Study on the Anticancer Effect of an Astragaloside- and Chlorogenic Acid-Containing Herbal Medicine (RLT-03) in Breast Cancer

Background. Although surgery, chemotherapy, radiotherapy, and endocrine therapy are widely used in clinical practice for breast cancer treatment, herbal medicines (HMs) are considered as an alternative to palliative treatments because of their coordinated intervention effects and relatively low side effects. Astragaloside (AS) and chlorogenic acid (CGA) are major active ingredients of Radix Astragali and Lonicera japonica, which have shown antitumorigenic properties in certain cancers, but the role of HMs containing both AS and CGA remains unclear in breast cancer. In this study, we explored an AS- and CGA-containing HM (RLT-03) extracted from Radix Astragali, Lonicerae Japonicae Flos, Trichosanthin, and Rhizoma imperatae. Methods. RLT-03 was extracted using water and n-butanol, and the AS and CGA ingredients in RLT-03 were identified by high-performance liquid chromatography (HPLC) and evaporative light-scattering detector (ELSD). 4T1, EMT6, BT-549, and MDA-MB-231 breast cancer cell lines were used, and an EMT6 xenograft model was established. Cell proliferation, migration, and apoptosis were measured in vitro, and tumor volume and weight were observed in vivo. The expression of VEGF, EGF, IL-10, TGF-β, and CD34 and cell apoptosis in tumors were examined. Results. RLT-03 inhibited cell viability and induced apoptosis in a dose- and time-dependent manner. In vivo, tumor volume and weight were reduced, and the expression of VEGF, EGF, IL-10, TGF-β, and CD34 was suppressed in the tumor microenvironment, while cell apoptosis was induced. Conclusion. RLT-03 exhibited therapeutic effects against breast cancer by regulating the expression of ligands of receptor tyrosine kinases (RTKs) and inflammatory factors. Thus, RLT-03 represents a potential supplementary HM that can be used in breast cancer therapy.

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Ruanjian Sanjie Decoction Induces Apoptosis in Triple-negative Breast Cancer Cells by Downregulating cIAP1/2 and XIAP

10.21203/rs.3.rs-97493/v1 ◽

2020 ◽

Author(s):

Xiumei Zhao ◽

Tongxing Wang ◽

Qiang Jia ◽

Luyao Wang ◽

Cheng Tan ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cell ◽

Breast Cancer Cell ◽

Breast Cancer Cell Lines ◽

Network Pharmacology ◽

Therapeutic Effects ◽

Dependent Manner ◽

Active Components ◽

The Core

Abstract Background: Traditional Chinese medicine (TCM) comprises a unique theoretical system developed over thousands of years. The previous study reported that Ruanjian Sanjie (RJSJ) exerts anti-tumor effects by inducing cell apoptosis. However, the mechanism is not clear. Methods: In this study, we investigated the possible mechanism by the strategy of combining network pharmacology analysis with experiment (in vitro and in vivo). First, four kinds of breast cancer cell lines were used to conduct proliferation, apoptosis and cell cycle analysis. Secondly, to study pathophysiological processes of breast cancer at the molecular network level, we for the first time constructed an “integrated apoptosis module network of breast cancer” by assembling the regulatory relationships of canonical apoptosis signaling pathways. Through the strategy of combining network analysis and experiments, we analyzed the main mechanism of RJSJ in breast cancer and screened out the core genes. We further studied the inhibitory effect of RJSJ combined with carboplatin (CBP) in vivo. Finally, the synergistic effect of RJSJ and CBP were analyzed and the potential active components in RJSJ were predicted.Results: This study demonstrated that RJSJ could significantly inhibit breast cancer cell proliferation and induce apoptosis in a concentration-dependent manner. The primary mechanism of RJSJ in the treatment of breast cancer was pro-apoptotic. The core apoptosis genes regulated by RJSJ were cIAP1/2 and XIAP. We also found that RJSJ in combination with CBP tended to synergistically induce apoptosis, which might mainly be achieved through the regulation of multiple targets and pathways. Alexandrin (BX05, XKC02, SCG01), baicalin (BX22), guanosine (BX32), arjunglucoside I (XKC10) etc. were predicted as potential active components.Conclusions: These findings provide the rationale for exploring the therapeutic effects of RJSJ against breast cancer and providing a bridge for the combined use of Chinese and Western medicine.

