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PMID: 

Carcinogenesis. 2018 05 3 ;39(5):700-707. PMID: 29546393

Abstract Title: 

Bufalin inhibits human breast cancer tumorigenesis by inducing cell death through the ROS-mediated RIP1/RIP3/PARP-1 pathways.

Abstract: 

Bufalin, a key active ingredient of the Chinese medicine Chan Su, inhibits breast cancer tumorigenesis in vitro and in vivo. Here, we found that the pan-caspase inhibitor zVAD-fmk failed to inhibit bufalin-induced cell death in MCF-7 and MDA-MB-231 human breast cancer cells, confirming that the cell death induced by bufalin is caspase-independent. Instead, bufalin increased the expression of the necroptosis mediators RIP1 and RIP3. Bufalin-induced cell death was prevented by small molecule inhibitors of RIP1 and poly (ADP-ribose) polymerase-1 (PARP-1) or genetic knockdown of RIP3 by shRNA transfection. In addition, ectopic RIP3 expression enhanced cell death by bufalin. We also found that bufalin increased intracellular reactive oxygen species levels; and cell death by bufalin was inhibited by the antioxidant NAC. In a mouse xenograft model of human breast cancer, bufalin induced PARP-1-dependent tumor cell death and inhibited tumor growth. These results demonstrated that bufalin inhibits human breast cancer tumorigenesis by inducing cell death through the reactive oxygen species-mediated RIP1/RIP3/PARP-1 pathways.

PMID: 

Colloids Surf B Biointerfaces. 2018 Jun 1 ;166:224-234. Epub 2018 Mar 19. PMID: 29602078

Abstract Title: 

Increasing the anticancer performance of bufalin (BUF) by introducing an endosome-escaping polymer and tumor-targeting peptide in the design of a polymeric prodrug.

Abstract: 

A well-defined multifunctional brush-type polymeric prodrug covalently linked with an anticancer drug (bufalin, BUF), a tumor-targeting peptide (RGD), and an endosome-escaping polymer, poly(N,N-diethylaminoethyl methacrylate-co-butyl methacrylate (P(DEA-co-BMA)), was developed. Its anticancer performance against colon cancer was investigated in vitro and in vivo. Reversible addition-fragmentation transfer (RAFT) polymerization of oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA), 2-((3-(tert-butoxy)-3-oxopropyl)thio)ethyl methacrylate (BSTMA), and 2-(2-bromoisobutyryloxy)ethylmethacrylate (BIEM) afforded the multifunctional random copolymer, P(OEGMA-co-BSTMA-co-BIEM), in which hydrophilic POEGMA can stabilize nanoparticles in water, PBSTMA can be converted into carboxyl groups, and PBIEM can be employed as a macromolecular atom radical transfer polymerization (ATRP) initiator. The ATRP of DEA and BMA using P(OEGMA-co-BSTMA-co-BIEM) as a macromolecular ATRP initiator led to the formation of the pH-responsive brush-type copolymer, P(OEGMA-co-BSTMA)-g-P(DEA-co- BMA). After hydrolysis by trifluoroacetic acid and post-functionalization the final polymeric prodrug, P(OEGMA-co-BUF-co-RGD)-g-P(DEA-co-BMA), was obtained with a drug content of∼7.8 wt%. P(OEGMA-co-BUF-co-RGD)-g-P(DEA-co-BMA) can be assembled into nanoparticles (BUF- NP-RGD) in aqueous solution with a diameter of 148.4 ± 0.7 nm and a zeta potential of -7.6 ± 0.4 mV. BUF-NP-RGD exhibited controlled drug release in the presence of esterase. Additionally, P(OEGMA-co- BSMA)-g-P(DEA-co-BMA) showed a significant hemolysis effect at a pH comparable to that of endosomes/lysosomes. Cell viability and a tumor-bearing nude mouse model were employed to evaluate the anticancer efficacy of BUF-NP-RGD. It was revealed that BUF-NP-RGD showed improved anticancer performance compared with that of free BUF both in vitro and in vivo. Histological and immunochemical analysis further demonstrated that BUF-NP-RGD exhibited improved cell apoptosis, angiogenesis inhibition, and an anti-proliferation effect.

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PMID: 

Oncol Lett. 2018 May ;15(5):6443-6449. Epub 2018 Feb 21. PMID: 29616114

Abstract Title: 

Bufalin induces DNA damage response under hypoxic condition in myeloma cells.

