Bruceine D and afatinib combination inhibits ovarian cancer cells proliferation and migration through DNA damage repair and EGFR pathway

Owing to the high rates of relapse and migration, ovarian cancer (OC) has been recognized as the most lethal gynecological malignancy worldwide. The activity of the epidermal growth factor receptor (EGFR) signaling pathway is frequently associated with OC cell proliferation and migration. Despite this knowledge, inhibition of EGFR signaling in OC patients failed to achieve satisfactory therapeutic effects. In this study, we identified that bruceine D (BD) and EGFR inhibitor, afatinib, combination resulted in synergistic anti-OC effects. The results indicated that compared with one of both drugs alone, the combination of BD and afatinib slowed the DNA replication rate, inhibition of cell viability, and proliferation and clone formation. This resulted in cell cycle arrest and cell apoptosis. In addition, the combination of BD and afatinib possessed a stronger ability to inhibit the OC cell adhesion and migration than treatment with BD or afatinib alone. Mechanistically, the combined treatment triggered intense DNA damage, suppressed DNA damage repair, and enhanced the inhibition of the EGFR pathway. These results demonstrated that compared with each pathway inhibition, combined blocking of both DNA damage repair and the EGFR pathway appears to more effective against OC treatment. The results support the potential of BD and afatinib combination as a therapeutic strategy for OC patients.


Introduction
Ovarian cancer (OC) is the seventh most common cancer among women worldwide and the most lethal gynecological malignancy. 1 Despite this, great efforts are still made to advance the treatment of OC, even though the prognosis of this condition is poor and its 5-year survival rate is only 46%. 2,3 Chemotherapy, which typically consists of platinum and taxol-based therapy, is the general treatment for OC; however, most women relapse 6-12 months after the last treatment and develop resistance. 4 This highlights the urgent need for the development of a new therapeutic strategy.
The human epidermal growth factor receptor (EGFR), which is a member of ErbB family of receptor tyrosine kinase, can regulate cell proliferation, angiogenesis, and migration via activated downstream pathways. 5 The EGFR pathway is amplified in most cancer cells and has previously been reported to be associated with resistance to chemotherapy and radiotherapy. 6 Small molecules that targeted EGFR have achieved significant efficacy in some cancer patients, 7 for example, in the setting of lung cancer with known EGFR mutations. 8 Recent studies have revealed that EGFR is overexpressed in nearly 70% of OC and had a positive correlation with poor prognosis. 9,10 This indicates that EGFR might be a potentially valuable target for the treatment of OC; however, clinical trials with several different EGFR inhibitors have shown only modest activity in the setting of OC. 11 In addition, cells would develop resistance quickly during treatment with an EGFR inhibitor. 12 On a more promising note, an EGFR inhibitor (e.g., afatinib) was able to sensitize the anticancer activity of chemotherapeutic drugs in some cancer cells, such as in the setting of pancreatic, colorectal, breast, and non-small cell lung cancers. 13 Together, these data suggest that the combination of an EGFR inhibitor and a chemotherapeutic drug might be an extremely valuable strategy for the treatment of OC.
Natural compounds have been considered an important source of anticancer drugs. 14 Bruceine D (BD), which is one of the active components of Brucea javanica, is widely used to treat cancer in China. 15 In the setting of the clinic, BD showed vital anticancer activity in lung cancer, 16 hepatocellular carcinoma, 17 human chronic myeloid leukemia, 18 and pancreatic cancer. 19 In addition, the anticancer mechanisms of BD have been studied in the setting of these cancers, such as the idea that BD-mediated pancreatic cell death may be due to NF-kB inhibition and p38-MAPK pathway activation. 20 BD also inhibits hepatocellular carcinoma growth both in vivo and in vitro by reducing Wnt translational activity, 17 Little has been reported about the anticancer effects of BD in the setting of OC; however, these mechanisms remain unclear.
In the current study, we investigated the anticancer effect of BD and afatinib combination in OC cells and explored the underlying antitumor pharmacological mechanisms. We demonstrated that compared to BD and/or afatinib treatment alone, the combination of BD and afatinib had a stronger ability to inhibit OC cell proliferation and migration. The possible mechanisms of BD and afatinib were discussed as well. This study might provide a strategy for OC treatment.