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SY-707, an ALK/FAK/IGF1R Inhibitor, Suppresses Growth and Metastasis of Breast Cancer Cells

10.21203/rs.3.rs-148993/v1 ◽

2021 ◽

Author(s):

Ping Liu ◽

Yinghui Sun ◽

Shuang Liu ◽

Jing Niu ◽

Xijie Liu ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Signaling Pathways ◽

Breast Cancer Cells ◽

Critical Role ◽

Xenograft Model ◽

Breast Cancer Cell Lines ◽

Dependent Manner ◽

Mouse Xenograft Model

Abstract Background Focal adhesion kinase (FAK), a multi-functional cytoplasmic tyrosine kinase, plays a critical role in cancer migration, proliferation and metastasis via regulating multiple signaling pathways. SY-707 is an ALK/FAK/IGF1R multi-kinase inhibitor and now evaluated in Phase II clinical trial for ALK positive non-small cell lung cancer (NSCLC).MethodsHTRF (Homogeneous Time-Resolved Fluorescence) assay was used to analyze kinase enzyme activity to determine inhibitory activities of SY-707 on other kinases. ATP content, PE-Annexin V and would healing assays were used to examine cell proliferation, cell cycle and migration when cells were treated with SY707. Then, SD rat and beagle dog models were used to evaluate the pharmacokinetics profile, and mouse xenograft model was used to evaluate the in vivo anti-cancer activities of SY707. ResultsIn this study, we assessed preclinical anti-growth and anti-metastasis potency of SY-707 in breast cancer cells. SY-707 was able to inhibit the growth of breast cancer cell lines and induced cell apoptosis by suppressing the FAK signaling pathways. Moreover, SY-707 exerted inhibition on cell migration and adhesion in a dose-dependent manner. In T47D xenograft mice, SY-707 had significant anti-tumor activities lonely or synergistically with Paclitaxel. Meanwhile, SY-707 also displayed significant suppression on spontaneous metastasis of tumor to the lung in 4T1 murine breast cancer xenograft model. ConclusionsSY-707 illustrated potent anti-proliferation and anti-migration potential in breast cancer in vitro and in vivo, implying its therapeutic application for the treatment of breast cancer in future clinical trials.

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SY-707, an ALK/FAK/IGF1R Inhibitor, Suppresses Growth and Metastasis of Breast Cancer Cells

10.21203/rs.3.rs-148993/v2 ◽

2021 ◽

Author(s):

Ping Liu ◽

Yinghui Sun ◽

Shuang Liu ◽

Jing Niu ◽

Xijie Liu ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Signaling Pathways ◽

