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

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

Cytochrome P450 Mining for Bufadienolide Diversification.

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

Hellebrigenin triggers death of promyelocytic leukemia cells by non-genotoxic ways.

Bufadienolides are digitalis-like aglycones mainly found in skin secretions of toads. Among their biological properties, the mechanisms of antiproliferative action on tumor cells remain unclear for many compounds, including against leukemia cells. Herein, it was evaluated the mechanisms involved in the antiproliferative and genotoxic actions of hellebrigenin on tumor cell lines and in silico capacity to inhibit the human topoisomerase IIa enzyme. Firstly, its cytotoxic action was investigated by colorimetric assays in human tumor and peripheral blood mononuclear cells (PBMC). Next, biochemical and morphological studies were detailed by light microscopy (trypan blue dye exclusion), immunocytochemistry (BrdU uptake), flow cytometry and DNA/chromosomal damages (Cometa and aberrations). Finally, computational modelling was used to search for topoisomerase inhibition. Hellebrigenin reduced proliferation, BrdU incorporation, viability, and membrane integrity of HL-60 leukemia cells. Additionally, it increased G2/M arrest, internucleosomal DNA fragmentation, mitochondrial depolarization, and phosphatidylserine externalization in a concentration-dependent manner. In contrast to doxorubicin, hellebrigenin did not cause DNA strand breaks in HL-60 cell line and lymphocytes, and it interacts with ATPase domain residues of human topoisomerase IIa, generating a complex of hydrophobic and van der Waals interactions and hydrogen bonds. So, hellebrigenin presented potent anti-leukemic activity at concentrations as low as 0.06 µM, a value comparable to the clinical anticancer agent doxorubicin, and caused biochemical changes suggestive of apoptosis without genotoxic/clastogenic-related action, but it probably triggers catalytic inhibition of topoisomerase II. These findings also emphasize toad steroid toxins as promising lead antineoplasic compounds with relatively low cytotoxic action on human normal cells.

Toad venom bufadienolides and bufotoxins: An updated review.

Bufadienolides, naturally found in toad venoms having steroid-like structures, reveal antiproliferative effects at low doses. However, their application as anticancer drugs is strongly prevented by their Na+ /K+ -ATPase binding activities. Although several kinds of research were dedicated to moderating their Na+ /K+ -ATPase binding activity, still deeper fundamental knowledge is required to bring these findings into medical practice. In this work, we reviewed data related to anticancer activity of bufadienolides such as bufalin, arenobufagin, bufotalin, gamabufotalin, cinobufotalin, and cinobufagin and their derivatives. Bufotoxins, derivatives of bufadienolides containing polar molecules mainly belonging to argininyl residues, are reviewed as well. The established structures of bufotoxins have been compiled into a one-page figure to review their structures. We also highlighted advances in the structure-modification of the structure of compounds in this class. Drug delivery approaches to target these compounds to tumor cells were discussed in one section. The issues related to extraction, identification, and quantification are separated into another section.

Bufadienolides from the Bufo viridis toad venom exert cytotoxic effects on cancer cells by inducing cell apoptosis and cell cycle arrest.

A series of bufadienolides were isolated from the Bufo viridis toad venom, and their cytotoxic activities against three human cancer cell lines (HeLa, HT-29, MCF7) and a non-cancer cell line (L-O2) were explored using the MTT assay in vitro. All of nine compounds exhibited cytotoxic activities against the three cancer cell lines, with compound D4 exhibiting potent cytotoxic activity against HeLa cells and was better than positive control. Herein, we further evaluated the effect of compound D4 on HeLa cells. The results revealed that compound D4 has excellent cytotoxic effect on HeLa cells by inhibiting cell colony formation and migration, promoting cell apoptosis, increasing reactive oxygen species (ROS) levels and arresting of HeLa cells in S and G2/M phases. These findings encourage further work on the chemistry and bioactivity of the Bufo viridis toad venom.

Discovery of Novel 3,11-Bispeptide Ester Arenobufagin Derivatives with Potential in Vivo Antitumor Activity and Reduced Cardiotoxicity.

