2-methylbenzoyl berbamine, a multi-targeted inhibitor, suppresses the growth of human osteosarcoma through disabling NF-κB, ERK and AKT signaling networks

Results

BBD24 inhibited the proliferation of osteosarcoma cell lines and primary tumor cells in vitro

We first sought to determine whether BBD24 suppressed the proliferation of human osteosarcoma cell lines in vitro. Two human osteosarcoma cell lines, HOS and MG63, were treated with BBD24 at various concentrations. Cell viability was measured using the MTT assay at 24, 48 and 72 hours. The concentration that inhibited 50% of target cells (IC₅₀) was calculated from dose-response curves. To investigate the superiority of BBD24 in the fight against OS, we also measured the IC₅₀ of BBM in HOS and MG63 cells. As shown in Figure 1, the proliferation of HOS and MG63 cells was greatly inhibited by BBD24 in a time- and dose-dependent manner. The IC₅₀ values for HOS cells treated with BBD24 for 24, 48, and 72 hours were 3.88 μg/ml, 2.19 μg/ml and 1.79 μg/ml, respectively (Figure 1A). Similarly, the IC₅₀ values for MG63 cells treated with BBD24 for 24, 48, and 72 hours were 3.99 μg/ml, 2.27 μg/ml and 1.90 μg/ml, respectively (Figure 1B). These results suggested that BBD24 had potent antitumor activity against osteosarcoma cell lines, which led us to further investigate whether this compound was also active against primary osteosarcoma cells. Primary osteosarcoma cells were treated with BBD24 at various concentrations for 48 hours. Cell viability was measured by MTT assay, and IC₅₀ values were calculated. As expected, BBD24 also significantly suppressed the proliferation of primary tumor cells from all 8 osteosarcoma specimens. The IC₅₀ values of BBD24 in the 8 primary tumor specimens ranged from 1.18 μg/ml to 2.17 μg/ml (Figure 1A). In contrast, BBD24 did not affect normal blood cells under the same conditions, and their IC₅₀ values ranged from 8.60 μg/ml to 9.75 μg/ml (n=4) (Figure 1A). These results suggested that BBD24 selectively suppressed the proliferation of tumor cells from both osteosarcoma cell lines and fresh primary tumor specimens but spared normal blood cells. The prototype drug BBM was also effective in suppressing OS cell proliferation (Figure 1). However, compared with BBD24, the IC₅₀ values of BBM for OS cell lines and primary OS cells were both higher, while the toxicity was similar. Our results indicated that BBD24 was more effective and safer than BBM.

Figure 1. Effects of BBD24 on proliferation of human osteosarcoma cells and normal blood cells. Cells were treated with BBD24 at indicated concentrations for indicated times. Cell viability was measured by MTT assay. (A) IC50 of BBD24 and BBM on human osteosarcoma cells and normal blood cells (peripheral blood mononuclear cells, MBMNCs). (B, C) Cell viability of HOS and MG63 after treating with different concentrations of BBD24 and BBM for different times.

Effect of BBD24 on NF-κB, ERK and AKT signaling networks

To further study the mechanism of BBD24 in OS, we investigated whether BBD24 affects the NF-κB and AKT signaling pathways in human OS cells. HOS cells were treated with BBD24 at various concentrations for 48 hours and then collected for analysis of nuclear and total NF-κB p65 protein levels using western blot. We found that treating OS cells with BBD24 caused a significant decrease in nuclear NF-κB p65 protein expression in a dose-dependent manner, but total NF-κBp65 protein expression was not changed substantially (Figure 2A–2C). These results indicated that BBD24 downregulated the NF-κB signaling network by blocking the cytoplasmic-to-nuclear translocation of NF-κB p65 in tumor cells. We next assessed the effect of BBD24 on ERK and AKT activation in human OS cells. HOS cells were treated with BBD24 at various concentrations for 48 hours, and total cellular proteins were extracted for western blotting analysis of total and phosphorylated ERK and AKT. The western blot results showed that BBD24 treatment caused a significant decrease in phosphorylated ERK and AKT protein levels in tumor cells in a dose-dependent manner but did not affect total ERK and AKT protein levels (Figure 2A, 2D, 2E). These results indicated that BBD24 treatment inhibited the activation of both the ERK and AKT signaling pathways.