Cell culture
The OC cell line A2780, CP70, and human normal lung line MRC-5 were purchased from Cell Resource Center of Peking Union Medical College. The cells were cultured at 37°C under 5% CO 2 , simultaneously maintained in DMEM/RPMI 1640/MEM (Gibco, USA), supplemented with 10% Fetal Bovine Serum (FBS) (Gibco, USA) and 1% penicillin-streptomycin.

Drugs administrations
BD (CAS:21499-66-1) and afatinib (CAS: 439081-18-2) were purchased from Shanghai Yuanye Biotechnology Company and were dissolved in dimethylsulfoxide (DMSO) to appropriate concentrations. The same amount of DMSO was also given to the control group to eliminate errors. Since previous studies had suggested that either BD or afatinib treatment for 48 h achieved good anticancer effect. 16,21 BD and afatinib were added to the A2780 and CP70 cells at the same time for 48 h for all experiments.

Cell viability assay
Cell viability was detected by 3-(4, 5-dimethylthiadiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT). 22 Cells (3000 cells/well in 96-well plates) were cultivated for 48 h, and then MTT was added with the final concentration of 0.5 mg/mL for 4 h at 37°C. The reaction product formazan was dissolved in DMSO after removing the medium. Cell viability was calculated by reading absorbance at 490 nm using an automated Thermo Fisher Multiskan FC Microplate. The results are shown as the mean 6 standard deviation of three measurements (n = 3).
Long-term proliferation studies A2780 and CP70 cells (2 3 10 5 cells/well in 6-well plates) were added in BD or afatinib or the combination of BD and afatinib for 48 h. During proliferation,

Significance of this Study WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT?
Ovarian cancer is characterized as the most lethal gynecological malignancy worldwide. EGFR is frequently associated with ovarian cancer cell proliferation and migration. EGFR inhibitors failed to achieve satisfactory therapeutic effects in ovarian cancer patients.

WHAT ARE THE NEW FINDINGS?
Compared with one of both drugs, the combination of bruceine D and afatinib resulted in cell cycle arrest and cell apoptosis. The combination inhibited the ovarian cancer cell adhesion and migration than treatment with bruceine D or afatinib alone. Mechanistically, the combined treatment triggered intense DNA damage, suppressed DNA damage repair, and enhanced the inhibition of the EGFR pathway.