Breast Cancer Cells ◽

Critical Role ◽

Xenograft Model ◽

Breast Cancer Cell Lines ◽

Dependent Manner ◽

Mouse Xenograft Model

Abstract Background Focal adhesion kinase (FAK), a multi-functional cytoplasmic tyrosine kinase, plays a critical role in cancer migration, proliferation and metastasis via regulating multiple signaling pathways. SY-707 is an ALK/FAK/IGF1R multi-kinase inhibitor and now evaluated in Phase II clinical trial for ALK positive non-small cell lung cancer (NSCLC).Methods HTRF (Homogeneous Time-Resolved Fluorescence) assay was used to analyze kinase enzyme activity to determine inhibitory activities of SY-707 on other kinases. ATP content, PE-Annexin V and would healing assays were used to examine cell proliferation, cell cycle and migration when cells were treated with SY707. Then, SD rat and beagle dog models were used to evaluate the pharmacokinetics profile, and mouse xenograft model was used to evaluate the in vivo anti-cancer activities of SY707. Results In this study, we assessed preclinical anti-growth and anti-metastasis potency of SY-707 in breast cancer cells. SY-707 was able to inhibit the growth of breast cancer cell lines and induced cell apoptosis by suppressing the FAK signaling pathways. Moreover, SY-707 exerted inhibition on cell migration and adhesion in a dose-dependent manner. In T47D xenograft mice, SY-707 had significant anti-tumor activities lonely or synergistically with Paclitaxel. Meanwhile, SY-707 also displayed significant suppression on spontaneous metastasis of tumor to the lung in 4T1 murine breast cancer xenograft model. Conclusions SY-707 illustrated potent anti-proliferation and anti-migration potential in breast cancer in vitro and in vivo, implying its therapeutic application for the treatment of breast cancer in future clinical trials.

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Effect of Auraptene on angiogenesis in Xenograft model of breast cancer

Hormone Molecular Biology and Clinical Investigation ◽

10.1515/hmbci-2021-0056 ◽

2021 ◽

Vol 0 (0) ◽

Author(s):

Mohammad Reza Shiran ◽

Elham Mahmoudian ◽

Abolghasem Ajami ◽

Seyed Mostafa Hosseini ◽

Ayjamal Khojasteh ◽

...

Keyword(s):

Breast Cancer ◽

Gene Expression ◽

Animal Model ◽

Protein Expression ◽

Western Blot ◽

Xenograft Model ◽

Dependent Manner ◽

Concentration Dependent Manner

Abstract Objectives Angiogenesis is the most important challenge in breast cancer treatment. Recently, scientists become interesting in rare natural products and intensive researches was performed to identify their pharmacological profile. Auraptene shows helpful effects such as cancer chemo-preventive, anti-inflammatory, anti-oxidant, immuno-modulatory. In this regard, we investigated the anti-angiogenesis effect of Auraptene in in-vitro and in-vivo model of breast cancer. Methods In this study, 4T, MDA-MB-231 and HUVEC cell lines were used. The proliferation study was done by MTT assay. For tube formation assay, 250 matrigel, 1 × 104 HUVEC treated with Auraptene, 20 ng/mL EGF, 20 ng/mL bFGF and 20 ng/mL VEGF were used. Gene expression of important gene related to angiogenesis in animal model of breast cancer was investigated by Real-time PCR. Protein expression of VCAM-1 and TNFR-1 gene related to angiogenesis in animal model of breast cancer was investigated by western-blot. Results Auraptene treatment led to reduction in cell viability of MDA-MB-231 in a concentration-dependent manner. Also, we observed change in the number of tubes or branches formed by cells incubated with 40 and 80 μM Auraptene. Auraptene effect the gene expression of important gene related to angiogenesis (VEGF, VEGFR2, COX2, IFNɣ). Moreover, the western blot data exhibited that Auraptene effect the protein expression of VCAM-1 and TNFR-1. Conclusions Overall, this study shows that Auraptene significantly suppressed angiogenesis via down-regulation of VEGF, VEGFR2, VCAM-1, TNFR-1, COX-2 and up-regulation of IFNγ.

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Blockade of CDK7 Reverses Endocrine Therapy Resistance in Breast Cancer

International Journal of Molecular Sciences ◽

10.3390/ijms21082974 ◽

2020 ◽

Vol 21 (8) ◽

pp. 2974 ◽

Cited By ~ 2

Author(s):

Yasmin M. Attia ◽

Samia A. Shouman ◽

Salama A. Salama ◽

Cristina Ivan ◽

Abdelrahman M. Elsayed ◽

...

