Journal of Cancer Research and Therapeutics

ORIGINAL ARTICLE
Year
: 2020  |  Volume : 16  |  Issue : 2  |  Page : 215--221

Berberine enhances the radiosensitivity of osteosarcoma by targeting Rad51 and epithelial–mesenchymal transition


Dapeng Wang1, Ke Fen Zhang2, Gang Du1, Jun Wang3, Jinmin Zhao1,  
1 Research Centre for Regenerative Medicine, Guangxi Key Laboratory of Regenerative Medicine, The First Affiliated Hospital of Guangxi Medical University, 530022 Guangxi, Taian, Shandong, China
2 Department of Pathology, Taishan Sanatorium, Taian, Shandong, China
3 Department of Oncology, The Central Hospital of Taian, Taian, Shandong, China

Correspondence Address:
Jinmin Zhao
Research Centre for Regenerative Medicine, Guangxi Key Laboratory of Regenerative Medicine, The First Affiliated Hospital of Guangxi Medical University, 530022 Guangxi
China

Abstract

Objective: Osteosarcoma is a malignant bone tumor and is generally treated with radiotherapy combined with radiosensitizers. The aim of the present study was to investigate the radiosensitization effects of berberine on osteosarcoma cells and the role of Rad51 in radiosensitivity by berberine. Materials and Methods: Cells from the human osteosarcoma cell line MG-63 were exposed to γ-ray irradiation (0, 2, 4, 6, and 8 Gy) and berberine (20 μM). Radiosensitivity was evaluated by determining cell viability using an MTT assay. Flow cytometry was used to determine cell cycle and apoptosis. Real-time PCR and western blot were performed to analyze the mRNA and protein expressions of Rad51. The protein levels of E-cadherin and vimentin were also measured to evaluate the epithelial–mesenchymal transition (EMT) process. Tumor invasion was determined by the Boyden chamber assay. Results: Berberine exacerbated the decline in viability of MG-63 cells exposed to γ-rays irradiation at various concentrations (25, 50, 75, and 100 μmol/L) and induced cell cycle arrest in the G2/M phase as well as apoptosis. The mRNA and protein expressions of Rad51 were significantly decreased by berberine in MG-63 cells. Inhibition of Rad51 by B02 enhanced the radiosensitivity of MG-63 cells. Berberine inhibited their invasive capability as well as increased E-cadherin and decreased vimentin protein levels; this indicated that berberine suppressed the EMT process in MG-63 cells exposed to γ-rays irradiation. Conclusion: Berberine enhances the radiosensitivity of MG-63 osteosarcoma cells. Rad51 is a potential target of berberine in the radiosensitization of osteosarcoma.



How to cite this article:
Wang D, Zhang KF, Du G, Wang J, Zhao J. Berberine enhances the radiosensitivity of osteosarcoma by targeting Rad51 and epithelial–mesenchymal transition.J Can Res Ther 2020;16:215-221


How to cite this URL:
Wang D, Zhang KF, Du G, Wang J, Zhao J. Berberine enhances the radiosensitivity of osteosarcoma by targeting Rad51 and epithelial–mesenchymal transition. J Can Res Ther [serial online] 2020 [cited 2020 Oct 26 ];16:215-221
Available from: https://www.cancerjournal.net/text.asp?2020/16/2/215/285181


Full Text



 Introduction



Osteosarcoma is a malignant bone tumor that produces osteoid or immature bones and is prevalent in children and young adults. The risk, development, and prognosis of osteosarcoma are linked to the genetic background of a patient, especially in the case of polymorphisms related to DNA repair.[1] Current standard treatments include intensive chemotherapy and surgical resection.[2] Ionizing radiation therapy is also an important treatment for osteosarcoma and shows minimal damaging effects to the surrounding normal tissue.[3] However, the clinical effects of radiotherapy as a local treatment are often limited due to radioresistance.[4] Therefore, radiosensitizers are usually administered in combination with radiotherapy in patients with osteosarcoma.[5]

Berberine is an isoquinoline alkaloid component of Chinese herbs that is usually prescribed in the treatment of gastrointestinal diseases.[6] Berberine has various biological functions; it can act as an antimicrobial, antidiarrheal, anti-inflammatory, and antidiabetic agents.[7] Berberine acts therapeutically on a variety of diseases and shows cytotoxic activity against cancer cells, including osteosarcoma.[8],[9] Furthermore, berberine may also be a radiosensitizer and could enhance radiosensitivity in nasopharyngeal cancer.[10] However, the radiosensitizing effects of berberine on osteosarcoma are still unclear.