Arenobufagin, one of the bufadienolides isolated from traditional Chinese medicine Chan'su, exhibits potent antitumor activity. However, serious toxicity and small therapeutic window limits its drug development. In the present study, to our knowledge, novel 3,11-bispeptide ester arenobufagin derivatives have been firstly designed and synthesized on the base of our previous discovery of active 3-monopeptide ester derivative. The in vitro antiproliferative activity evaluation revealed that the moiety at C3 and C11 hydroxy had an important influence on cytotoxic activity and selectivity. Compound ZM350 notably inhibited tumor growth by 58.8 % at a dose 10 mg/kg in an A549 nude mice xenograft model. Therefore, compound ZM350 also presented a concentration-dependent apoptosis induction and low inhibitory effect against both hERG potassium channel and Cav1.2 calcium channel. Our study suggests that novel 3,11-bispeptide ester derivatives will be a potential benefit to further antitumor agent development of arenobufagin.

Chemistry and the Potential Antiviral, Anticancer, and Anti-Inflammatory Activities of Cardiotonic Steroids Derived from Toads.

Cardiotonic steroids (CTS) were first documented by ancient Egyptians more than 3000 years ago. Cardiotonic steroids are a group of steroid hormones that circulate in the blood of amphibians and toads and can also be extracted from natural products such as plants, herbs, and marines. It is well known that cardiotonic steroids reveal effects against congestive heart failure and atrial fibrillation; therefore, the term "cardiotonic" has been coined. Cardiotonic steroids are divided into two distinct groups: cardenolides (plant-derived) and bufadienolides (mainly of animal origin). Cardenolides have an unsaturated five-membered lactone ring attached to the steroid nucleus at position 17; bufadienolides have a doubly unsaturated six-membered lactone ring. Cancer is a leading cause of mortality in humans all over the world. In 2040, the global cancer load is expected to be 28.4 million cases, which would be a 47% increase from 2020. Moreover, viruses and inflammations also have a very nebative impact on human health and lead to mortality. In the current review, we focus on the chemistry, antiviral and anti-cancer activities of cardiotonic steroids from the naturally derived (toads) venom to combat these chronic devastating health problems. The databases of different research engines (Google Scholar, PubMed, Science Direct, and Sci-Finder) were screened using different combinations of the following terms: "cardiotonic steroids", "anti-inflammatory", "antiviral", "anticancer", "toad venom", "bufadienolides", and "poison chemical composition". Various cardiotonic steroids were isolated from diverse toad species and exhibited superior anti-inflammatory, anticancer, and antiviral activities in in vivo and in vitro models such as marinobufagenin, gammabufotalin, resibufogenin, and bufalin. These steroids are especially difficult to identify. However, several compounds and their bioactivities were identified by using different molecular and biotechnological techniques. Biotechnology is a new tool to fully or partially generate upscaled quantities of natural products, which are otherwise only available at trace amounts in organisms.

Cytotoxic compounds from the leaf of Bersama abyssinica subspecies abyssinica.

From the leaves of Kenyan medicinal plant Bersama abyssinica Subspecies abyssinica, four previously undescribed compounds namely, three bufadienolides, 10ß-formylpaulliniogenin B, 10ß-formylpaulliniogenin A and 1ß-acetoxy-3ß,5ß-dihydroxy-15-methoxy-16,19-dioxobufa-14(15),20,22-trienolide, and a phenolic compound 2,6,4'-trihydroxybenzophenone-4-O-(6‴-cinnamoyl)-ß-D-glucoside were isolated together with four known compounds. The structural elucidation of the compounds was based on 1D and 2D NMR spectroscopy and HRMS data analyses. The relative configurations were defined by NOESY correlations. Cytotoxic activities on L929 and KB3.1 cell lines of the isolated compounds were investigated using MTT assay. The 1ß-acetoxy-3ß,5ß-dihydroxy-15-methoxy-16,19-dioxobufa-14(15),20,22-trienolide showed significant cytotoxic activity against KB3.1 cell lines with IC50 of 3.9 ± 0.99 µM.

