Proanthocyanidin B2 inhibits proliferation and induces apoptosis of osteosarcoma cells by suppressing the PI3K/AKT pathway

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumour in children and adolescents. The long‐term survival rate of OS patients is stubbornly low mainly due to the chemotherapy resistance. We therefore aimed to investigate the antitumoral effects and underlying mechanisms of proanthocyanidin B2 (PB2) on OS cells in the current study. The effect of PB2 on the proliferation and apoptosis of OS cell lines was assessed by CCK‐8, colony formation, and flow cytometry assays. The target gene and protein expression levels were measured by qRT‐PCR and Western blotting. A xenograft mouse model was established to assess the effects of PB2 on OS proliferation and apoptosis in vivo. Results from in vitro experiments showed that PB2 inhibited the proliferation and induced apoptosis of OS cells, and also increased the expression levels of apoptosis‐related proteins. Moreover, PB2 induced OS cell apoptosis through suppressing the PI3K/AKT signalling pathway. The in vivo experiments further confirmed that PB2 could inhibit OS tumour growth and induce its apoptosis. Taken together, these results suggested that PB2 inhibited the proliferation and induced apoptosis of OS cells through the suppression of the PI3K/AKT signalling pathway.

diseases and local or distant recurrence. 7,8 Furthermore, due to the toxicity of multi-agent chemotherapy, OS patients often experience acute and late complications, such as myelosuppression and peripheral neuropathic pain, which seriously affects their quality of life. 9 Therefore, there is an urgent need to explore new chemotherapeutic agents that not only kill the primary tumour but also reduce the adverse effects of chemotherapy.
Recently, natural drugs have become a popular researching focus since drug discovery is a high-risk and high-cost process. 10,11 Evidence has suggested that several natural drugs, for example, vincristine, taxanes and camptothecin, are effective anti-tumour agents. [12][13][14] Furthermore, some natural compounds have been demonstrated to own anti-tumour effects on OS cell lines with few adverse influences. [15][16][17] Therefore, researches on natural drugs may provide new ideas for the treatment of OS.
Proanthocyanidin B2 (PB2) is a polyphenolic compound derived from common dietary foods such as grape seed and cranberry. 18,19 Evidence has indicated that PB2 has a variety of pharmacological bioactivities, including anti-oxidation, anti-lipid peroxidation and anti-inflammation. [20][21][22] Specifically, it has been frequently reported that PB2 also possesses anti-tumour properties without affecting the viability of normal cells. [23][24][25][26][27] Feng et al 23 reported that PB2 could suppress aerobic glycolysis and induce apoptosis of hepatocellular cells by disrupting the PKM2/HSP90/HIF-1α axis. Engelbrecht et al 28 revealed that PB2 could inhibit the proliferation and induce apoptosis of colon cancer cells through inactivating the PI3K/PKB signalling pathway. However, there still lack studies involving the biological activities of PB2 for the treatment of OS. Therefore, we set out to assess the potential antitumoral effects of PB2 on OS and to explore the underlying molecular mechanisms. Information about primary antibodies was listed in Table 1.

| Cell proliferation assay
The effects of PB2 on OS cell proliferation were evaluated with CCK-8 and colony formation assays. Cells were seeded in 96-well culture plates at a density of 3 × 10 3 cells/well in 100 μL complete culture medium for 24 hours. Then, the cells were cultured in DMEM or in DMEM with different concentrations of PB2 (30- captured using a digital camera. The colonies were counted, and the clone formation rate was calculated according to the formula: clone formation rate = number of clone/number of seeded cell × 100%.

| Apoptosis analysis by flow cytometry
The effects of PB2 on OS cell apoptosis were analysed with an Annexin V-FITC apoptosis detection kit. 143B and MNNG cells were

| Mitochondrial membrane potential analysis
Disruption of mitochondrial membrane potential (MMP, ΔΨm) is a landmark event of early apoptosis. The JC-1 mitochondrial membrane potential assay kit was used to measure the changes of MMP.

