cinobufagin and Osteosarcoma

Cinobufagin (Huachansu), an aqueous extract from the dried skin of the toad Bufo bufo gargarizans Cantor (frog skin), is a biologically active ingredient of a traditional Chinese medicine cinobufacini that can treat multiple bone pathological conditions such as bone pain, bone tumors, and osteosarcoma.. The study aimed to explore the roles and molecular mechanisms of cinobufagin underlying osteosarcoma development and doxorubicin (ADR) resistance.. Cell viability, migration, and invasion were examined by CCK-8, wound healing, and Transwell invasion assays, respectively. RNA sequencing analysis was performed in MNNG/HOS cells treated with or without cinobufagin. The relationships of cinobufagin, forkhead box O1 (FOXO1), and Fc fragment of IgG binding protein (FCGBP) were examined by luciferase reporter, immunofluorescence (IF), RT-qPCR, and chromatin immunoprecipitation (ChIP) assays together with weighted gene co-expression network analysis (WGCNA) analysis. Epithelial-mesenchymal transition (EMT) marker levels were examined through the Western blot assay. The function and molecular basis of cinobufagin in osteosarcoma were further investigated by mouse xenograft experiments.. Cinobufagin reduced cell viability, weakened ADR resistance, and inhibited cell migration/invasion/EMT in osteosarcoma cells. Cinobufagin enhanced FOXO1-mediated transcription of downstream genes including FCGBP. FCGBP knockdown partly abrogated the effect of cinobufagin on osteosarcoma cell development. Cinobufagin inhibited the growth of mouse osteosarcoma xenografts in vivo. Cinobufagin reduced the expression of Ki-67 and MMP9 and facilitated caspase-3 expression in osteosarcoma xenografts.. Cinobufagin suppressed tumor progression and reduced ADR resistance by potentiating FOXO1-mediated transcription of FCGBP in osteosarcoma.

Topics: Amphibian Venoms; Animals; Bone Neoplasms; Bufanolides; Cell Adhesion Molecules; Cell Line, Tumor; Cell Movement; Cell Proliferation; Doxorubicin; Forkhead Box Protein O1; Gene Expression Regulation, Neoplastic; Humans; Mice; Osteosarcoma

Current clinical treatments for osteosarcoma are limited by disease recurrence and primary or secondary chemoresistance. Cancer stem-like cells have been proposed to facilitate the initiation, progression, recurrence and chemoresistance of osteosarcoma. Furthermore, previous studies have reported that IL-6-STAT3 pathway is overexpressed in various types of cancer and contributes to cell proliferation, apoptosis, invasion/migration, chemoresistance and modulation of stemness features.. To examined the effect of cinobufagin on cancer progression and modulation of stemness features in osteosarcoma, and investigated the molecular mechanisms underlying such effects.. Human osteosarcoma cell lines U2OS/MG-63 were recruited in this study. Cell proliferation, migration, and invasion were determined by MTT assay, colony formation assay,wound healing assay, and cell invasion assay respectively. Its effect on stemness was assessed by flow cytometry and mammosphere formation. The protein expression levels of related proteins were detected by Western blot. The xenograft model, immunofluorescence staining and immunohistochemistry were used to determine the effect of cinobufagin on tumorigenicity in vivo experiment.. We found that cinobufagin suppressed the viability of U2OS/MG-63 spheroids/parent cells in a time-and dose-dependent manner. Notably, cinobufagin had no effect on the viability of hFOB 1.19 cells. Moreover, cinobufagin induced apoptosis, increased the width of wounds, reduced invasive osteosarcoma spheroids/parent cell numbers and reduced EMT phenotype and OPN levels in U2OS/MG-63 spheroids as well as U2OS/MG-63 parent cells lines. Noticeablely, we found that OPN levels were higher in spheroids group than that in parent cells. In addition, cinobufagin ameliorated the proportion of CD133-positive cells, the size of spheroids and Nanog, Sox-2 and Oct3/4 protein levels. Our in vivo experiments showed that cinobufagin consistently reduced tumor volume,the expressions of OPN, Sox-2, Oct3/4, Nanog and p-STAT3 by the immuno histochemistry staining as well as CD133 expression in tumor tissues by immunofluorescence analysis. From a mechanistic point of view, cinobufagin was shown to inhibit IL-6-OPN-STAT3 signaling pathway. Exogenous IL-6/OE-OPN/overexpression STAT3 attenuated the induction of cinobufagin-mediated apoptosis and the suppression of stemness properties respectively.. Collectively, our data demonstrated that cinobufagin inhibited the viability and tumorigenesis capability of osteosarcoma cells by blocking IL-6- OPN-STAT3 signaling pathway. Cinobufagin may therefore represent a promising therapeutic agent for osteosarcoma management.

