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Introduction

Osteosarcoma is a malignant tumor which seriously affects the physical and mental health of young adults. Current major treatment methods for osteosarcoma include surgical resection and neoadjuvant chemotherapy. However, the side effects and drug resistance to chemotherapy have not been eradicated yet, and this has affected the development of chemotherapy to a certain extent.

Drug resistance is a complicated process consisting of multiple factors. One of the key factors is aberrant apoptosis. But in recent years, autophagy alone or accompanied by apoptosis affects cancer chemotherapy sensitivity. Autophagy is a highly conserved (1,2) process in which the cytoplasm, including excess or aberrant organelles, is sequestered into double-membrane vesicles and delivered to degradative organelles, lysosomes/vacuoles, for breakdown and eventual recycling of the resulting macromolecules. Therefore, autophagy is essential for maintaining cell homeostasis and seems to play a pro-survival role as well. Excessive autophagy could lead to autophagic cell death, also known as type II programmed cell death (3,4).

Under drug stimuli or stress conditions, generation of reactive oxygen species (ROS) (5) and/or phosphorylation of the MAPK signaling pathway (6) are vital for the induction of autophagy. Conventional MAPKs are constituted by ERK, JNK, and p38. Wang et al (7) reported that N-acetyl cysteine (NAC), an ROS inhibitor, blocked rhArg-induced autophagy which plays a cytoprotective role in triple-negative breast cancer cells in vitro. Furthermore, Li et al (8) found that palmitate-induced autophagy protected H9c2 cells against apoptosis through ROS-dependent JNK and p38 MAPK. Consistent with these findings, Zhou et al (9) also demonstrated that the ROS-mediated JNK signaling pathway modulated cytoprotective autophagy in Ciclopirox-treated rhabdomyosarcoma.

Cinobufagin is one of the active ingredients in the anticancer Chinese medicine called 'Chan Su', which contains bufalin, resibufogenin and cinobufagin. Chan Su and its derivatives (10) have been reported to have anticancer activities in various types of cancer such as leukemia (11), hepatocellular cancer (12), lung cancer (13), prostate cancer (14), colon cancer (15), and cervical cancer (16). Data showed that cinobufagin was able to induce mitochondrial-dependent apoptosis (17) in hepatocellular cancer cells, exert pro-apoptotic effects in multiple myeloma (MM) U266 cells (18) through modulation of the ROS-mediated MAPK signaling pathway and was also able to induce osteosarcoma cell apoptosis (19) via the GSK-3β/NF-κB pathway.

Recently, it has been reported that bufalin induced cytoprotective autophagy in glioma cells through endoplasmic reticulum stress (20). Xie et al (21) demonstrated that ROS/JNK activation contributes to bufalin-induced autophagic cell death in colon cells. However, the role played by autophagy depends on the concentration and duration of stimuli as well as the type of cancer cells. To date, there is no literature reporting that cinobufagin induces autophagy in osteosarcoma cells. This study aimed to clarify several points: i) whether cinobufagin could induce autophagy in osteosarcoma cells; and ii) the interplay between autophagy and apoptosis and its potential mechanism.

Materials and methods

Cinobufagin was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China) and dissolved in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Grand Island, NY, USA) to each working dose. Fetal bovine serum (FBS) was purchased from Hangzhou Sijiqing Biological Engineering Material Co., Ltd. (Hangzhou, Zhejiang, China). Chloroquine (CQ), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-acetyl cysteine (NAC) and 3-methyladenine (3-MA) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The Annexin V-FITC Apoptosis Detection Kit I was obtained from BD Biosciences (San Diego, CA, USA). Hoechst 33258 was purchased from Promega Corp. (Madison, Wi, USA). Lipofectamine 2000 transfection reagent was purchased from Invitrogen Life Technologies (Carlsbad, CA, USA). Antibodies against caspase-3, caspase-8, caspase-9, cleaved PARP, phospho-JNK, total JNK, phospho-p38, total p38, LC3B, p62, GAPDH, enhanced chemiluminescence (ECL) and western blotting kits were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Cell culture and viability assay

The U2OS cells were purchased from Shanghai Institute of Biochemistry and Cell Biology (Chinese Academy of Sciences, Shanghai, China) and cultured in DMEM containing 10% FBS, 100 µg/ml penicillin and 100 µg/ml streptomycin at 37°C in a 5% CO2 incubator.