Keyword(s):

Breast Cancer ◽

Tumor Volume ◽

Apoptotic Cell ◽

Therapy Resistance ◽

Tamoxifen Treatment ◽

Clinical Samples ◽

Breast Cancer Cell Lines ◽

Breast Cancer Patients

Cyclin-dependent kinase (CDK)-7 inhibitors are emerging as promising drugs for the treatment of different types of cancer that show chemotherapy resistance. Evaluation of the effects of CDK7 inhibitor, THZ1, alone and combined with tamoxifen is of paramount importance. Thus, in the current work, we assessed the effects of THZ1 and/or tamoxifen in two estrogen receptor-positive (ER+) breast cancer cell lines (MCF7) and its tamoxifen resistant counterpart (LCC2) in vitro and in xenograft mouse models of breast cancer. Furthermore, we evaluated the expression of CDK7 in clinical samples from breast cancer patients. Cell viability, apoptosis, and genes involved in cell cycle regulation and tamoxifen resistance were determined. Tumor volume and weight, proliferation marker (Ki67), angiogenic marker (CD31), and apoptotic markers were assayed. Bioinformatic data indicated CDK7 expression was associated with negative prognosis, enhanced pro-oncogenic pathways, and decreased response to tamoxifen. Treatment with THZ1 enhanced tamoxifen-induced cytotoxicity, while it inhibited genes involved in tumor progression in MCF-7 and LCC2 cells. In vivo, THZ1 boosted the effect of tamoxifen on tumor weight and tumor volume, reduced Ki67 and CD31 expression, and increased apoptotic cell death. Our findings identify CDK7 as a possible therapeutic target for breast cancer whether it is sensitive or resistant to tamoxifen therapy.

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Isoform-specific promotion of breast cancer tumorigenicity by TBX3 involves induction of angiogenesis

Laboratory Investigation ◽

10.1038/s41374-019-0326-6 ◽

2019 ◽

Vol 100 (3) ◽

pp. 400-413

Author(s):

Milica Krstic ◽

Haider M. Hassan ◽

Bart Kolendowski ◽

M. Nicole Hague ◽

Pieter. H. Anborgh ◽

...

Keyword(s):

Breast Cancer ◽

Molecular Subtype ◽

Xenograft Model ◽

Tumor Formation ◽

Transcriptional Analysis ◽

Breast Cancer Cell Lines ◽

Tumor Vascularity ◽

Mouse Xenograft Model

Abstract TBX3 is a member of the highly conserved family of T-box transcription factors involved in embryogenesis, organogenesis and tumor progression. While the functional role of TBX3 in tumorigenesis has been widely studied, less is known about the specific functions of the different isoforms (TBX3iso1 and TBX3iso2) which differ in their DNA-binding domain. We therefore sought to investigate the functional consequence of this highly conserved splice event as it relates to TBX3-induced tumorigenesis. By utilizing a nude mouse xenograft model, we have identified differential tumorigenic potential between TBX3 isoforms, with TBX3iso1 overexpression more commonly associated with invasive carcinoma and high tumor vascularity. Transcriptional analysis of signaling pathways altered by TBX3iso1 and TBX3iso2 overexpression revealed significant differences in angiogenesis-related genes. Importantly, osteopontin (OPN), a cancer-associated secreted phosphoprotein, was significantly up-regulated with TBX3iso1 (but not TBX3iso2) overexpression. This pattern was observed across three non/weakly-tumorigenic breast cancer cell lines (21PT, 21NT, and MCF7). Up-regulation of OPN in TBX3iso1 overexpressing cells was associated with induction of hyaluronan synthase 2 (HAS2) expression and increased retention of hyaluronan in pericellular matrices. These transcriptional changes were accompanied by the ability to induce endothelial cell vascular channel formation by conditioned media in vitro, which could be inhibited through addition of an OPN neutralizing antibody. Within the TCGA breast cancer cohort, we identified an 8.1-fold higher TBX3iso1 to TBX3iso2 transcript ratio in tumors relative to control, and this ratio was positively associated with high-tumor grade and an aggressive molecular subtype. Collectively, the described changes involving TBX3iso1-dependent promotion of angiogenesis may thus serve as an adaptive mechanism within breast cancer cells, potentially explaining differences in tumor formation rates between TBX3 isoforms in vivo. This study is the first of its kind to report significant functional differences between the two TBX3 isoforms, both in vitro and in vivo.