Figure 2. BBD24 down-regulated NF-κB, ERK and AKT signaling pathways of HOS cells. The cells were treated with BBD24 at indicated concentrations for 48 hours, and then nuclear and cytoplasmic proteins were extracted for western blot analysis. β-actin and histone 1 were used as cytoplasmic and nuclear loading controls, respectively. (A–E) BBD24 treatment reduced nuclear NF-κB p65 protein level, and inhibited activation of ERK and AKT of HOS cells in dose-dependent manners. Results were expressed as means ± SD of three independent experiments. * P<0.05.

BBD24 induced OS cell apoptosis and autophagy

To establish a link between the above cancer-associated signaling networks and cell death pathways, we further examined OS cell death-related proteins after treatment with BBD24. HOS cells were treated with BBD24 at 2 μg/ml for 0, 12, 24, and 48 hours and then collected for analysis of cell viability, apoptosis and necrosis using flow cytometry (FCM). We found that BBD24 treatment markedly reduced the viability of HOS cells in a time-dependent manner, and we observed a significantly increased apoptosis rate (Figure 3A). Notably, we also found that HOS cells treated with BBD24 for 48 hours displayed greatly increased necrosis (Figure 3A–3C). Western blot analysis provided more information. BBD24 induced Bax overexpression in HOS cells (Figure 3D, 3E). In addition, cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 were significantly upregulated in BBD24-treated OS cells (Figure 3D, 3F–3H). To determine whether autophagy was also involved in BBD24-induced cell death, we subsequently evaluated the expression level of LC-3I/II in HOS cells after treatment with BBD24 for 48 hours. Interestingly, we also observed a marked increase in LC3 II after BBD24 treatment at ≥ 0.5 μg/ml for 48 h (Figure 3D, 3I). These results indicated that apoptosis pathways were mainly activated along with necrosis and autophagic death pathways in osteosarcoma cells after treatment with BBD24. These findings suggested that BBD24 exerted its antitumor actions by triggering multiple death pathways.

Figure 3. BBD24 induced multiple cell death pathways of HOS cells. Human osteosarcoma HOS cells were treated with BBD24 at 2 μg/ml for indicated times. The cells were harvested by trypsinization and collected by centrifugation for analysis of cell viability, apoptosis and necrosis using flow cytometry assay. HOS osteosarcoma cells were treated with BBD24 at the indicated concentrations for 48 hours, followed by Western blot analysis for caspase family and LC3-II. (A–C) Flow cytometry assay indicated that BBD24 induced apoptosis and necrosis of HOS cells with time dependence. (D–I) BBD24 promoted activation of caspases and autophagy in HOS cells in a dose-dependent manner. Results were expressed as means ± SD of three independent experiments. * P<0.05.

BBD24 suppressed OS cell migration and invasion

MG63 and HOS cells (1×10⁵) were seeded on a Transwell insert to assess migratory and invasive abilities. After treatment with different concentrations of BBD24, the migration and invasion of OS cells were reduced in a dose-dependent manner (Figure 4). Particularly in the 2 μg/ml group, both the migration and invasion of OS cells were almost completely suppressed (Figure 4).

Figure 4. BBD24 suppressed OS cells migration and invasion. 1×10⁵ MG63 and HOS cells were seeded on a transwell insert, then treating with different concentrations of BBD24. (A–C) BBD24 reduced the migration and invasion of MG63 and HOS cells in a dose dependent manner. Results were expressed as means ± SD of three independent experiments. * P<0.05.

BBD24 inhibited the growth and increased the cisplatin sensitivity of OS cells in vitro and in vivo