MTT assay

The cells were maintained in 96-well flat bottom microtiter plates at 1×104 cells/well overnight, and then treatment with cinobufagin at concentrations of 0, 15, 30 60 and 120 mg/l, respectively. MTT (20 µl) solution (5 g/l) was added to the cells and incubation was carried out for 4 h at 37°C. Automatic multiwell spectrophotometer was used to calculate the absorbance value/well at 570 nm. All MTT assays were performed three times. The inhibitory rate of U2OS cell proliferation was calculated according to the formula: (1 − experimental absorbance value/control absorbance value) × 100%. IC50 values (50% inhibition concentration) were then calculated using the Statistical Package for the Social Sciences (SPSS, Inc., Chicago, IL, USA).

Detection of apoptosis

U2OS cells were washed with PBS, and then stained following the manufacturer's instructions. The number of apoptotic cells was measured by flow cytometry and analyzed using Cell Quest ™ software (Becton-Dickinson, San Jose, CA, USA). Each group was calculated in triplicate.

Hoechst 33258 staining

After being fixed with 3.7% paraformaldelyde for 30 min, the cells were stained with 10 mg/l Hoechst 33258 at 37°C for 15 min. Apoptotic cells were detected using a fluorescence microscope equipped with a UV filter. The presence of condensed or fragmented nuclei stained bright blue were observed in the cells.

Green fluorescent protein (GFP)-LC3 dot assay

Cells were transfected with GFP-LC3 plasmids following the manufacturer's manual. After transfection for 24 h, the cells were treated with cinobufagin at various times. The induction of autophagy was identified by counting the percentage of cells emitting green fluorescence.

Western blot analysis

Cells were washed in phosphate buffered saline, and resuspended at room temperature. After treatment on ice for 30 min, the lysate was centrifuged at 14,0009 × g at 4°C. Protein concentration was measured with the Bradford protein assay reagent and bovine serum albumin as a standard. The membranes were incubated overnight at 4°C with the designated primary antibodies and secondary antibodies at room temperature for 2 h. GAPDH was used as the control.

Transmission electron microscopy

Cells were fixed in 2% paraformaldehyde, and then with 1% osmium tetroxide overnight at 4°C. The cells were then dehydrated in an increased dose of alcohol and embedded in Agar 100 epoxy resin. Ultrathin sections were mounted on Cu grids and stained first with uranyl acetate and post-stained with lead citrate.

ROS detection

After cinobufagin treatment at various times, U2OS cells were incubated with 5 mM DCFH-DA for 30 min in the dark, and washed three times. The fluorescence was excited at the wavelength of 485 nm and the corresponding emission wavelength was 520 nm. ROS fluorescence intensity was examined under a Leica DM2500 fluorescence microscope using a suitable analysis software system.

RNA interference

The cells were transfected with 60 nM of specific or nontargeting siRNA using Lipofectamine 2000 (Invitrogen Life Technologies) according to the manufacturer's instructions. Cells were treated with 120 mg/l cinobufagin at various times and used in other experiments. The siRNAs were obtained from GenePharma, Ltd. (Shanghai, China). The si-beclin-1 targeting sequence was 5′-UUCAACACUCUUCAGCUCAUCAUCC-3′ and the scrambled siRNA sequence was 5′-UUCUCCGAACGUGUCACGUTT-3′.

Statistical analysis

All data are presented as the mean ± SD. The differences between groups were analyzed using the Student's t-test and statistical analyses were performed using SPSS software version 16.0 (SPSS, Inc.). P<0.05 was considered to indicate a statistically significant difference.

Results

Cinobufagin inhibits U2OS cell proliferation and induces caspase-dependent apoptosis

As shown in Fig. 1, the MTT assay results proved that cinobufagin inhibited the proliferation of U2OS cells in a dose- and time-dependent manner after treatment with 0, 15, 30, 60 and 120 mg/l for 12, 24 and 48 h. The IC50 value of cinobufagin treatment at 24 h was 120 mg/l. To further confirm cinobufagin-triggered apoptosis in U2OS cells, flow cytometry, Hoechst 33258 and western blotting were performed in turn.

Figure 1 Inhibitory effect of cinobufagin on U2OS cell proliferation (n=3).