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Effects of a Matrine- and Sophoridine-Containing Herbal Compound Medicine (AH-05) on Liver Cancer

Natural Product Communications ◽

10.1177/1934578x20935227 ◽

2020 ◽

Vol 15 (7) ◽

pp. 1934578X2093522

Author(s):

Yanchu Li ◽

Lu Chen ◽

Rong Pu ◽

Lu Zhou ◽

Xufeng Zhou ◽

...

Keyword(s):

T Cell ◽

Liver Cancer ◽

Tumor Volume ◽

Cell Polarization ◽

Hematological Toxicity ◽

Therapeutic Effects ◽

Dependent Manner ◽

Herbal Compound ◽

T Cell Polarization

Herbal medicine can present an alternative way of treating liver cancer. Here, we explored a matrine- and sophoridine-containing herbal compound medicine (AH-05) extracted from Adenophora capillaris, Sophora flavescens, Astragalus, and other plants. H22 and HepG2 cell models, as well as an H22 xenograft model, were established. Cell proliferation and apoptosis were measured in vitro, and tumor volume and weight were observed in vivo. The activation of AKT/mTOR and nuclear factor-κB (NF-κB) pathways in tumor cells and the polarization of CD4/CD8 T cells in the spleen were tested. To assess safety, hematological toxicity and pathology of the liver, kidney, spleen, and intestine were evaluated. AH-05 inhibited cell viability in a dose- and time-dependent manner. In vivo, tumor volume and weight were reduced, and the activation of NF-κB p50, NF-κB p65, AKT, p-AKT Ser473, and mTOR was suppressed. In addition, AH-05 promoted CD4+ T cell polarization in the spleen. With regard to safety, slight intestinal mucosa edema was observed, but no severe pathological or hematological toxicity was detected. AH-05 exhibited its therapeutic effects against liver cancer by regulating the AKT/mTOR and NF-κB signaling pathways, and the immune environment, by promoting CD4+ T cell polarization in the spleen. Thus, AH-05 represents a potential supplementary herbal compound medicine for liver cancer.

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Oncostatin M suppresses oestrogen receptor-α expression and is associated with poor outcome in human breast cancer

Endocrine Related Cancer ◽

10.1530/erc-11-0326 ◽

2012 ◽

Vol 19 (2) ◽

pp. 181-195 ◽

Cited By ~ 44

Author(s):

Nathan R West ◽

Leigh C Murphy ◽

Peter H Watson

Keyword(s):

Breast Cancer ◽

Mrna Expression ◽

Oestrogen Receptor ◽

Oncostatin M ◽

Breast Cancer Cell Lines ◽

Dependent Manner ◽

Cancer Management ◽

Er Expression

The most important clinical biomarker for breast cancer management is oestrogen receptor alpha (ERα). Tumours that express ER are candidates for endocrine therapy and are biologically less aggressive, while ER-negative tumours are largely treated with conventional chemotherapy and have a poor prognosis. Despite its significance, the mechanisms regulating ER expression are poorly understood. We hypothesised that the inflammatory cytokine oncostatin M (OSM) can downregulate ER expression in breast cancer. Recombinant OSM potently suppressed ER protein and mRNA expression in vitro in a dose- and time-dependent manner in two human ER+ breast cancer cell lines, MCF7 and T47D. This was dependent on the expression of OSM receptor beta (OSMRβ) and could be blocked by inhibition of the MEKK1/2 mitogen-activated protein kinases. ER loss was also necessary for maximal OSM-induced signal transduction and migratory activity. In vivo, high expression of OSM and OSMR mRNA (determined by RT-PCR) was associated with reduced ER (P<0.01) and progesterone receptor (P<0.05) protein levels in a cohort of 70 invasive breast cancers. High OSM and OSMR mRNA expression was also associated with low expression of ESR1 (ER, P<0.0001) and ER-regulated genes in a previously published breast cancer gene expression dataset (n=321 cases). In the latter cohort, high OSMR expression was associated with shorter recurrence-free and overall survival in univariate (P<0.0001) and multivariate (P=0.022) analyses. OSM signalling may be a novel factor causing suppression of ER and disease progression in breast cancer.