Rad51 is a DNA repair protein that is involved in double-strand breaks (DSBs) induced by radiation.[11] Rad51 mediates DNA DSBs repair in the mammalian cells through homologous recombination (HR).[12] Rad51 gene polymorphisms are associated with increased risks of developing some tumors, such as colorectal cancer.[13] Overexpression of Rad51 was correlated with advanced progression and poor prognoses of colorectal cancer.[14] Downregulation of RAD51 expression could enhance radiosensitivity through suppression of HR-mediated DNA repair in osteosarcoma.[15] However, whether Rad51 affects the radiosensitivity of berberine in osteosarcoma remains unknown.

In this study, we evaluated cell viability, cell cycle, apoptosis, and the invasion of osteosarcoma cells with irradiation by berberine. We also investigated the mRNA and protein expressions of Rad51 by berberine, the role of Rad51 in epithelial–mesenchymal transition (EMT), and the radiosensitizing effect of berberine on osteosarcoma. Our study will provide evidence for Rad51 as a target, responsible for radiosensitizing effects of berberine.

 Materials and Methods



Reagents and antibodies

Berberine was purchased from Sigma-Aldrich Chemical Corp (St. Louis, MO, USA). The MTT kit was purchased from Sigma Chemical Co. (St. Louis, MO, USA). Reverse transcriptase was purchased from Superscript III enzyme (Life Technologies, USA). The quantitative reverse transcription-polymerase chain reaction (qRT-PCR) kit was purchased from SYBR Green reagent (TaKaRa, Japan). Antibodies were purchased from the following sources: mouse monoclonal antibodies against human Rad51, E-cadherin, and vimentin were purchased from Cell Signaling Technology (Danvers, MA, USA). Rad51 inhibitor B02 was purchased from MedChem Express (No. HY-101462, New Jersey, USA).

Cell culture

The human osteosarcoma cell line MG-63 was obtained from American Type Culture Collection (ATCC; Rockville, MD, USA) and routinely maintained in DMEM (Invitrogen-GIBCO, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (Invitrogen-GIBCO). Cells were cultured in an atmosphere of 95% humidified air and 5% CO2 at 37°C. The medium was changed once every 2 days, and the cells were digested for passage after reaching confluence.

Irradiation condition

The MG-63 cells were cultured in a dish to 70%–80% confluence. Then, the cells were exposed to Cobalt-60 γ-ray (dose rate: 2.4 Gy/min) with irradiation doses of 0, 2, 4, 6, and 8 Gy in a cobalt-60 therapy machine (XINHUA, CHINA). The irradiation parameters were set as follows: Source-skin-distance = 80cm; depth = 1 cm. After irradiation, cells were incubated with berberine (0, 20, and 40 μM) for 24 h.

Cell viability assay

MG-63 cells were seeded in a 96-well plate at a density of 1 × 104 cells/well (100 μL). After 24 h, cells were transferred to fresh DMEM containing berberine of 0, 20, 40, 60, 80, and 100 μM for 24 and 48 h. In another experiment, MG-63 cells were incubated with berberine (20 and 40 μM) or Rad51 inhibitor B02 (50 μM). Cell viability was measured by adding 10 μL MTT solution (5 mg/mL) into each well, followed by incubation at 37°C for 4 h. The cell suspension underwent centrifugation at 1000 g for 10 min, and after removing the supernatant, the formazan pellet was dissolved in 100 μL DMSO. The viable cells were quantified by measuring the absorbance at a 570 nm wavelength in an ELISA plate reader. The viable cells of each group were normalized to those of the controls.

Cell cycle analysis

MG-63 cells were incubated with serum-free medium for 24 h to induce synchronization. Then, cells were incubated with berberine (0, 20 μM) and (or) irradiation (4 Gy) for 24 h in complete medium. Cells were harvested and added to cold 70% ethanol for resuspension, followed by incubation with propidium iodide (PI) solution (20 μg/mL) for 30 min. Cell cycle analysis was performed by flow cytometry (FACScan, Becton Dickinson, San Francisco, CA, USA), and the data were analyzed using Lysis II Software (Becton Dickinson).