As shown in Fig. 2A and B, the percentage of apoptotic cells in the control group was 2.71±0.24%, and the apoptotic ratio gradually increased with prolonged treatment time. Then morphological changes in the cell nuclei were observed under a fluorescence microscope by Hoechst 33258 staining. Shrunken, condensed nuclei with bright blue fluorescence were tagged as apoptotic cells. As shown in Fig. 2C and D, more apoptotic cells with brighter fluorescence were observed as the treatment of cinobufagin progressed. To further characterize apoptosis, western blotting was performed. As shown in Fig. 2E and F, caspase-3, -8, -9 and cleaved PARP were upregulated following treatment. In the presence of the specific caspase inhibitors Z-IETD-fmk, Z-LEHD-fmk and Z-VAD-fmk, respectively, cell viability was upregulated (Fig. 2G). The aforementioned results indicated that cinobufagin is cytotoxic against U2OS cells and induces caspase-dependent apoptosis.

Figure 2 Cinobufagin induces caspase-dependent apoptosis in U2OS cells. (A and B) Apoptosis was measured by Annexin V-FITC (AV-FITC)/propidium iodide (PI) staining following treatment of the cells with 120 mg/l cinobufagin for 0, 12, 24 and 48 h. (C and D). The morphological changes in U2OS cells treated with 120mg/L cinobufagin were observed by Hoechst 33258 stain. Concentrated cell nuclei with bright blue fluorescence were marked as apoptotic cells. Data from three independent experiments were compared. (E and F) Caspase-3, -8, -9 and cleaved PARP levels were examined by western blot analysis in U2OS cells incubated with 120 mg/l cinobufacin for 12, 24 and 48 h. GAPDH served as the loading control. (G) The cell viability of U2OS cells was determined following inbubation with 120 mg/l cinobufagin with/without caspase-inhibitors; ΔP<0.05 compared with control group.

Cinobufagin triggers U2OS cell autophagy flux

We clearly observed a multitude of autophagic vacuoles engulfing damaged organelles and macromolecular substances triggered by 120 mg/l cinobufagin under TEM (Fig. 3A; black arrows). In order to further elucidate cinobufagin-induced autophagy, GFP-LC3 transient transfection and western blot analysis were employed.

Figure 3 Cinobufagin triggers autophagy in U2OS cells. (A) Ultrastructural observations were made in U2OS cells using a transmission electron microscope. (a) Untreated U2OS cells exhibited normal morphology. (b) U2OS cells treated with 120 mg/l cinobufagin for 24 h showed typical autophagosomes with a double membrane which included the damaged organelles and extra protein. (B and C) Cells transiently transfected with GFP-LC3 plasmids were exposed to 120 mg/l cinobufagin for 0 h (control), 12, 24 and 48 h. A confocal microscope was used to measure GFP-LC3 fluorescence distribution. The punctate GFP-LC3 was indicative of autophagosomes. ΔP<0.05 compared with control group. (D and E) Western blot analysis of LC3I, LC3II and p62 expression after U2OS cells were treated with 120 mg/l cinobufagin for 24, 48 and 72 h, respectively. ΔP<0.05 compared with the control group. (F and G) LC3II level was examined by western blot analysis in U2OS cells after treatment with 120 mg/l cinobufagin in the absence or presence of 10 µl chloroquine (CQ) for 24 h.

At a low level of autophagy, autophagy marker protein GFP-LC3 plasmids are dispersed. GFP-LC3 fluorescent particles during activation of autophagy are increased in both brightness and density with increased treatment time (Fig. 3B and C). During autophagy, microtubule-associated protein 1 light chain 3 (LC3)/Atg8 converts 18-kDa LC3-I to 16-kDa LC3-II. The ratio of LC3-II/LC3-I correlates with activation of autophagic activity. p62 (22) accumulates the ubiquitin protein selectively to increase autophagy, and eventually to complete the protein degradation by fusion with the lysosomes. Consistent with the GFP-LC3 plasmid results, western blot analysis indicated an increase in the levels of LC3-II/LC3-I, and a reduction in p62 expression in a time-dependent manner (Fig. 3D and E).