| RNA extraction, reverse transcription and quantitative real-time PCR
143B and MNNG cells were incubated in 6-well plates and treated with PB2 at different concentrations of 0, 50, 70 and 90 μmol/L for 48 hours. Total RNA was extracted from the cells using TRIzol reagent and reverse-transcribed into cDNA by using a reverse transcription kit. The levels of gene expression were determined by quantitative real-time PCR (qRT-PCR) using a 7500 real-time PCR system (Applied Biosystems). The β-actin was used as an internal reference to verify equal amounts of cDNA, and the levels of different genes expression were quantified using the 2 −ΔΔC t method. The primers used for PCR were presented in Table 2.

| Western blotting analysis
143B and MNNG cells were treated with different concentrations of PB2 (0, 50, 70 and 90 μmol/L) for 48 hours. Total protein was extracted from cells using RIPA lysis buffer containing a protease inhibitor, the mitochondrial and cytoplasmic proteins were extracted using a cell mitochondrial isolation kit. The protein was quantified by BCA protein assay and separated by SDS-PAGE (7.5%, 10% and 12.5%) for electrophoresis, then transferred to PVDF membranes.
Next, the membranes were blocked with 5% non-fat milk and then incubated with primary antibodies and second antibodies. The protein expression levels were determined with an Odyssey two-colour infrared laser imaging system (LI-COR Biosciences). Quantitative analysis was conducted by Image J software.

| Animal experiments
The animal protocol was approved by the Ethics Committee of randomly assigned into either an experimental group that received PB2 treatment (100 mg/kg, intragastrical gavage) or a normal control (NC) group that received the same volume of saline, once a day for 3 weeks. After the last treatment of PB2 or saline, the mice were sacrificed by cervical dislocation after intraperitoneal injection of 1.25% pentobarbital (40 mg/kg). The xenograft tumours and major organs, including liver, lung, kidney, and heart were harvested from the mice in both groups and immersed in 4% PFA for immunohistochemistry staining.

| Immunohistochemistry staining
Haematoxylin and eosin (H&E) staining, Ki-67 staining, and the TdT-UTP nick end labelling (TUNEL) staining were carried out, and the staining processes were performed according to the standard protocols. PFA-immersed tumours were embedded in paraffin and cut into 4 μm-thick sections. For H&E staining, slides were stained with haematoxylin for 10 minutes and eosin for 5 minutes to visualize the tissue injuries. For Ki-67 staining, slides were deparaffinized and rehydrated. Antigen retrieval was performed using microwave for 5 minutes, and slices were incubated with 3% hydrogen peroxide for 10 minutes. After that, the slices were incubated with anti-Ki-67 antibody overnight at 4°C with gentle shaking and then incubated with secondary antibody at room temperature for 1 hour. For TUNEL assays, sections were dehydrated with ethanol after being deparaffinized twice in xylene, digested with 20 μg/mL proteinase K without DNase at room temperature for 15-30 minutes and then incubated in the TUNEL reaction mixture at room temperature for 1 hour.
Finally, sections were mounted on microscope slides with a coverslip and observed using a microscope (Leica). The brown-stained cells were regarded as Ki-67 and TUNEL-positive cells.

| Statistical analysis
Data were expressed as mean ± standard deviation (SD used to analyse the data, and P values less than .05 were considered statistically significant.

| PB2 inhibits the proliferation of OS cells
To investigate the anti-proliferative effects of PB2, OS cell lines (143B, MNNG, SJSA, and MG-63) and osteoblast cells (hFOB1. 19) were incubated and treated with PB2 in a series of concentrations (30-100 μmol/L) for 24, 48, and 72 hours. The CCK-8 assay was used to measure the influence of PB2 on cell proliferation, and the growth curves were plotted. As shown in Figure 1A and  Figure S1F).
Finally, we calculated the half-maximal inhibitory concentration (IC50) at 48 hours for each cell line, respectively. 143B and MNNG cell lines were specifically chosen for the subsequent experiments because these two cell lines were relatively more sensitive to PB2 according to the IC50 (Table 3).
Moreover, in order to further verify the role of PB2 in the proliferation of OS cells, colony formation assays and proliferating cell nuclear antigen (PCNA), an indicator reflected the cell proliferation, were employed. Results in Figure 1B,C showed that the clone forma-  Figure 1D,E).
Collectively, these results confirmed that PB2 could inhibit the proliferation of OS cells.  Figure 2A,B). Meanwhile, results from Western blotting ( Figure 2C) and qRT-PCR ( Figure 1E) showed an increased protein abundance