Cell apoptosis assay

MG-63 cells were treated with berberine (0, 20 μM) and (or) irradiation (4 Gy) for 24 h. After centrifugation at 1000 g for 5 min, the cells were incubated in 100 μL 1 × binding buffer containing Annexin V and PI (both final concentration; 10 μg/mL) in the dark. After 15 min, the cells were analyzed by flow cytometry (FACScan, Becton Dickinson, San Francisco, CA, USA), and the data were analyzed by CELLQuest Software (Becton Dickinson, New Jersey).

Cell invasion assay

Cell invasion capability was evaluated by the Boyden chamber assay. MG-63 cells were seeded on the upper side of the filter membrane in 100 μL DMEMs of 24-well culture plates (5 × 104 cells/well) and were treated with berberine (20 μM), irradiation (4 Gy), or berberine + irradiation for 24 h. After wiping cells from the upper surface of the filter membrane, the migrated cells on the lower part of filter membrane (coated with 0.5 mg/mL type I collagen) were stained with hematoxylin and eosin, and they were counted under light microscopy (×200).

Quantitative reverse transcription-polymerase chain reaction

Total RNA was extracted from MG-63 cells incubated with berberine (0, 20, 40, 60, 80, and 100 μM) for 24 h and was reverse transcribed to cDNA to determine Rad51 mRNA level in the StepOne Plus Real-time PCR System (Applied Biosystems, USA). The PCR condition was as follows: initial denaturation at 95°C for 5 min, followed by 40 amplification cycles of denaturation at 95°C for 15 s and annealing at 60°C for 15 s. The primer sequences were as follows: Rad51: forward: 5'-CAG TGG CTG AGA GGT ATG GTC T-3'; reverse: 5'-GGT CTG GTG GTC TGT GTT GAA-3'; GAPDH: Forward: 5'-ATC ACT GCC ACC CAG AAG AC-3'; reverse: 5'-ATC ACT GCC ACC CAG AAG AC-3'. Rad51 mRNA expression was calculated by 2−ΔΔCt method. Relative expression of Rad51 mRNA was calculated based on the mean GAPDH expression. All experiments were performed in triplicate.

Western blotting

Proteins were extracted from MG-63 cells, followed by sonication and centrifugation at 12,000 g for 30 min at 4°C. Proteins (50 μg) were separated by 12% SDS-PAGE gels and then were transferred to PVDF membranes (Bio-Rad, Hercules, CA, USA). Membranes werefirst blocked with 5% nonfat milk in TBS-T buffer at room temperature, followed by incubation with mouse monoclonal antibodies against human Rad51, E-cadherin, or vimentin (all 1:500 dilutions). After washing with PBS solution, membranes were incubated with horseradish peroxidase-conjugated rabbit anti-mouse secondary antibodies (1:1000 dilutions) at room temperature for 1 h. The bands were incubated in enhanced chemical luminescence (ECL, Pierce® ECL Plus, IL, USA). β-actin was used as a loading control.

Statistical analysis

Data were expressed as the mean ± standard deviation from experiments that were repeated more than three times. Statistical analysis was performed using SPSS 19.0 (SPSS Inc., Chicago, IL, USA). A two-tailed unpaired Student's t-test was performed to measure the differences between the two groups. One-way analysis of variance was performed to measure the differences between multiple groups. P < 0.05 was considered indicative of statistically significant differences.

 Results



Berberine sensitizes osteosarcoma cells to radiation

We performed an MTT assay to measure the effect of berberine on osteosarcoma cell growth. MG-63 cells were treated with different concentrations of berberine (0, 20, 40, 60, and 80 μM) for 24 or 48 h. The results showed that berberine decreased the viability of MG-63 cells in a concentration- and time-dependent manner [Figure 1]a. This indicates that berberine has inhibitory effects on MG-63 cell proliferation. We then evaluated the radiosensitivity of berberine on MG-63 cells. Cells were treated with irradiation (2, 4, 6, and 8 Gy) alone or irradiation with berberine (20 and 40 μM) for 24 h. The results showed that berberine significantly reduced the cell viability of MG-63 cells following various doses of irradiation (all P <0.05) [Figure 1]b and [Figure 1]c. This indicates that berberine can enhance the radiosensitivity of osteosarcoma cells.{Figure 1}

