for 12 weeks. Primary mouse peritoneal macrophages challenged with oxidized low-density lipoprotein (oxLDL) were used for in vitro mechanistic study. The effectiveness of luteolin in the ApoEmouse model of atherosclerosis was estimated in the aortic sinus and enface, and the underlying mechanisms were explored by molecular modeling study and siRNA-induced gene silencing.Results: Our results showed that luteolin remarkably attenuated atherosclerosis in high-fat diet-induced ApoEmouse via alleviating inflammation. We further found that luteolin decreased oxLDL-induced inflammation by inhibiting signal transducer and activator of transcription 3 (STAT3) in vitro, respectively. Further molecular modeling analysis indicated that luteolin interacted with STAT3 primarily through hydrogen bond interaction.Conclusion: Luteolin could be a promising candidate molecule for atherosclerosis, and STAT3 may be a potential therapeutic target that could prevent the development of atherosclerosis.

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PMID: 

Mol Brain. 2019 Dec 26 ;12(1):114. Epub 2019 Dec 26. PMID: 31878979

Abstract Title: 

Anxiolytic effects of the flavonoid luteolin in a mouse model of acute colitis.

Abstract: 

Anxiety related disorders commonly occur in association with major depressive disorder (MDD) in individuals suffering from peripheral inflammation, with a higher prevalence among IBS patients. We have previously shown that the bioflavonoid luteolin has pronounced analgesic and antidepressant-like effects in mice with dextran sodium sulfate (DSS)-induced colitis. Here, we further evaluate the biological effect of luteolin as a possible anxiolytic agent in DSS treated mice. Anxiolytic action was evaluated using the open field test (OF), the novelty suppressed feeding test (NSFT) and the elevated plus maze test (EPM). Luteolin increased the number of crossings in the center of the OF apparatus, reduced the latency to interact with the food pellet in the NSFT, and increased the time spent in the open arms in the EPM. These results suggest luteolin as a possible natural anxiolytic molecule without sedative effects, thus reinforcing its therapeutic potential for the comorbidities involving peripheral inflammation, pain, mood and anxiety-related disorders.

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PMID: 

Oncol Lett. 2018 Sep ;16(3):3826-3832. Epub 2018 Jul 12. PMID: 30127995

Abstract Title: 

Bufalin induced apoptosis of bladder carcinoma cells through the inactivation of Na+K+-ATPase.

Abstract: 

Bufalin has been demonstrated to possess a wide range of pharmacological effects. Among these is its antitumour effect, which has been confirmed in multiple organs or tissues and provoked many concerns. However, its cytostatic effect and underlying mechanism in bladder cancer has not thoroughly been elucidated. This study aimed to investigate the hypothesis that Bufalin induces cell apoptosis and inhibits cell growth in bladder cancer through the inactivation of Na+/K+-ATPase (NKA). In the current study, it was demonstrated that Bufalin remarkably inhibited cell viability and induced cell apoptosis in bladder cancer cell line T24. Subsequently, we found that the expression of NKA was significantly supressed in Bufalin-treated cells and the NKA-α3 isoform was most sensitive to Bufalin among all α subunits of NKA. By transfection with NKA-α3 overexpressing plasmids, the expression of the NKA-α3 subunit was upregulated and NKA-α3 overexpression was found to markedly attenuated Bufalin-induced cell apoptosis in T24 cells, suggesting NKA-α3 played a critical role in Bufalin-induced cell apoptosis. Taken together, the present study confirmed that Bufalin promotes tumour apoptosis and inhibits tumour growth in bladder cancer, and this antitumour effect may be ascribed to the inactivation of NKA.

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