Topics: Antineoplastic Agents; Bone Neoplasms; Bufanolides; Cell Proliferation; Dose-Response Relationship, Drug; Drug Screening Assays, Antitumor; Humans; Interleukin-6; Molecular Structure; Osteopontin; Osteosarcoma; STAT3 Transcription Factor; Structure-Activity Relationship; Tumor Cells, Cultured

Osteosarcoma is a common primary malignant bone tumor that mainly occurs in childhood and adolescence. Despite developments in the diagnosis and treatment of osteosarcoma, the prognosis is still very poor. Cinobufagin is an active component in the anti-tumor Chinese medicine called "Chan Su", and we previously revealed that cinobufagin induced apoptosis and reduced the viability of osteosarcoma cells; however, the underlying mechanism remains to be elucidated. Herein, the present study was undertaken to illuminate the molecular mechanism of cinobufagin-induced apoptosis of osteosarcoma cell.. U2OS and 143B cells were treated with different concentrations of cinobufagin. Cell viability, colony formation ability and morphological changes were assessed by a CCK-8 assay, a clonogenic assay and light microscopy, respectively. Cell apoptosis was detected by Hoechst 33258 and Annexin V-FITC/PI staining. Reactive oxygen species (ROS) and mitochondrial membrane potential (ΔΨm) were determined by flow cytometry. Glutathione (GSH) levels were detected by a GSH and GSSG assay kit. The levels of apoptosis-related proteins were determined by western blotting, and 143B cells were introduced to establish a xenograft tumor model. The effect of cinobufagin on osteosarcoma was further investigated in vivo.. Our results showed that cinobufagin significantly reduced the viability of U2OS and 143B cells in vitro in a dose-and time-dependent manner. In addition, cinobufagin-induced apoptosis in U2OS and 143B cells was concentration-dependent. Moreover, we found that cinobufagin treatment increased the level of intracellular ROS, decreased ΔΨm, reduced GSH and inhibited GSH reductase (GR). The effects of cinobufagin on cell proliferation, apoptosis, ROS generation and ΔΨm loss were dramatically reversed when the cells were pretreated with the thiol-antioxidants NAC or GSH. Moreover, cinobufagin treatment increased the expression of the pro-apoptotic protein Bax and decreased the expression of the anti-apoptitic protein Bcl-2, thus altering the ratio of Bax to Bcl-2. Furthermore, Cinobufagin treatment caused cytochrome c release from the mitochondria to cytoplasm, thus increasing the protein levels of cleaved-caspase family members to induce apoptosis. Ac-DEVD-CHO or Z-LEHD-FMK significantly reduced cinobufagin-induced apoptosis. Finally, a subcutaneous xenograft animal study verified that cinobufagin also significantly suppressed osteosarcoma growth in vivo.. Our present data demonstrated that cinobufagin triggered cell apoptosis in osteosarcoma cells via the intrinsic mitochondria-dependent apoptosis pathway by the accumulation of ROS and the loss of ΔΨm. In an in vivo subcutaneous xenograft model, cinobufagin exhibited excellent tumor inhibitory effects. These results suggest that cinobufagin might potentially be further developed as an anti-tumor candidate for treating osteosarcoma patients in the clinic.

Topics: Animals; Apoptosis; Bone Neoplasms; Bufanolides; Caspase Inhibitors; Cell Line, Tumor; Cell Survival; Glutathione; Glutathione Reductase; Humans; Male; Membrane Potential, Mitochondrial; Mice; Mice, Inbred BALB C; Mice, Nude; Mitochondria; Oligopeptides; Osteosarcoma; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Signal Transduction