Network Pharmacology Prediction: The Possible Mechanisms of Cinobufotalin against Osteosarcoma.

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.

Surfactant Assisted Rapid-Release Liposomal Strategies Enhance the Antitumor Efficiency of Bufalin Derivative and Reduce Cardiotoxicity.

BACKGROUND: BF211, a derivative of bufalin (BF), shows significantly improved solubility and potent antitumor efficiency compared to BF. Unfortunately, the unwanted toxicity such as cardiotoxicity caused by unspecific distribution has hindered its clinical use. METHODS: PEGylated BF211 liposomes (BF211@Lipo) were designed and optimizely prepared based on the pre-prescription research. In vitro and in vivo cardiotoxicity was evaluated. In vivo pharmacokinetics and biodistribution of BF211@Lipo were investigated. In vivo antitumor activity and toxicity were evaluated in HepG2 cell xenograft models. The rapid-release triggered by Poloxamer 188 (P188) was assessed in vitro and in vivo. RESULTS: The optimized BF211@Lipo displayed a spherical morphology with a size of (164.6 ± 10.3) nm and a high encapsulation efficiency of (93.24 ± 2.15) %. The in vivo concentration-time curves of BF211 loaded in liposomes showed a prolonged half-life in plasma and increased tumor accumulation. No obvious abnormality in electrocardiograms was observed in guinea pigs even at 9 mg/kg. Moreover, to improve the efficient release of BF211@Lipo, a surfactant-assisted rapid-release strategy was developed, and the release-promoting mechanism was revealed by the fluorescence resonance energy transfer (FRET) and fluorescence nanoparticle tracking analysis (fl-NTA) technology. Sequential injection of BF211@Lipo and P188 could ignite the "cold" liposomes locally in tumor regions, facilitating the burst release of BF211 and enhancing the therapeutic index. CONCLUSION: Our progressive efforts that begin with preparation technology and dosage regimen enable BF211 to like a drug, providing a promising nano platform to deliver the cardiac glycosides and alleviate the side effects by decreasing unspecific biodistribution.

A hybrid membrane coating nanodrug system against gastric cancer via the VEGFR2/STAT3 signaling pathway.

Although drug combination has proved to be an efficient strategy for clinic gastric cancer therapy, how to further improve their bioavailability and reduce the side effects are still challenges due to the low solubility and untargeted ability of drugs. Recently, newly emerging nanotechnology has provided an alternative for constructing new drug delivery systems with high targeting ability and solubility. In this study, a pH-responsive liposome (Liposome-PEO, LP) loaded with apatinib (AP) and cinobufagin (CS-1) was used for combinational therapy against gastric cancer after coating with a hybrid membrane (R/C). The results indicated that the constructed nanocomplex LP-R/C@AC not only efficiently killed tumor cells in vitro by inducing apoptosis, autophagy, and pyroptosis, but also significantly inhibited tumor invasion and metastasis via the VEGFR2/STAT3 pathway. Moreover, it showed stronger anti-tumor activity in gastric cancer-bearing mouse models, as compared to the sole drugs. A naturally-derived hybrid cell membrane coating bestowed nanocomplexes with enhanced biointerfacing including prolonged circulation time and targeting ability.

Bufadienolides and ecdysteroids from the whole plants of Helleborus niger and their cytotoxicity.

A new bufadienolide (1), two new bufadienolide glycosides (2 and 3), a new ecdysteroid (4), and four known compounds (5-8), were isolated from the whole plants of Helleborus niger L. (Ranunculaceae). The structures of the new compounds (1-4) were determined by spectroscopic analysis, including 2D NMR spectral data, and hydrolytic studies. Compounds 1-6 showed cytotoxicity against HL-60 human leukemia cells, A549 human lung adenocarcinoma cells, and SBC-3 human small-cell lung cancer cells, with IC50 values ranging from 0.0055 to 1.9 µM. HL-60 cells treated with either 3 or 4 showed apoptosis characteristics, such as nuclear chromatin condensation, accumulation of sub-G1 cells, and activation of caspase-3/7.