HOW MIGHT IT IMPACT ON CLINICAL PRACTICE IN THE FORESEEABLE FUTURE?
Bruceine D and afatinib combination can potentially be a therapeutic strategy for ovarian cancer patients.
the number of cells in each well was counted by the cell counter. Subsequently, the cells (2 3 10 5 ) were moved into a 6-well plate to continue to cultivate, and then count the cell numbers of each group until the cells were less than 2 3 10 5 . 2 PDs = M/N (PDs: population doublings, M: number of every count cells, and N = number of cells implanted). 23 Western blot assay A2780 and CP70 cells (2 3 10 5 cells/well in 6-well plates) were added in BD or afatinib or the combination of BD and afatinib for 48 h. After that, the cells were dissolved, and the concentrations were determined by using the Bradford assay (Bio-Rad, CA, USA). Then cells were boiled for 10 min as samples. The proteins (30 mg/sample), which were loaded 20 mL per lane were separated via 12% Sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE), moved to Polyvinylidene fluoride (PVDF) membranes and blocked using 5% skim milk at room temperature for 1. Colony formation assay A2780, CP70, and MRC-5 (3000 cells/well in 12-well plates) were added in BD or afatinib or the combination of BD and afatinib for 48 h. When the colonies had grown completely in the control group, every well of plates was cleaned with Phosphate buffer saline (PBS), then the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and subsequently stained with crystal violet for 5 min at room temperature. Colonies in each plate were photographed under a light microscope. Ultimately the crystals were dissolved using 500 mL acetic acid (33%), and the results were calculated by reading absorbance at 560 nm by using an automated Thermo Fisher Multiskan FC Microplate. 25 EdU staining assay A2780 and CP70 cells (5 3 10 4 cells/well in 12-well plates with coverslips) were added in BD or afatinib or the combination of BD and afatinib for 48 h. The 5-Ethynyl-2'deoxyuridine (EDU) Staining Proliferation Kit (Beyotime, China) was used to examine the cells. Ultimately, using Nikon Fluorescence Microscope to observe the cells.
Cell cycle distribution analysis A2780 and CP70 cells (2 3 10 5 cells/well in 6-well plates) were added in BD or afatinib or the combination of BD and afatinib for 48 h. After that, the cells were trypsinized, washed, treated with RNase at 37°C for 30 min, and stained with propidium iodide (PI) at room temperature for 15 min, subsequently assessed on a flow cytometer (BD FACSCalibur, BD Biosciences) for the cell cycle distribution, and data were analyzed by FlowJo 7.6. 22 Annexin V/PI apoptosis assay A2780 and CP70 cells (2 3 10 5 cells/well in 6-well plates) were added in BD or afatinib or the combination of BD and afatinib for 48 h. Cells were collected for Annexin V/ PI apoptosis assay via the protocol of the Annexin V/PI apoptosis Kit (Sigma), then the cells were detected by a flow cytometer (BD FACSCalibur, BD Biosciences), and data were analyzed by FlowJo 7.6. 23 Immunofluorescence assays A2780 and CP70 cells (5 3 10 4 cells/well in 12-well plates with coverslips) were added in BD or afatinib or the combination of BD and afatinib for 48 h. After that, cells were fixed with 4% paraformaldehyde for 15 min at room temperature and then permeabilized in 0.5% Triton X-100 for 30 min. Subsequently, the cells were blocked with 5% GS (Gibco) at room temperature for 1 h and incubated with primary antibody against 53BP1 (cat. no. SAB4503016, Millipore) diluted 1:1600 at 4°C overnight, which detected DNA damage criterion. The next day, the cells were cleaned by PBS-Tween 0.1%, incubated with a secondary antibody at room temperature against DyLight 488-conjugated anti-Rabbit (cat. no. Sa00013-2, Proteintech) diluted 1:50 for 1.5 h, and mounted with 4', 6-diamidino-2-phenylindole (DAPI) finally. The fluorescent images were captured by using Nikon Fluorescence Microscope. 23 Comet assays DNA damage was detected via neutral or alkaline comet assay. First, A2780 and CP70 cells (2 3 10 4 cells/well in 12-well plates) were added in BD or afatinib or the combination of BD and afatinib for 48 h. The cells were mixed with 0.5% low melting temperature agarose and layered on slides precoated by 1.5% normal agarose. Then the slides were lysed in 3% DMSO, 100 mM EDTA,10 mM Tris (pH 8.0), 0.5% Triton X-100, 2.5 M NaCl, and 1% N-lauroylsarcosine. After that, the cells were electrophoresed in 1% DMSO, 300 mM sodium acetate and 100 mM Tris-HCl at 1.5 V/cm, and mounted with PI solution ultimately. The slides were visualized by a Nikon Fluorescence Microscope, and the results were analyzed with CASP. 25 The calculation of combination index of drug interaction The combination index (CI) of BD interaction with afatinib was measured via the Chou-Talalay method. 25 The results were performed with the CompuSyn software, where CI \ 1, = 1, and . 1 indicate synergistic, additive, and antagonistic effects, respectively.
Cell adhesion assay A2780 and CP70 cells (2 3 10 5 cells/well in 6-well plates) were added in BD or afatinib or the combination of BD and afatinib for 48 h. And 2.5 mg/mL human fibronectin in PBS (Millipore, CA) was precoated in a 96-well plate at 4°C overnight. After that, the cells (5 3 10 4 /well) were planted into serum-free medium and cultivated at 37°C for 20 min. Then the cells were fixed with 4% paraformaldehyde for 15 min at room temperature and stained with crystal violet for 5 min at room temperature after the medium was removed. In the end, the crystals were dissolved by 100 mL acetic acid (33%), and the results were determined by reading absorbance at 560 nm using an automated Thermo Fisher Multiskan FC microplate. The following formula was used to calculate the relative number of cells attached to the extracellular matrix: average of treated cells OD/average OD control unit. The relative number of cells attached to the extracellular matrix was calculated using the following equation: (mean OD of treated cells/mean OD of control cells) 3 100%. 22 Transwell assay A2780 and CP70 cells (2 3 10 5 cells/well in 6-well plates) were added in BD or afatinib or the combination of BD and afatinib for 48 h. After that, the treated cells were digested, resuspended, and seeded with serum-free medium at a density of 1 3 10 5 /100 mL. Transwell chambers were inserted into a 24-well plate and followed with 600 mL of medium 10% FBS added into the lower chamber. The cells were cultivated at 37°C for 48 h. Then the fluid and cells in the chamber were thrown away, and the edge of chamber was washed by PBS three times. The cells were fixed with 4% paraformaldehyde for 15 min at room temperature, and then the cells on the chamber membrane were rinsed with pure water. After that, the cells were stained with crystal violet at room temperature for 5 min and washed by pure water. The crystals were dissolved by 100 mL acetic acid (33%), and the absorbance was shown at 560 nm using an automated Thermo Fisher Multiskan FC Microplate. The following formula was used to calculate cell migration rate: (average of treated cells OD/ average OD control unit) 3 100%. 22