Osteosarcoma (OS) is a major cause of cancer-related mortality in children and young adults worldwide. Due to preexisting or acquired chemoresistance, the current standard neoadjuvant chemotherapy regimens show only moderate activity against OS. In the current study, we explored the potential anti-OS Cinobufagin in vitro and in vivo, and investigated its underlying mechanisms. The antitumor potential of Cinobufagin was assessed using cell viability assays, and cycle and apoptosis were determined. In a cell-based assay, the mRNA and protein expression of Notch-1, Hes-1, Hes-5 and Hey-1 were determined by quantitative polymerase chain reactions and western blotting. The involvement of Notch signaling in Cinobufagin-induced apoptosis was confirmed using gain and loss-of function assays. A xenograft OS model was established and the antitumor effect and biosafety of Cinobufagin were evaluated. Cinobufagin suppressed OS cells growth in a dose- and time-dependent manner, involving both cell cycle arrest at the S phase and programmed cell death. Cinobufagin treatment decreased the expression of Notch-1, and Hes-1, Hes-5 and Hey-1 gene expression in OS cell lines. Furthermore, Notch activation attenuated the Cinobufagin-induced apoptosis, while Notch inhibition enhanced this effect. Using a mouse xenograft model, we found that Cinobufagin inhibited OS cell growth in vivo. The mice showed excellent tolerance to Cinobufagin treatment. Taken together, our data suggested that Cinobufagin inhibited cell survival and induced apoptosis in OS cells both in vitro and in vivo, and these effects were partly mediated through the Notch pathway.

Topics: Animals; Antineoplastic Agents; Apoptosis; Bufanolides; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Cell Survival; Gene Expression Regulation, Neoplastic; Humans; Male; Mice; Osteosarcoma; Receptors, Notch; Signal Transduction; Xenograft Model Antitumor Assays

The main objective of this study was to explore whether autophagy could be triggered by cinobufagin, and to clarify the role of autophagy in the antitumor effects of cinobufagin on U2OS cells and the underlying mechanisms. U2OS cells were exposed to 15, 30, 60 and 120 mg/l cinobufagin for 0, 12, 24 and 48 h. An MTT assay was used to measure cell viability. FITC-Annexin Ⅴ/PI staining and flow cytometry were used to analyze the apoptotic ratio, while apoptotic morphological changes were assessed by PI and Hoechst 33258 viable cell staining. The effects of autophagy on the cells were investigated with GFP-LC3b green fluorescence plasmid transfection and transmission electron microscopy. The levels of caspase-3, -8, - 9, cleaved PARP, LC3-II/LC3-I, p62 and the activation of JNK/p-38 were detected by western blot analysis. Reactive oxygen species (ROS) fluorescence intensity was examined under fluorescence microscopy with an analysis software system. Cell proliferation was obviously inhibited by cinobufagin in a dose- and time-dependent manner. The apoptosis ratio was gradually increased with treatment time as evidenced by flow cytometric analysis and Hoechst 33258 staining. Exposure to cinobufagin resulted in the activation of caspase-3, -8, -9, as well as cleaved PARP which indicated that cinobufagin induced caspase-dependent apoptosis. Autophagy was confirmed in the cinobufagin-treated cells as evidenced by formation of autophagosomes, accumulation of GFP-LC3 fluorescence particles as well as the upregulation of LC3-II/LC3-I levels. Inhibition of autophagy diminished apoptosis as detected by the MTT assays. Moreover the percentage of apoptotic cells decreased following pretreatment with 3-MA, CQ and si-beclin-1. Cinobufagin also induced phosphorylation of the JNK and p38 signaling pathway as well as ROS generation. The JNK and p38 inhibitors significantly attenuated coexistence of apoptosis and autophagy-related proteins. The ROS scavenger also prevented phosphorylation of the JNK and p38 signaling pathway. Our research proved that cinobufagin triggered apoptosis and autophagic cell death via activation of the ROS/JNK/p-38 axis.

Topics: Amphibian Venoms; Antineoplastic Agents; Apoptosis; Autophagy; Bone Neoplasms; Bufanolides; Caspase 3; Caspase 8; Caspase 9; Cell Line, Tumor; Cell Proliferation; Cell Survival; Humans; JNK Mitogen-Activated Protein Kinases; MAP Kinase Signaling System; Osteosarcoma; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Poly(ADP-ribose) Polymerases; Reactive Oxygen Species; RNA Interference; RNA-Binding Proteins; RNA, Small Interfering