Furthermore autophagy is a dynamic, multi-step process. And when blocked at the final stage, LC3-II/LC3-I is also accumulated. To further confirm the induction of autophagy flux by cinobufagin, we verified the accumulation of LC3-II/LC3-I with or without autophagic inhibitor CQ. As shown in Fig. 3F and G, when compared to the control group, the CQ group alone exhibited no statistical significant difference. The levels of LC3-II/LC3-I were increased in the cinobufagin+CQ group following pretreatment with CQ for 1 h compared to the cinobufagin group. All the above results indicated that cinobufagin induced autophagy flux in the U2OS cells.

The influence of autophagy inhibition on cinobufagin-induced apoptosis and cell viability in U2OS cells

Autophagy is a multi-step process and can be inhibited at different stages. To determine the role of autophagy induced by cinobufagin, autophagy inhibitors (CQ and 3-MA) were used in the following experiments. 3-MA blocks class III phosphatidylinositol 3-kinases (PI-3Ks) leading to inhibition of autophagosome (23) formation at an early stage. CQ is generally used to block autophagosome combination (24) with lysosomes and finally degradation obstruction in the late stage. As shown in Fig. 4A and B, preincubation with 3-MA resulted in the decrease of LC3-II/LC3-I. Meanwhile, inhibition of autophagy using CQ enhanced LC3-II/LC3-I compared with cinobufagin alone. However, a marked reduction in cinobufagin-induced cell growth inhibition was also observed when autophagy was inhibited by CQ and 3-MA (Fig. 4C). Consistently, we also found that pretreatment with CQ decreased the proportion of apoptotic cells (56.11%) compared with the cinobufagin group alone (28.19%) (Fig. 4D and E).

Figure 4 Suppression of autophagy attenuates cinobufagin-induced apoptosis and growth inhibition of U2OS cells. (A and B) LC3II/LC3I levels were examined by western blot analysis in U2OS cells after treatment with 120 mg/l cinobufagin for 24 h in the absence or presence of 10 µl chloroquine (CQ) and 5 µl 3-methyladenine (3-MA). GAPDH served as the loading control. All results are represented as mean ± SD. ΔP<0.05 compared with the control group, ※P<0.05 compared with the cinobufagin group. (C) Cell viability following treatent with 120 mg/l cinobufagin as determined by MTT assay (n=3, means ± SD) in the presence or absence of CQ and 3-MA. (D and E) Apoptosis was measured by Annexin V-FITC (AV-FITC)/propidium iodide (PI) staining following treatment of 120 mg/l cinobufagin for 24 h with or without CQ. ΔP<0.05 compared with the control group, ※P<0.05 compared with the cinobufagin group. (F and G) U2OS cells were transiently transfected with beclin-1 siRNA and then treated with 120 mg/l cinobufagin. The levels of beclin-1 and LC3II/LC3I were detected by western blot analysis. (H) Viability was measured by MTT assay. ΔP<0.05 compared with the si-control group, ※P<0.05 compared with the si-control+cinobufagin group. Cinobufagin (120 mg/l)-induced cell viability as determined by MTT assay (n=3, means ± SD) in the presence or absence of si-beclin-1.

We further inhibited autophagy by silencing the beclin 1 gene, as beclin-1 is an important autophagy-related gene. siRNA was used. Transfection with si-beclin-1 resulted in a marked reduction in beclin-1 and LC3-II/LC3-I protein expression, while the scrambled siRNA-control group showed no change (Fig. 4F and G). As shown in Fig. 4H, beclin-1 knockdown markedly reduced the viability of the cells treated with cinobufagin. Thus, beclin-1 has a positive effect on the growth of U2OS cells.

ROS participates in cinobufagin treatment in U2OS cells

As shown in Fig. 5A and B, the intensity of green fluorescence representing the degree of ROS became more intense as treatment time progressed, which indicated that ROS were involved in cinobufagin treatment. NAC, an ROS exclusive inhibitor, was preincubated for 1 h to clarify the effects of ROS on cinobufagin-induced cytotoxicity in U2OS cells. The western blot analysis assay demonstrated that the levels of LC3-II/LC3-I, cleaved caspase-3 and cleaved PARP were reduced in the cinobufagin+NAC group compared to the cinobufagin group (Fig. 5C and D) indicating that NAC antagonized cinobufagin treatment. All these results revealed that ROS are the crucial factor of cinobufagin-induced apoptosis and autophagy.