Statistics analysis
GraphPad Prism 5 was used for statistical analysis. Results are shown as means 6 SD, and the unpaired Student's two-tailed t-test and/or two-way ANOVA was used to determine the statistical significance (ns, not significant; *p \ 0.05; **p \ 0.01; ***p \ 0.001).

Synergistic anticancer effect between BD and afatinib on OC cells
To verify the relevance between EGFR and prognosis of OC, transcriptome data from 1836 OC tissues were obtained from The Cancer Genome Atlas (TCGA) database and analyzed. The results showed that patients expressing higher EGFR showed lower survival rates compared to patients with low EGFR expression (Figure 1(a)). Therefore, afatinib, a second-generation EGFR inhibitor, 26 which binds irreversibly to the EGFR receptor to reduce resistance and recurrence, 27 was selected for analysis in this study. First, MTT was performed to evaluate the cytotoxicity of BD and afatinib against human OC cell lines A2780 and CP70. The results demonstrated that the IC 50 values of BD against A2780 and CP70 cells were 1.10 and 0.71 mM, respectively (Figure 1(b)). In addition, afatinib was also able to inhibit the cell viability of A2780 and CP70 cells, with IC 50 values of 7.29 and 5.10 mM, respectively (Figure 1(b)). Furthermore, the inhibition of cell growth in the combination group was significantly higher than those in other two single drug groups (Figure 1(c) and (d)). Finally, the interaction of BD and afatinib was verified by CI values. Results demonstrated that BD combined with afatinib exhibited a synergistic effect against both OC cell lines A2780 and CP70 (Figure 1

Effects of BD and afatinib combination treatment on OC cell proliferation and DNA replication
To investigate whether the synergistic effects of afatinib and BD extended to inhibit cell proliferation, a colony formation assay and a long-term proliferation assay were performed. The results showed that both the single-drug group and the combined-drug group could inhibit the cell proliferation of OC cell lines A2780 and CP70, whereas the effect of the combined-drug group is more significant (Figure 2(a) and (b)). Results from the colony formation assay further supported the conclusion that compared to the single-drug group, the combination of afatinib and BD resulted in fewer clones of A2780 and CP70 (Figure 2(c) and (d)). It is of note that the synergistic effects of afatinib and BD failed in MRC-5 cells (Figure 2(c) and (d)). In addition, quantitative analysis of EdU assay indicated that the combination of BD and afatinib significantly inhibited DNA replication in A2780 cells (Figure 3(a) and (b)). Similar results were also obtained in CP70 cells (Supplemental Figure S2).