Identification of Na+/K+-ATPase α/ß isoforms in Rhinella marina tissues by RNAseq and a molecular docking approach at the protein level to evaluate α isoform affinities for bufadienolides.

Na+/K+-ATPase (NKA) function is inhibited by Bufadienolides (BD), a group of cardiotonic steroids (CTS) primarily produced by anurans of the Bufonidae family, such as Rhinella marina. This study characterized the presence of α and ß NKA subunit isoforms in R. marina via RNAseq in four tissues: oocytes, skin, heart, and skeletal muscle. Transcripts encoding three α-like isoforms (α1, α2, α3) and three ß-like isoforms (ß1, ß2, ß4) were identified. The amino acid sequence of α1-like isoform shared 99.4% identity with the α1 isoform previously published for R. marina. Sequences for α2, α3, and ß4 from R. marina were previously unavailable. The first extracellular loop in the α2-like isoform in R. marina showed similar substitutions to those found in their susceptible homologues in other taxa (L/Q111T and S119T); in contrast, this same loop in α3-like isoform showed similar substitutions (Q111L and G120R) to those reported for toad-eating animals such as snakes, which suggests relatively lower affinity for CTS. Docking results showed that all three α-like isoforms identified in R. marina transcriptomes have low affinity to CTS compared to the susceptible α1 isoform of Sus scrofa (pig), with α1-like isoform being the most resistant. The tissue-specific RNAseq results showed the following expression of NKA α-like and ß-like subunit isoforms: Oocytes expressed α1 and ß1; skin α1, ß1, and low levels of ß2; heart α1, α3, and ß1; skeletal muscle α1, ß4, with low levels of α2, α3, and ß1. R. marina could be used as an important model for future structural, functional and pharmacological studies of NKA and its isoforms.

Bufothionine induces autophagy in H22 hepatoma-bearing mice by inhibiting JAK2/STAT3 pathway, a possible anti-cancer mechanism of cinobufacini.

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 LC3Ⅱ 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.

Novel Strategies for Solubility and Bioavailability Enhancement of Bufadienolides.

Toad venom contains a large number of bufadienolides, which have a variety of pharmacological activities, including antitumor, cardiovascular, anti-inflammatory, analgesic and immunomodulatory effects. The strong antitumor effect of bufadienolides has attracted considerable attention in recent years, but the clinical application of bufadienolides is limited due to their low solubility and poor bioavailability. In order to overcome these shortcomings, many strategies have been explored, such as structural modification, solid dispersion, cyclodextrin inclusion, microemulsion and nanodrug delivery systems, etc. In this review, we have tried to summarize the pharmacological activities and structure-activity relationship of bufadienolides. Furthermore, the strategies for solubility and bioavailability enhancement of bufadienolides also are discussed. This review can provide a basis for further study on bufadienolides.

1ß-OH-arenobufagin induces mitochondrial apoptosis in hepatocellular carcinoma through the suppression of mTOR signaling pathway.

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.

Response Surface Methodology to Optimize the Combination Treatment of Paclitaxel, Bufalin and Cinobufagin for Hepatoma Therapy.

BACKGROUND: Hepatoma is a common malignancy in the world with high morbidity and mortality. The treatment of hepatoma is limited by its poor response to many chemotherapeutic agents. Although paclitaxel (PTX) is widely used in clinical chemotherapy, the low sensitivity to hepatoma restricts its application. Combination therapy is a promising approach to resolve this dilemma. OBJECTIVE: To evaluate the interaction between paclitaxel, bufalin (BFL) and cinobufagin (CBF), and explore the optimum combination efficiently. METHODS: HepG2 cells were treated with PTX, BFL and CBF individually or in combination. Their interactions were evaluated by two classical models (Chou-Talalay model and Bliss independence). Response surface methodology (RSM) was used to explore the optimum combination. Furthermore, the optimum drug combination was verified by the morphological experiment. RESULTS: Synergistic effects were observed when cells were exposed to binary mixtures of PTX+CBF and BFL+CBF. Although the interaction of PTX and BFL was summative, a strong synergistic effect was observed when cells were exposed to ternary mixtures of PTX+BFL+CBF. The interaction results of RSM were consistent with classical models, but more efficient. Moreover, the optimum combination dose was given by RSM without the combinatorial explosion of exhaustive testing. CONCLUSION: The combination of BFL and CBF synergistically enhanced the potency of PTX against HepG2 cells. RSM could give an accurate evaluation for drug interactions and efficient prediction of optimum combination.