Abstract: 

Hypoxia serves a crucial role in the development of drug resistance in various cancer cells. Therefore, many attempts targeting hypoxia are underway to overcome the drug resistance mediated by hypoxia. This strategy is useful for multiple myeloma (MM) cells, as MM cells reside within the bone marrow, where oxygen concentrations are relatively low. A natural compound library was screened to identify compounds exerting cytotoxicity in MM cells under hypoxic conditions. Bufalin exhibited marked cytotoxicity to MM cells under normoxic and hypoxic conditions. No significant toxicity was observed in lymphocytes obtained from healthy donors. Under normoxic conditions, bufalin induced a DNA double strand break (DSB) response, ROS induction and apoptosis within 24 with a rapid response compared with melphalan. Interestingly, the bufalin-induced DSB response was not impaired by low oxygen concentrations while the DSB response by melphalan was reduced. Furthermore, treatment with bufalin abolished HIF-1α expression under hypoxia, suggesting that bufalin exerts cytotoxicity under hypoxia by regulating HIF-1α. These results indicate that bufalin induces apoptosis in MM cells through DSB under hypoxic conditions by inhibiting HIF-1α, suggesting that bufalin could be useful for eradication of drug-resistant MM cells in the hypoxic microenvironment.

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PMID: 

Int J Oncol. 2018 Jun ;52(6):2051-2060. Epub 2018 Apr 2. PMID: 29620259

Abstract Title: 

Synergistic anti-hepatoma effect of bufalin combined with sorafenib via mediating the tumor vascular microenvironment by targeting mTOR/VEGF signaling.

Abstract: 

Sorafenib inhibits tumor growth primarily by inhibiting vessel formation, however, its efficacy requires improvement, therefore, the development of strategies which augment its antiangiogenic effect are of primary concern. Bufalin inhibits tumor cell proliferation and metastasis, and induces apoptosis. In our previous study, it was demonstrated that the antiangiogenic effect of sorafenib was improved by bufalin in human umbilical vein endothelial cells (HUVECs). However, whether bufalin synergizes with sorafenib by affecting the tumor vascular microenvironment remains to be elucidated. In the present study, it was found that hepatocellular carcinoma (HCC) cell proliferation was inhibited by either bufalin or sorafenib following incubation for 24 h, and the inhibition was enhanced upon treatment with a combination of the two. Conditioned medium (CM), comprising supernatant from HCC cells was collected from each of the treatment groups. The migration and tubule formation were suppressed the most in the combination-CM treated HUVECs. The secretion of vascular endothelial growth factor (VEGF) was decreased in HCC cells treated with the combination-CM, as determined by an angiogenesis array, enzyme-linked immunosorbent assay (ELISA) and western blot analysis. The inhibition of tube formation in HUVECs treated with the combination-CM was reversed by incubation with VEGF. The in vivo experiments demonstrated that subcutaneous HCC cell tumors from mice treated with the combination treatment expressed the lowest levels of VEGF, as evidenced by immunohistochemistry and ELISA. Additionally, the level of phosphorylated mechanistic target of rapamycin (mTOR) was reduced in HUVECs pretreated with the phosphoinositide 3-kinase inhibitor PI103. Furthermore, the migration of HCC cells and HUVEC tube formation were attenuated by PI103 pretreatment. In conclusion, the results revealed a synergistic anti-hepatoma effect of bufalin combined with sorafenib via affecting the tumor vascular microenvironment by targeting mTOR/VEGF signaling.

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PMID: 

Indian J Hematol Blood Transfus. 2018 Apr ;34(2):268-272. Epub 2017 Sep 30. PMID: 29622868

Abstract Title: 

Bufalin Enhances the Cytotoxity of Human Multiple Myeloma Cells H929 to AKT Inhibitor MK2206: The Role of Protein AKT Phosphorylation.

Abstract: 

This study was purposed to investigate bufalin combined with AKT inhibitor MK2206 on growth inhibition and apoptosis of multiple myeloma cell line H929. CCK-8 assay and Annexin/PI staining were used to access the effects of bufalin and MK2206 in single or in combination, on inhibition of proliferation and induction of apoptosis in H929 cells. The apoptotic cells markedly increased after treated with nM bufalin andμM MK2206, including caspase3 and PARP1 proteins activated. The difference was statistically significant ( 

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PMID: 

J Endocrinol. 2018 06 ;237(3):255-269. Epub 2018 Apr 10. PMID: 29636364

Abstract Title: 

Bufalin suppresses endometriosis progression by inducing pyroptosis and apoptosis.