Effects of BD and afatinib combination treatment on cell cycle arrest and apoptosis
To determine whether the growth inhibition of OC cells by BD in combination with afatinib is a result of cell cycle arrest, cell cycle distribution was assessed. As expected, when compared to BD and afatinib treated alone, the combination of BD and afatinib significantly blocked A2780 and CP70 cells in the G0/G1 phase (Figure 3(c) and (d)). To investigate the molecular mechanism of cell cycle arrest by BD and afatinib   combination, related cell cycle proteins were detected by western blot analysis. The results suggested that G1 phase protein levels of CDK2 and cyclin D1 were decreased in the combination treatment group, whereas BD and afatinib treatment alone resulted in no change in amount of CDK2 and cyclin D1 in A2780 cells (Figure 3(e) and (f)). Similar results were also observed in CP70 cells (Supplemental Figure S3).
To further investigate the fate of BD and afatinib combination treated A2780 and CP70 cells, we monitored the apoptotic cells after 48 h of drug treatment. As shown in Figure 4, the amount of BD-induced apoptotic cells increased from 6.3% to 13.2% and the amount of afatinib-induced apoptotic cells increased from 6.3% to 15.8% in A2780 cells. The number of apoptotic cells dramatically increased from 6.3% to 48.9% in the BD and afatinib combination treatment group (Figure 4(a) and (b)). Similar results were also found in CP70 cells (Supplemental Figure S4(a) and S4(b)). This is consistent with the flow cytometry apoptosis results. More cleaved caspase 3 and cleaved PARP were generated with the decreased expression of Bcl2 in the BD and afatinib combination treatment groups of A2780 and CP70 cells (Figure 4(c) and (d) and Supplemental Figure S4(c) and (d)). These results suggested that the combination of BD and afatinib could effectively enhance the anticancer activity of BD against OC cells by promoting cell apoptosis.

Effects of BD and afatinib combination treatment on cell adhesion and migration
Since cell adhesion plays an important role in OC progression and peritoneal metastases, 28 therefore, a cell adhesion assay was conducted to test the effects of afatinib and BD combination treatment on the adhesion of OC cells. The results showed that compared to BDor afatinib-treated cells, cells treated with both BD and afatinib displayed decreased adhesion to the extracellular matrix (Figure 5(a) and (b)). It is widely known that cell adhesion is closely related to cell migration. In this context, decreased cell adhesion might reduce cell migration. A transwell assay was performed to better determine the abilities of cell migration. These results indicated that BD and afatinib combination treated cells displayed a weakened ability to traverse a membrane from the serum-free to serum side (10% FBS) compared to untreated cells, or those that were treated with only one drug ( Figure 5(c) and (d)).
Effects of BD and afatinib combination treatment on EGFR signaling, DNA damage, and DNA damage repair.
To further explore the underlying mechanism of the combination treatment of BD and afatinib against OC and the synergistic interaction between BD and afatinib, we first evaluated the EGFR signaling pathway in OC cells A2780 and CP70 after treatment with BD and afatinib either alone or in combination. As shown in Figure 6(a) to (d), treatment with afatinib and/or BD alone resulted in a decreased level of p-EGFR and p-Akt; however, the total protein content of EGFR and Akt remained unchanged in both A2780 and CP70 cell lines at 48 h. As expected, the inhibition effect of p-EGFR caused by BD was weaker than afatinib ( Figure 6(a)-(d)). Surprisingly, the combined treatment with both BD and afatinib resulted in further inhibition of p-Akt (Figure 6(a) to (d)).
Considering the positive correlation between p-Akt and DNA damage, comet assays were performed to investigate the DNA damage in cells treated with BD or afatinib alone, and in BD and afatinib combination treated cells. The results showed that the DNA damage was the most obvious in the combined treatment group, with the DNA fragments that leave the genome forming a ''tail'' (Figure 6(e)). Quantitative analysis revealed that combined treatment triggered more DNA damage than BD or afatinib treatment alone (Figure 6(f)). Immunofluorescence (IF) assays using 53BP1 as a DNA double-strand break marker 29 were performed to verify this result. The results found that 53BP1 foci per cell accumulated significantly in the combination treatment group as compared to the single-drug treatment in both A2780 and CP70 cells (Figure 7(a)-(d)). These results suggest that there might be some DNA damage repair problems in both BD-and afatinib-treated cells. Consistent with this, the results from western blot analysis showed that in the combination group, g-H2AX was significantly increased, whereas the proteins related to DNA repair, such as RPA and Ku70, were decreased significantly (Figure 7(e)-(h)). Together, all results demonstrated that BD combined with afatinib blocked EGFR signaling, promoted DNA damage, and inhibited DNA damage repair in OC cells.