Three bufadienolides induce cell death in the human lung cancer cell line CL1-5 mainly through autophagy.

The effects of 3 bufadienolides, namely kalantuboside B, kalantuboside A, and bryotoxin C, isolated from Kalanchoe tubiflora (Harvey) were evaluated and characterized in CL1-5 highly metastatic human lung cancer cells. In contrast to their apoptosis-promoting activity in other cancer cells, these bufadienolides only slight or did not induce apoptosis in CL1-5 cancer cells. Instead, they activated an autophagy pathway, as indicated by increased autophagosome formation. Autophagy induced by these bufadienolides was demonstrated to be linked to the down-regulation of p-mTOR and the up-regulation of LC3-II, ATG5, ATG7, and Beclin-1. Our findings revealed an autophagy as the alternative mechanism of drug action by bufadienolides in CL1-5 lung cancer cells and provided evidence that bufadienolides are a potential therapeutic strategy for highly metastatic human lung cancer.

Gamabufotalin suppressed osteosarcoma stem cells through the TGF-ß/periostin/PI3K/AKT pathway.

AIMS: To investigate the effect of gamabufotalin (GBT) on metastasis and modulation of stemness features in osteosarcoma, and the molecular mechanisms underlying such effects. METHODS: Human osteosarcoma U2OS/MG-63 cell lines were used in this study. Cell proliferation, migration, and invasion were determined by MTT assay, wound healing assay, and cell invasion assay, respectively. The inhibitive effect of GBT on stemness was assessed by flow cytometry and mammosphere formation. The protein levels of related proteins were detected by western blotting analysis. The effect of GBT on tumorigenicity and metastasis was determined by immunofluorescence staining and immunohistochemistry in vivo experiments. RESULTS: We found that GBT suppressed the viability of U2OS/MG-63 cells in a time- and dose-dependent manner. Notably, GBT had no effect on the viability of human fetal osteoblastic (hFOB) 1.19 cells. Moreover, GBT increased the width of wounds, reduced the number of invasive osteosarcoma cells and reversed the epithelial-mesenchymal transition phenotype. Notably, we found that, compared with hFOB1.19 cells, the levels of transforming growth factor-ß (TGF-ß), periostin, phosphorylated-AKT (p-AKT), and phosphorylated-PI3K (p-PI3K) were higher in spheroids group than in parent cells. In addition, GBT reduced the ratio of CD133+ cells, the size of spheroids and Nanog, as well as the protein levels of SRY-box transcription factor 2 (SOX2), and octamer-binding protein 3/4 (OCT3/4). Our in vivo experiments showed that GBT consistently reduced lung metastasis lesions, the expression levels of matrix metalloproteinase 2 (MMP2), TGF-ß, periostin, p-AKT, and p-PI3K (immunohistochemistry staining), as well as that of CD133 in tumor tissues (immunofluorescence analysis). From a mechanistic point of view, exogenous TGF-ß/periostin/PI3K/AKT overexpression neutralized the reduction of GBT-decreased invasion/migration and the suppression of stemness properties. CONCLUSION: Collectively, our data demonstrated that GBT inhibited the viability and tumorigenesis capability of osteosarcoma cells by blocking the TGF-ß/periostin/PI3K/AKT signaling pathway. Therefore, GBT may represent a promising therapeutic agent for the management of osteosarcoma.