Abstract: 

The steroid receptor coactivator (SRC)-1 isoform/estrogen receptor (ER)-β axis has an essential role in endometriosis progression. In this context, therefore, bufalin was employed as a 'tool compound' to evaluate inhibitors of SRC in alternative endometriosis treatment. Bufalin effectively suppressed the growth of primary human endometrial stroma cells isolated from endometriosis patients compared to women without endometriosis and immortalized human endometrial epithelial and stromal cells expressing the SRC-1 isoform compared to their parental cells, compared to the vehicle, bufalin treatment significantly suppressed the growth of endometriotic lesions in mice with surgically induced endometriosis because bufalin disrupted the functional axis of SRC-1 isoform/ERβ by increasing SRC-1 isoform protein stability, hyperactivating the transcriptional activity of the SRC-1 isoform and degrading the ERβ protein by proteasome 26S subunit, non-ATPase 2 in endometriotic lesions. Bufalin treatment elevated the apoptosis signaling in epithelial cells of endometrioticlesions. In stromal cells of endometriotic lesions, bufalin treatment increased the levels of pyroptosis markers (caspase 1 and the active form of interleukin 1β) and reduced proliferation. In addition, bufalin treatment increased the expression levels of endoplasmic reticulum-stress (ERS) markers(PKR-like ER kinase, protein disulfide isomerase and binding immunoglobulin) in endometriotic lesions. Collectively, the bufalin-induced disruption of the SRC-1 isoform/ERβ axis might induce apoptosis, pyroptosis and ERS signaling in endometriotic lesions, causing the suppression of endometriosis.Therefore, future generations of SRC-modulators could be employed as an alternative medical approach for endometriosis treatment.

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PMID: 

Biomed Pharmacother. 2018 Jul ;103:204-215. Epub 2018 Apr 24. PMID: 29653366

Abstract Title: 

Bufalin inhibits glioblastoma growth by promoting proteasomal degradation of the Na/K-ATPaseα1 subunit.

Abstract: 

Chansu is a traditional Chinese medicine that is generally recognized as a specific inhibitor of Na/K-ATPase. Bufalin, an active component of Chansu, is an endogenous steroid hormone with great potential as a cancer treatment. However, the mechanism by which it exerts its antitumor activity requires further research. Currently, theα1 subunit of Na/K-ATPase (ATP1A1) is known to exert important roles in tumorigenesis, and the precise mechanisms underlying the effect of Bufalin on the Na/K-ATPaseα1 subunit was therefore investigated in this study to determine its role in glioblastoma treatments. The effect of ATP1A1 on the sensitivity of glioblastoma cells to Bufalin was investigated using MTT assays, RT-PCR and siRNA. Western blot was also used to explore the important roles of the ubiquitin-proteasome pathway in the Bufalin-mediated inhibition of ATP1A1. Xenografted mice were used to examine the anti-tumor activity of Bufalin in vivo. LC-MS/MS analysis was performed to determine the ability of Bufalin to traverse the blood-brain barrier (BBB). The results indicated that Bufalin inhibited the expression of ATP1A1 in glioblastoma by promoting the activation of proteasomes and the subsequent protein degradation of ATP1A1, while Bufalin had no effect on ATP1A1 protein synthesis. Bufalin also inhibited the expression of ATP1A1 in xenografted mice and significantly suppressed tumorgrowth. These data should contribute to future basic and clinical investigations of Bufalin. In conclusion, Bufalin significantly inhibited the expression of ATP1A1 in glioblastoma cells by activating the ubiquitin-proteasome signaling pathway. Bufalin may therefore have the potential to be an effective anti-glioma drug for human glioblastoma in the future.

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PMID: 

Mol Med Rep. 2018 Jun ;17(6):8101-8110. Epub 2018 Apr 24. PMID: 29693132

Abstract Title: 

Synergistic anticancer effects of bufalin and sorafenib by regulating apoptosis associated proteins.