Discussion
Previous studies have shown that the expression of EGFR had a positive correlation with OC and that its inhibitors could enhance the anticancer activity of chemotherapy drugs in the setting of some cancers. 30 However, whether a combination of afatinib, an EGFR inhibitor, and BD, a traditional Chinese medicine antitumor drug, would achieve success in OC is not yet known. In this study, we first evaluated the anticancer activity of the combination of BD and afatinib in OC. Our results demonstrated that compared to treatment with BD or afatinib alone, the combination treatment possessed a stronger ability to influence cell    proliferation and migration inhibition, which led to more cell apoptosis. Furthermore, we revealed that this stronger, anticancer effects of the combination treatment is the consequence of the dual blocking of the EGFR and DNA damage repair signaling pathway ( Figure 8).
Although EGFR inhibitors have been widely used in the clinical treatment of EGFR-mutated cancers, their application was limited due to the drug resistance. 31 Since EGFR inhibitors have the anticancer activity and enhancing effects of other chemotherapy drugs, this combination treatment strategy is another important development to determine the full effects of an EGFR inhibitor; however, EGFR mutations in the setting of OC are rare. 32 The combination effect on OC along with the underlying mechanism is an important question to solve. We found that BD has anti-proliferation and anti-migration effects on OC cell lines A2780 and CP70. When combined with afatinib, the effect of antimigration and anti-cloning is more significant (Figures 2 and 5). This suggests that combination treatment may result in a simultaneous blockade of multiple survival and migration pathways, including EGFR signaling. Consistently, our results indicated that in addition to blocking the EGFR signaling pathway, BD combined with afatinib triggered intense DNA damage and provoked stronger DNA damage but suppressed the DNA damage repair signaling. Both strategies lead to catastrophic DNA damage that causes cell death. 33 DNA-PKcs is phosphorylated at Thr2609 to respond to DNA damage caused by anti-cancer drugs, which is a significant step for the ability of DNA repair. 34 To repair damaged DNA, DNA-PKcs recruits other proteins, like Ku70. 35 Some studies suggested that EGFR binds to DNA-PKcs and induces its phosphorylation. 35 We found that the combination of BD and afatinib can truly inhibit Ku70 and EGFR expression (Figures 6(a)-(d) and 7(e)-(h)). So, it is reasonable to assume that BD combined with afatinib may block DNA damage repair signaling by EGFR-mediated Ku70 expression.

Conclusions
In summary, our study not only suggests a possible role of BD in anti-OC but also shows that combination with afatinib significantly inhibits tumor growth and migration. BD combined with afatinib blocked EGFR signaling thus blocking MAPK signaling, DNA damage, and DNA damage repair in OC cells. This combination may provide benefits in the treatment of OC. Further research in vivo is also warranted to fully elucidate the value of BD combined with afatinib as a treatment option for OC.