Figure 5 Reactive oxygen species (ROS) mediate cinobufagin-induced autophagy and apoptosis in U2OS cells. (A and B) The average fluorescence density of ROS induced by cinobufagin in the U2OS cells. Data are presented as mean ± SD from three independent experiments. P<0.05. Mean levels of ROS are shown in the histogram (right); ΔP<0.05 compared with the control group. (C and D) Western blot analysis of the protein levels of LC3II/LC3I, caspase-3 and cleaved PARP in the U2OS cells incubated with cinobufagin in the presence or absence of NAC. Densitometric analysis of LC3II/LC3I, cleaved caspase-3, cleaved PARP expression is shown in the histogram (right). GAPDH served as the loading control. All data are represented as mean ± SD. ΔP<0.05 vs. the control group, ※P<0.05 vs. the cinobufagin group.

The activation of the JNK signaling pathway following cinobufagin treatment in U2OS cells

As shown in Fig. 6A and B, western blot analysis was employed to assess the expression of JNK, p-JNK, p-p38 and p38. Cinobufagin cleaved the phosphorylation of the JNK and p-38 signaling pathway. In addition, pretreatment with the JNK and p-38 inhibitors SP600125 and SB203580 significantly attenuated LC3-II/LC3-I, cleaved caspase-3 as well as cleaved PARP levels acccompanied by downregulation of p-JNK/p-p38 compared to the cinobufagin group (Fig. 6C and D). These data indicated that the JNK/p38 signaling pathway was upstream in cinobufagin cytotoxicity. To study the relationship between ROS and JNK/p38 signaling pathway, the ROS inhibitor NAC was used. As expected, NAC suppressed the level of p-JNK and p-p38 (Fig. 6E and F). Thus, the ROS/JNK/p38 signaling pathway plays a central role in cinobufagin-triggered apoptosis and autophagy.

Figure 6 Reactive oxygen species (ROS)-activated MAPK and JNK/P38 pathways contribute to cinobufagin-induced autophagy and apoptosis. (A and B) U2OS cells were incubated with 120 mg/l cinobufagin at various times and measurement of p-JNK, JNK, p-38 and p-p38 by western blot analysis followed. ΔP<0.05 vs. the control group. (C and D) U2OS cells were pretreated with SP600125 (10 µM) or SB203580 (0.5 µM) for 1 h, and then incubated with 120 mg/l cinobufagin for 24 h to determine the level of autophagy-related protein LC3II/LC3I, apoptotic-related protein cleaved caspase-3, cleaved PARP as well as p-JNK, t-JNK, p-p38, t-P38. ΔP<0.05 vs. the control group, ※P<0.05 vs. the cinobufagin group. (E and F) NAC attenuated cinobufagin-induced JNK and p-38 activation. U2OS cells were pretreated with NAC (5 mM) for 1 h, and then incubated with 120 mg/l of cinobufagin for 24 h to detect the effects of ROS on JNK/p38 signaling. GAPDH was used as loading control. Data are represented as the mean ± SEM of three independent experiments. ΔP<0.05 vs. the control group, ※P<0.05 vs. the cinobufagin group.

Discussion

In our research, the data showed that cinobufagin suppressed the proliferation of U2OS cells in a concentration- and time-dependent manner. Research showed that cinobufagin/bufalin induced osteosarcoma apoptosis by triggering the mitochondrial pathway (25) and downregulating Hsp27 (26). Yin et al (19) demonstrated that cinobufagin-induced apoptosis was attributed to the activation of the GSK-3β/NF-κB pathway in osteosarcoma cells. Our experimental results revealed that the percentage of apoptotic cellswas increased at the indicated treatment times by flow cytometry and Hoechst 33258 staining (Fig. 2A–D). The process of apoptosis is carried out by two pathways: i) extrinsic stimuli through cell surface death receptor (caspase-8) pathway or ii) intrinsic stimuli through the mitochondrial signaling pathway (caspase-9). However, for either pathway, caspase-3/cleaved-PARP are both activated and are related to cell structure, cell cycle regulation, DNA repair and other related proteins, ultimately causing irreversible cell death (27). Cao et al found that cinobufagin (28) induced osteosarcoma cell mitochondrial-dependent apoptosis accompanied by caspase-7 activation. However, Xie et al (21) did not find any increase in caspase-3 and PARP cleavage following bufalin treatment in colon cells. In our experiments, cleaved caspase-3, -8, -9 as well as cleaved PARP were simultaneously upregulated. Following pretreatment with caspase inhibitors, cell viability increased to different degrees. In short, cinobufagin triggered caspase-dependent apoptosis in our experiment.