Enhanced radiosensitivity was associated with cell cycle arrest, apoptosis, and invasion

We stained MG-63 cells with PI using flow cytometry to investigate whether enhanced radiosensitivity by berberine was associated with cell cycle regulation. Berberine (20 μM) significantly increased the percentage of cells in the G2/M phase after irradiation with 4 Gy for 24 h (P < 0.05) [Figure 2]a. However, the percentage of G0/G1 phase was significantly decreased after irradiation (P < 0.05), and the percentage of S phase remained unchanged. This indicates that berberine could induce cell cycle arrest in the G2/M phase in MG-63 cells with irradiation.{Figure 2}

We further measured apoptosis of MG-63 cells with irradiation after berberine treatment by double staining with Annexin V-FITC and PI. Berberine significantly increased the cell apoptotic rate of MG-63 cells with irradiation (P < 0.05) [Figure 2]b and [Figure 2]c. This indicates that decreased cell viability might be associated with enhanced apoptosis after berberine treatment.

We then performed the Boyden chamber assay to measure the tumor invasion capability of berberine in MG-63 cells with irradiation. The number of invasive MG-63 cells was significantly decreased by berberine compared to that of control cells (P < 0.05). Berberine also significantly decreased cell invasion in MG-63 cells with irradiation (P < 0.05) [Figure 2]d. This result indicates that berberine might increase radiosensitivity through inhibiting tumor invasion in osteosarcoma cells exposed to irradiation.

Rad51 was a target of berberine and was involved in radiosensitization

To explore the molecular mechanism underlying berberine-enhanced radiosensitivity, qRT-PCR and western blot were carried out to determine the Rad51 mRNA and protein expressions in MG-63 cells with various berberine concentrations. The results demonstrated that berberine (0, 20, 40, 60, and 80 μM) decreased Rad51 mRNA and protein expressions in a concentration-dependent manner after 24-h incubation (P < 0.05) [Figure 3]a and [Figure 3]b.{Figure 3}

To investigate whether decreased Rad51 expression contributed to the enhanced radiosensitivity of osteosarcoma cells, we analyzed the effect of B02, a specific Rad51 inhibitor, on the radiosensitivity of MG-63 cells. Cells were treated with irradiation (2, 4, 6, and 8 Gy) alone or irradiation with B02 (50 μM) for 24 h. B02 significantly reduced the cell viability of MG-63 cells at all doses of irradiation (P < 0.05) [Figure 3]c. This indicates that the inhibition of Rad51 expression might be involved in increasing the radiosensitizing effect of berberine.

Berberine suppresses epithelial–mesenchymal transition though decreasing Rad51 expression in osteosarcoma cells

To explore the mechanism of berberine-increased radiosensitivity, we measured E-cadherin and vimentin protein expressions, which are two EMT markers. Compared with the control group, berberine increased the E-cadherin expression and decreased the vimentin expression (both P <0.05) [Figure 4]a and [Figure 4]b. E-cadherin is an epithelial phenotype marker, and vimentin is a mesenchymal phenotype marker, and this indicates that berberine could inhibit the EMT process in osteosarcoma cells. In MG-63 cells with irradiation, berberine or B02 could significantly increase the presence of E-cadherin protein and decrease the levels of vimentin protein (both P < 0.05). Irradiation could make MG-63 cells more sensitive to EMT inhibition by berberine or B02. These findings indicate that berberine could inhibit the EMT process by targeting Rad51 in osteosarcoma cells.{Figure 4}

 Discussion



In this study, we have shown berberine to increase the radiosensitivity of osteosarcoma cells as evidenced by a reduction in viable cells after irradiation at various doses. Berberine also induced cell cycle arrest in the G2/M phase, enhanced apoptosis, and inhibited the invasive capability in MG-63 cells with irradiation. Berberine decreased Rad51 mRNA and protein expression in MG-63 cells, which was associated with its radiosensitizing effect. Furthermore, in MG-63 cells experiencing irradiation, berberine increased E-cadherin and decreased vimentin protein expressions, which was dependent on decreased Rad51 expression. This indicates that Rad51 might contribute to the enhanced radiosensitivity and decreased EMT process brought on by berberine in irradiated osteosarcoma cells.