To investigate the effects of cinobufagin on the apoptosis in U-2OS osteosarcomas cells (U-2OS cells) and explore its potential mechanism.. The cytostatic effects of cinobufagin (10, 20, 50, 100, 200, and 400 nmol/L) on U-2OS cells were evaluated by MTT assay at 24, 48, and 72 hours after culture; simple U-2OS cells served as control group. The impact of cinobufagin (100 nmol/L) on the apoptosis in U-2OS cells was determined by flow cytometry at 48 hours after culture, which were treated with cinobufagin (experimental group) or with cinobufagin plus Z-VAD-FMK (control group), and simple U-2OS cells served as blank control group. The Caspase-3 activity was measured by Caspase-3 activity assay kit at 48 hours after culture, which were treated with cinobufagin (20, 50, and 100 nmol/L), and simple U-2OS cells served as control group. The expression of apoptosis signal pathway related proteins in U-2OS cells treated with cinobufagin were detected by Western blot at 48 hours after culture, which were treated with cinobufagin (20, 50, and 100 nmol/L), and simple U-2OS cells served as control group.. The results of MTT assay showed that cinobufagin inhibited the proliferation of U-2OS cells in a dose- and time-dependent manners. At each time point, the growth rate of U-2OS cells was significantly reduced with the increasing cinobufagin concentration, and as time prolonged, the growth rate of U-2OS cells behaved the same way in the same group. There were significant differences among different time points and groups (P < 0.05). The apoptotic rate of experimental group (46.87% +/- 11.23%) was significantly higher than that of the control group (2.34% +/- 0.98%) and blank control group (1.04% +/- 0.25%) (P < 0.05). The Caspase-3 activity in 20, 50, and 100 nmol/L groups were 1.14 +/- 0.32, 1.31 +/- 0.41, and 1.92 +/- 0.54, respectively, which were significantly higher than that in control group (P < 0.05). Compared with 20and 50 nmol/L groups, 100 nmol/L group significantly increased the Caspase-3 activity in U-2OS cells (P < 0.05). Compared with the control group, the expressions of cleaved Caspase-3, cleaved Caspase-9, and Bax were obviously up-regulated; the Bcl-2 expression was down-regulated; and the ratio of Bax/Bcl-2 was increased in different cinobufagin-treated groups (P < 0.05). The same tendency was seen in different cinobufagin-treated goups, showing significant differences among groups (P < 0.05).. Cinobufagin can inhibite the proliferation of U-2OS cells, and induce cell apoptosis. The potential mechanism of cinobufagin-induced apoptosis may be related to the mitochondria-mediated pathway.

Topics: Amino Acid Chloromethyl Ketones; Animals; Antineoplastic Agents; Apoptosis; bcl-2-Associated X Protein; Bufanolides; Caspase 3; Cell Line, Tumor; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Flow Cytometry; Gene Expression Regulation, Neoplastic; Humans; Materia Medica; Osteosarcoma

Cinobufagin, a major component of cinobufacini (huachansu), is an important cardenolidal steroid. Several studies have suggested that cinobufagin has potent anti-cancer effects. The present study examines the apoptosis-inducing activity and the underlying mechanism of action of cinobufagin in osteosarcoma (OS) cells. Our results showed that cinobufagin potently inhibited the proliferation of U2OS, MG63 and SaOS-2 cells. Significant increases in G2/M cell-cycle arrest and apoptosis in OS cells were also observed. The expression levels of several apoptotic proteins were assessed after cinobufagin treatment in U2OS cells. Among them, xIAP, cIAP-1, survivin and Bcl-2 levels decreased remarkably, while the levels of Bax and cleaved-PARP increased. Furthermore, we validated the inhibition of GSK-3β/NF-κB signaling following cinobufagin treatment. Western blots showed a decrease in nuclear p65 protein expression after exposure to different concentrations of cinobufagin, while the phosphorylation of GSK-3β was simultaneously increased. Transduction with constitutively active forms of GSK-3β could protect against the downregulation of p65 and upregulation of cleaved-PARP that are induced by cinobufagin treatment. However, combined treatment with cinobufagin and SB216367 resulted in a significant reduction in p65 and an increase in cleaved-PARP in U2OS cells. Altogether, these results show that cinobufagin is a promising agent for the treatment of OS. These studies are the first to reveal the involvement of the GSK-3β/NF-κB pathway in cinobufagin-induced apoptosis.

Topics: Amphibian Venoms; Antineoplastic Agents; Apoptosis; Apoptosis Regulatory Proteins; Bufanolides; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Down-Regulation; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Humans; NF-kappa B; Osteosarcoma; Phosphorylation; Poly(ADP-ribose) Polymerases; Signal Transduction; Transcription Factor RelA; Transfection; Up-Regulation