HOS cells were treated with different concentrations of BBD24, cisplatin or both for 72 h, and the MTT assay was used to measure cell viability. We found that both BBD24 and cisplatin alone inhibited the viability of HOS cells, while combining cisplatin with 2 μg/ml BBD24 significantly increased the cisplatin sensitivity of HOS cells (Figure 5A–5D). We further applied additional concentrations of BBD24. The results showed that cisplatin combined with 2 μg/ml BBD24 achieved better results than 1 μg/ml BBD24 combined with less than half the dose of cisplatin (Figure 5E, 5F). To determine whether BBD24 could inhibit osteosarcoma in vivo, we treated nude mice with BBD24 and/or cisplatin for 20 consecutive days. We found that tumor volume was significantly reduced after treatment with BBD24 and/or cisplatin, while cisplatin combined with BBD24 achieved the best result (Figure 6A, 6B). Immunohistochemical staining showed similar results; both BBD24 and cisplatin induced cleaved caspase-3 expression, while the BBD24+cisplatin group had the highest rate of positive staining (Figure 6C, 6D). Ki-67 and PNCA expression was also inhibited in the BBD24 and cisplatin groups, and BBD24+cisplatin further inhibited Ki-67 and PCNA staining compared to that with BBD24 or cisplatin alone (Figure 6C, 6E, 6F). When comparing BBD24 with cisplatin, BBD24 was more effective in inhibiting tumor growth and PCNA expression (Figure 6A, 6C, 6F). BBD24 also induced more cleaved caspase-3 expression than cisplatin (Figure 6C, 6D). Taken together, our results indicated that BBD24 was able to improve the cisplatin sensitivity of OS in vitro and in vivo.

Figure 5. BBD24 improved cisplatin sensitivity in vitro. HOS cells were treated with BBD24 and/or cisplatin for 72h, then MTT assay was used to measure the cell viability. (A, B) HOS cells were treated with different concentrations of BBD24 or cisplatin. (C) HOS cells were treated with BBD24 (2 μg/ml) and/or cisplatin (1 μg/ml). (D) Cell viability of HOS in cisplatin group and cisplatin+BBD24 group. (E, F) IC50 of BBD24, cisplatin, cisplatin+BBD24 (1 μg/ml) and cisplatin+BBD24 (2 μg/ml). Results were expressed as means ± SD of three independent experiments. * P<0.05.

Figure 6. in vivo anti-tumor activity of BBD24. Nude mice were treated with BBD24 and/or cisplatin for 20 consecutive days. (A, B) Tumor volumes in control, cisplatin, BBD24 and BBD24+cisplatin groups. (C–F) Immunohistochemical staining of Cleaved-caspase-3, Ki-67 and PCNA in different groups. Results were expressed as means ± SD of three independent experiments. * P<0.05.

Materials and Methods

Reagents and antibodies

Berbamine (BBM) and BBD24 were obtained from Hangzhou Bensheng Pharmaceutics (Hangzhou, China), and their chemical structures and formulas are shown in Figure 7. The compound was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 10 mg/ml. NF-κB p65 and histone 1 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). LC3I/II and β-actin antibodies were from Sigma (St Louis, MO, USA). Caspase family, AKT, p-AKT, ERK, p-ERK, PCNA and Ki-67 antibodies were purchased from Cell Signaling Technology. The annexin V-FITC/PI apoptosis detection kit was from BD Biosciences.

Figure 7. Chemical structure and formula of BBD24.

Human osteosarcoma cell lines and culture

Two human osteosarcoma cell lines, MNNG/HOS and MG63, which were provided by Cancer Institute Zhejiang University, were used in this study. OS cells were grown in DMEM supplemented with 10% FBS at 37°C in a 95% air, 5% CO₂ humidified incubator. All cell lines were used within 20 passages.

Primary osteosarcoma cells and normal hematopoietic cells

Institutional Review Board (IRB) approval was given by the Ethical Committee of the Second Affiliated Hospital, Zhejiang University School of Medicine (Approval number: 2017-043). Primary osteosarcoma specimens were obtained from patients who had given informed consent. Normal blood cells were also obtained from the peripheral blood of healthy volunteer donors. Mononuclear cells were isolated from the samples using Ficoll-Plaque density gradient separation. Primary osteosarcoma cells were cultured in DMEM with 10% FBS, and normal blood cells were cultured in RPMI-1640 medium with 10% FBS at 37°C in a 95% air, 5% CO2 humidified incubator. All primary cells were used within 5 passages.

Cell viability determination

For the cell viability assay, different OS cells and blood cells were seeded at a density of 1×10⁴ cells/ml. Cells were incubated with different concentrations of BBD24 and/or cisplatin for 72 h. Cell viability was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide).