Abstract: 

As one of the most recognized and well‑known drugs for hepatocellular carcinoma (HCC), the antitumor effect of sorafenib against HCC remains to be improved. Bufalin has displayed an antitumor effect in HCC; however, whether the enhanced antitumor effect may be generated with their combined treatment remains unclear. Therefore, in thepresent study, their combined effects on HCC proliferation and apoptosis were investigated. It was revealed that either bufalin or sorafenib suppressed PLC/PRF/5 and SMMC‑7721 cell proliferation in a concentration‑dependent manner following incubation for 24 h, and the inhibitory effect was augmented with their combined treatment. The synergistic effect peaked in HCC cells treated with 20 nM bufalin and 10 µM sorafenib. In addition, cell cycle and terminal deoxynucleotidyl transferase dUTP nick‑end labelling assays revealed that bufalin also enhanced sorafenib‑induced apoptosis. Colony formation assay demonstrated that combined treatment significantly suppressed HCC proliferation compared with treatment with either of them alone. Furthermore, B‑cell lymphoma 2‑associated X protein, caspase 7 and poly‑(adenosine diphosphate‑ribose) polymerase were upregulated in HCC cells with combined treatment. Taken together, the results of the present study revealed that the treatment of sorafenib combined with bufalin synergistically suppressed HCC proliferation and induced apoptosis. Therefore, bufalin combined with sorafenib may be a favorable treatment strategy for patients with HCC.

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PMID: 

Mol Clin Oncol. 2018 May ;8(5):631-636. Epub 2018 Mar 21. PMID: 29732152

Abstract Title: 

Molecular mechanisms underlying the antimetastatic activity of bufalin.

Abstract: 

Bufalin is a monomer compound extract from Chansu, which is a traditional Chinese medicine obtained from the skin and parotid venom glands of toads, such as Bufo bufo gargarizans Cantor and Bufo melanostictus Schneider. Chansu had been used in traditional Chinese medicine for>1,000 years due to its cardiac, anti-inflammatory and anticancer properties. Previous studies identified bufalin as the main anticancer compound of Chansu, and recent evidence has corroborated its anticancer properties. Bufalin inhibits cancer cell proliferation, induces cell cycle arrest, induces cancer cell apoptosis, inhibits neovascularization, induces cell differentiation, inhibits cancer metastasis and invasion, and enhances chemotherapeutic drug sensitivity. However, the function and mechanism of bufalin in metastatic cancer cells have not yet been expounded. The aim of the present review was to discuss the recent progress and prospects of bufalin in the prevention of cancer metastasis, particularly in inhibiting epithelial-to-mesenchymal transition.

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PMID: 

Oncoimmunology. 2018 ;7(5):e1426434. Epub 2018 Feb 8. PMID: 29721392

Abstract Title: 

Bufalin Suppresses Migration and Invasion of Hepatocellular Carcinoma Cells Elicited by Poly (I:C) Therapy.

Abstract: 

The Toll-like receptor 3 (TLR3) agonists as polyriboinosinic-polyribocytidylic acid (poly (I:C)) have been implicated as potential immunotherapy adjuvant for cancer whereas the exact roles of TLR3 agonists in hepatocellular carcinoma (HCC) treatment have not been clearly evaluated. In consistent with previous reports, we found that poly (I:C) triggering of TLR3 inhibited cell proliferation and induced apoptosis in HCC cells. However, poly (I:C), when used at lower concentration that cannot remarkably inhibit proliferation and induce apoptosis in HCC cells, enhanced the migration and invasion in vitro and the metastasis in vivo. More importantly, we found that bufalin, a prominent component of toad venom, could suppress poly (I:C)-inspired migration, invasion and metastasis of HCC cells despite that bufalin could not potentiate poly (I:C)-induced inhibition of proliferation and induction of apoptosis. In MHCC97 H cells, bufalin impaired poly (I:C)-induced activation of Tank-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3) pathway and NF-κB pathway. Inhibitor for TBK1 but not NF-κB suppressed poly (I:C)-inspired migration and invasion, which was further supported by using TBK1 deficient () cells. In another model using poly (I:C) transfection, bufalin could also suppress the migration and invasion of HCC cells, which was not observed inMHCC97 H cells. Our data suggest that bufalin can suppress the metastasis of HCC cells in poly (I:C) therapy by impairing TBK1 activation, indicating that bufalin may be used in combination with poly (I:C) therapy in HCC treatment for the sake of reversing poly (I:C)-triggered metastasis of HCC cells.

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