This study shows that berberine might be a new option as a radiosensitizer for osteosarcoma. Berberine has been reported to enhance radiosensitivity in various types of cancers, including nasopharyngeal carcinoma and prostate cancer.[16],[17] Berberine also induced G2/M cell cycle arrest and apoptosis in osteosarcoma cells with irradiation, which might be caused by DNA double-strand breaks.[18] In fact, radiation is a potent inducer of DNA damage, and exposure to γ-rays can induce chromosome aberrations and mutations.[19] Therefore, berberine and radiation might share common cellular pathways and therefore underlie the radiosensitizing effects of berberine. Furthermore, the investigation into DNA damage-related proteins is needed; it will increase our understanding of the molecular mechanisms of berberine radiosensitization.

In this study, wefirst found that berberine can decrease Rad51 mRNA and protein expression in osteosarcoma cells with irradiation, and inhibiting Rad51 can enhance the radiosensitivity of osteosarcoma cells. Rad51 is a DNA repair protein and promotes HR, which could modulate sensitivity to DNA-damaging drugs.[20] Inhibition of Rad51-mediated DNA damage repair enhanced the radiosensitivity of various tumors, including breast cancer, pancreatic cancer, and osteosarcoma.[21],[22],[23] Furthermore, berberine downregulated Rad51 expression in radiosensitized esophageal squamous cell carcinomas and breast cancer.[24],[25] This indicates that decreased levels of Rad51 protein might mediate radiosensitization of osteosarcoma cells. In fact, berberine is a genotoxic alkaloid and could effectively induce double-strand DNA breaks.[26] Hence, berberine may aggravate DNA damage induced by radiation and make it irreparable, thereby contributing to radiosensitization.

Our study also found that berberine markedly inhibited tumor invasion as well as the EMT process in osteosarcoma cells. EMT is a cellular process that confers decreased adhesion and increased motility of tumor cells, and EMT also can be induced by radiation itself to confer radioresistance in tumor cells.[27],[28] Therefore, EMT is a potential target for radiotherapy, and the suppression of EMT could enhance radiosensitivity of tumor cells that have been confirmed in our study.[29] Furthermore, EMT might also be a downstream target of berberine as evidenced by decreased EMT by berberine in prostate cancer.[30] The relationship between Rad51 and EMT remains largely unknown, and our study shows the association between decreased Rad51 expression and reversion of EMT. MiR-506 is a potent inhibitor of EMT and could help sensitize cancer cells to chemotherapy through the direct targeting of Rad51.[31] A recent report showed that reversing the EMT phenotype led to reduced efficiency of HR and Rad51 foci formation.[32] This suggests that EMT might cause increased Rad51 expression. In fact, cancer cells undergoing the EMT process acquire a cancer stem cell phenotype, and these radioresistant cells exhibit more efficient HR repair, which is a function of Rad51.[33] However, in our study, inhibition of Rad51 activity by B02 could reverse the EMT process as evidenced by increased E-cadherin and decreased vimentin proteins. This suggests that RAD51 might be an enhancer of EMT in cancer cells. This hypothesis is further supported by a recent study that showed the silencing of RAD51AP1, a DNA-binding protein that stimulates RAD51 activity, suppressed EMT in lung cancer cells.[34] This indicates that Rad51 could also regulate the EMT process, and reduced Rad51 expression might involve the suppression of EMT and enhanced radiosensitivity by berberine.

 Conclusion



This study demonstrates the radiosensitizing effects of berberine in osteosarcomas and further shows that the underlying mechanisms might be associated with G2/M cell cycle arrest, apoptosis, and inhibition of Rad51 expression, invasion, and the EMT process. Our study reports on the regulation of Rad51 by berberine in osteosarcomas and suggests that targeting Rad51 may be one important strategy for radiosensitization in osteosarcoma and other cancers. However, the present study did not determine whether the downregulation of Rad51 is directly associated with the radiosensitizing effects of berberine and the overexpression of Rad51 should be performed in osteosarcoma cells treated by berberine. To further explore the clinical relevance, an animal model experiment is needed to study the radiosensitizing effect of berberine and further elucidate the role of Rad51 in osteosarcoma radiosensitivity.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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