Autophagy, also known as type II cell death, is a non-caspase-dependent cell death form distinct from apoptosis. According to the stage of the tumor as well as the tissue or the cells targeted (29), autophagy can exert a positive or negative effect on the growth of tumors. In our experiments, we clearly observed activation of autophagy flux by cinobufagin in ostersarcoma U2OS cells for the first time as confirmed by the following results. i) An abundance of autophagosomes were observed under TEM (Fig. 3A). ii) GFP-LC3 green fluorescent particles aggregated with the progression of treatment time (Fig 3B and C). iii) The expression of LC3-II/LC3-I accumulated following pretreatment with CQ (Fig. 3D and E). However, what role autophagy plays has been largely debated. According to the autophagy inhibitors 3-MA and CQ, respectively the inhibition mechanism and the level of LC3-II/LC3-I changed compared to the cinobufagin group alone (Fig. 4A and B). Consistently, cell proliferation of the cinobufagin+3-MA/CQ group was synchronously attenuated compared to the cinobufagin group (Fig. 4C). Flow cytometry results also revealed that the number of apoptotic cells was diminished in the cinobufagin+CQ group more when compared with that in the cinobufagin group alone revealing that cinobufagin triggered autophagic cell death (Fig. 4D and E).

To further clarify the viewpoint above, the siRNA approach was used. Beclin-1 (a key autophagy regulator) was silenced, and LC3-II/LC3-I were downregulated in the cinobufagin+si-beclin-1 group compared with the scrambled control (SCR) siRNA transfection group (Fig. 4F and G). We also observed that silencing of beclin-1 decreased cinobufagin-induced suppression of cell viability when compared with the siSCR group (Fig. 4H). Induction of autophagy, at least in part, was responsible for cinobufagin cytotoxicity in osteosarcoma which was in line with the findings of Xie et al (21).

ROS include a variety of oxygen-containing, reactive and short-lived molecules (30) which are thought to operate as signaling molecules in signal transduction pathways regulating cell growth, differentiation, survival, inflammation and immune response. The MAPK pathway can be activated by extracellular stimuli, such as Gi protein-coupled receptors (GPCR), ultraviolet irradiation (31,32), genotoxic agents and oxidative stress. In our study, generation of ROS was activated in a time-dependent manner. Consequently, we confirmed that JNK/p-38 phosphorylation decreased by pretreatment with ROS inhibitor NAC. Following preincubation with the ROS inhibitor NAC, JNK inhibitor SP600125 and p-38 inhibitor SB230580 respectively, LC3-II/LC3-I, caspase-3, cleaved PARP as well as p-JNK and p-p38 were decreased to different degrees compared with the cinobufagin group, indicating that ROS activated the JNK/p38 signaling pathway and that cinobufagin induced autophagy and caspase-dependent apoptosis. In agreement with our results, Li et al (33) reported that suppression of celastrol-induced autophagy diminished caspase-dependent apoptosis. However, Xie et al demonstrated that bufalin induced autophagic cell death without cleaved caspase-3 and cleaved PARP generation. Shen et al (20) found that bufalin-induced autophagy played a cytoprotective role in bufalin-induced ER stress and cell death. Different types of cells caused stimulation to different levels of severity.

However, there is no doubt that the underlying mechanisms of cinobufagin in osteosarcoma therapy still requires further investigation. For example, in this study we did not address expression of the Bax/Bcl-2 and PI3kt/mTOR/Akt signaling pathways, and moreover whether endoplasmic reticulum stress occurs in cinobufagin treatment of osteosarcoma. Thus, further exploration is needed to reveal the underlying mechanisms in the crosstalk among apoptosis, autophagy and ER stress.

In conclusion, our study elucidated a detailed mechanism involved in cinobufagin-induced apoptosis and autophagy in osteosarcoma which will pave the way for further development of the clinical application of this compound for treating osteosarcoma.

References