| PB2 stimulates the release of mitochondrial apoptogenic factors by disrupting mitochondrial membrane permeability
The

| PB2 induces OS cell apoptosis by suppressing the PI3K/AKT signalling pathway
The PI3K/AKT signalling pathway is a major intracellular signalling cascade, regulating tumour cell proliferation, apoptosis, and migration. 33 Previous studies have reported that aberrant activation of the PI3K/AKT pathway was closely associated with the negative regulation of tumour cell apoptosis. 34 Therefore, we further analysed whether PB2-induced apoptosis of OS cells was through suppressing the PI3K/AKT signalling pathway. The qRT-PCR and Western blotting results indicated that mRNA levels of PI3K and AKT were significantly decreased ( Figure 1E), and similarly, protein levels of p-PI3K and p-AKT were significantly reduced in 143B and MNNG cell lines after the treatment with different concentrations of PB2 ( Figure 4A).
Taken together, these results demonstrated that PB2 could induce OS cell apoptosis through inactivating the PI3K/AKT signalling pathway.

| Activation of the PI3K/AKT pathway using 740Y-P reverses the effects of PB2 on OS cell proliferation and apoptosis
To

| PB2 inhibits the growth of OS xenografts in vivo
In addition to the findings from in vitro experiments, we also con- harvested from the PB2-treated group were remarkably inhibited as compared to the NC group. Specifically, the tumour growth rate in the PB2-treated group was significantly slower as compared to that in the NC group (P < .001, Figure 5B), while the bodyweights of mice in both groups were comparative ( Figure 5C). We then further investigated the underlying pathological changes by immunohistochemistry staining ( Figure 5D). We observed that there were more necrotic lesions in the tumour tissues in the PB2-treated group, while numerous tumour cells existed in the NC group by H&E staining. In consistence, there were fewer proliferative cells and more apoptotic cells in the PB2-treated group than that in the NC group revealed by Ki-67 and TUNEL assays. Finally, we assessed the effects of PB2 on major organs including liver, heart, lung, and kidney, and the results from H&E staining showed that PB2 exerted no significant toxicity on these normal organs ( Figure 5E). Overall, these findings confirmed that PB2 suppressed the growth of OS xenografts in vivo by inhibiting OS cell proliferation and inducing apoptosis.

| D ISCUSS I ON
Current therapeutic strategies for OS include surgical resection and chemotherapy. 6,35 There exist challenges of performing tumour resection surgery for young children with an immature skeleton, and   55 We will conduct clinical studies to further investigate whether PB2 assisted with F I G U R E 6 The potential mechanisms of PB2-induced apoptosis in OS cells. PB2 affects the balance of anti-apoptotic and pro-apoptotic proteins through suppressing the PI3K/AKT signalling pathway. Moreover, PB2 increases the mitochondrial membrane permeability and causes the release of cyto-C and AIF from mitochondrial into the cytoplasm, resulting in the activation of intrinsic apoptotic pathways and nuclear DNA agglutination nanomaterials could facilitate its bioavailability, and thus improve its therapeutic effects on osteosarcoma patients in the future.
In conclusion, our findings suggested that PB2 can suppress proliferation and induce apoptosis in 143B and MNNG cells via inactivating the PI3K/AKT signalling pathway. The antitumoral activities of PB2 may provide a new chemotherapeutic route for the treatment of OS in future clinic.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author on reasonable request.