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Chidamide Combined with Doxorubicin Induced p53-Driven Cell Cycle Arrest and Cell Apoptosis Reverse Multidrug Resistance of Breast Cancer

10.21203/rs.3.rs-67615/v1 ◽

2020 ◽

Author(s):

Lixia CAO ◽

Shaorong Zhao ◽

Qianxi Yang ◽

Zhendong Shi ◽

Jingjing Liu ◽

...

Keyword(s):

Breast Cancer ◽

Cell Cycle ◽

Cell Cycle Arrest ◽

Cell Apoptosis ◽

Breast Cancer Cell Lines ◽

Tunel Staining ◽

Drug Resistant ◽

Cycle Arrest ◽

Resistant Cells

Abstract Background The multidrug-resistant (MDR) phenotype is usually accompanied by an abnormal expression of histone deacetylase (HDAC). Given that HDAC is vital in chromatin remodeling and epigenetics, inhibiting the role of HDAC has become an important approach for tumor treatment. However, the effect of HDAC inhibitors on MDR breast cancer has not been elucidated. This study aimed to evaluate the resistance of two MDR breast cancer cell lines to the HDAC-selective inhibitor chidamide (CHI). Methods Cell viability, cell cycle and apoptosis were detected by CCK8, crystal violet staining, EDU staining, TUNEL assay, flow cytometry. The expression of HDAC1, H3K9, H3K18, p53, p21, caspase3/7/9 and the Bcl family was analyzed by western blotting and Quantitative real-time PCR. MDR breast cancer growth suppression by CHI and/or doxorubicin (DOX) in vivo was investigated in a tumor xenograft mouse model. Results The results showed that, CHI combined with DOX showed significant cytotoxicity to MDR breast cancer cells in vitro and in vivo compared with the CHI monotherapy. The cell cycle distribution results showed that CHI caused G0/G1 cell cycle arrest and inhibited cell growth regardless of the addition of DOX. At the same time, Annexin V staining and TUNEL staining results showed that CHI enhanced the number of cell apoptosis in drug-resistant cells. The western blot analysis found that p53 as a tumor suppressor was in a silent state in drug-resistant cells. However, p53 was activated in the CHI-treated and combined treatment groups, which, in turn, activated the p53 up-regulated apoptosis regulator recombinant protein (Puma) and pro-apoptotic protein Bax, downregulated the apoptotic proteins Bcl-xL and Bcl-2, and activated the caspase cascade to induce apoptosis. Conclusion The irreversible cell stress induced by CHI combined with DOX reduced the expression of HDAC1 and activated caspase-dependent apoptosis and p21-mediated growth arrest pathway, which might have been driven by the activation of p53. This provided a strong theoretical basis for exploring the treatment strategy of the combined use of CHI in patients with breast cancer who did not respond to chemotherapy or had cancer progression.

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Cinobufagin Induces Apoptosis in Osteosarcoma Cells Via the Mitochondria-Mediated Apoptotic Pathway

Cellular Physiology and Biochemistry ◽

10.1159/000488842 ◽

2018 ◽

Vol 46 (3) ◽

pp. 1134-1147 ◽

Cited By ~ 14

Author(s):

Guo Dai ◽

Di Zheng ◽

Weichun Guo ◽

Jian Yang ◽

An-yuan Cheng

Keyword(s):

Cell Apoptosis ◽

Xenograft Model ◽

Childhood And Adolescence ◽

Osteosarcoma Cell ◽

Dependent Manner ◽

Osteosarcoma Cells ◽

Apoptosis Pathway ◽

Subcutaneous Xenograft ◽

Induced Apoptosis

Background/Aims: 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. Methods: 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. Results: 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. Conclusions: 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.

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