Flow cytometry analysis

HOS cells were treated with 2 μg/ml BBD24 for 12, 24 or 48 h. Following compound treatment, the cells were harvested by trypsin, washed with PBS, and resuspended in 1× annexin-binding buffer. Cells were stained with annexin V-FITC and propidium iodide (Becton-Dickinson Company, USA). Five microliters of annexin-V-fluorescein isothiocyanate and 5 μL of propidium iodide were added to the buffer and incubated for 15 min in the dark. After that, flow cytometry analysis was performed to detect apoptotic and necrotic cells. In the flow cytometry image, the lower right quadrant represents apoptotic cells, and the upper left and upper right quadrants signify necrotic cells. We compared the apoptosis rate and necrosis rate between groups.

Western blot analysis

HOS cells were treated with different concentrations of BBD24 for 48 h. After treatment, cellular protein was extracted using Mammalian Protein Extraction Reagent. The protein concentration was quantified using a BCA Protein Assay Kit (Thermo Scientific, USA). Nucleoprotein was used to assess the expression of nuclear p65 and histone 1. Total protein was used to assess the expression of total p65, p-ERK, ERK, p-AKT, AKT, Bcl-2, Bax, caspases, LC3I/II and β-actin. Cell homogenates were loaded into SDS-PAGE gels with concentrations ranging from 4% to 15%, separated by electrophoresis and transferred onto polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% fat-free milk at room temperature for 1 h and subsequently incubated overnight at 4°C with primary antibodies. Then, the membranes were washed with TBST three times and then exposed to appropriate secondary antibodies that were conjugated with IRDye 800CW at room temperature for 1 h. An Odyssey infrared imaging system (LI-COR) was used to detect the positive bands. β-Actin and histone 1 were used as cytoplasmic and nuclear loading controls, respectively.

Migration and invasion assays

OS cells (1×10⁵) were seeded on a polycarbonate membrane insert with or without Matrigel coating (6.5 mm in diameter with 8.0 μm pores) in a Transwell apparatus (Costar, Cambridge, MA). DMEM containing 10% FBS was added to the lower chamber, while DMEM containing 0.2% BSA was added to the upper chamber. After incubation for 12 h at 37°C, the insert was washed with PBS, and cells on the top surface of the insert were removed by wiping with a cotton swab. Cells that migrated to the bottom surface of the insert were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. Cells were counted by using a microscope (Nikon Eclipse 80i; Nikon, Tokyo, Japan) and NIS-Elements D 4.50 (Nikon Instruments, Tokyo, Japan) at 200× magnification.

Animal model and treatment

The animal model of osteosarcoma was generated as reported previously [30]. Four-week-old female nude mice (BALB/c, nu/nu; SIPPR-BK Laboratory Animal Co., Ltd., Shanghai, China) were housed under pathogen-free conditions at 26–28°C with 50–65% humidity. All animal experiments were approved by the Animal Ethics Committee of Shanghai Ninth People’s Hospital, Shanghai Jiao Tong University School of Medicine (A-2016-017). HOS cells were harvested, counted, and resuspended in PBS to a final concentration of 2 × 10⁷ cells/mL. The animals were anesthetized with 3.5% pentobarbital, and then 1 × 10⁶ HOS cells in 50 μL of PBS were injected into the proximal tibia of mice using a 25-gauge needle. After 24 hours, mice were randomly assigned to 4 groups (control group, cisplatin group, BBD24 group and BBD24+cisplatin group). BBD24 was administered orally at a dose of 5 mg/kg twice a day for 20 consecutive days. Cisplatin was intraperitoneally injected twice a week at a dose of 15 mg/kg for 20 consecutive days. The mice in the control group were given equal volumes of water. Tumor weight was measured at the end of the experiments. The tumor volumes were calculated using the formula: volume=0.2618×L×W×(L+W), where W and L represent the average width and length of the tumor, respectively [6].

Immunohistochemistry

At the end of the experiments, the mice were sacrificed. Tumor samples were isolated from each mouse and fixed in 4% paraformaldehyde and then immunohistochemically stained for cleaved caspase-3, PCNA and Ki-67 as described previously [6].

Statistical analysis

The results are expressed as the mean ± S.D. Differences were evaluated by one-way analysis of variance (ANOVA), followed by Duncan’s post hoc test using SPSS 21.0 (IBM, Inc.), and P values less than 0.05 were considered statistically significant.

Conflicts of Interest

These authors declare no conflicts of interest.

Funding

This work was supported in part by the National Natural Science Foundation of China (81802228, 81702225). The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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