Abstract
Background/Aim: Castration-resistant prostate cancer (CRPC) contributes to the deaths of most men from prostate cancer. Focal adhesion kinase (FAK) is abnormally up-regulated in CRPC. Chalcone possesses potent anticancer activity with clinical potential. However, it remains unknown whether its derivatives can be exploited as promising oncotherapeutic agents in CRPC treatment by inhibiting FAK-related signaling pathway. Aim: This study aimed to investigate the anticancer effects and the underlying mechanisms of action of chalcone derivatives against CRPC cells. Materials and Methods: Two chalcone derivatives (compounds 1 and 2) were synthesized, and their anti-CRPC activity toward DU145 and PC3 cells was evaluated. The effect of chalcone derivatives on CRPC cells was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, colony-formation, 5-ethynyl-2′-deoxyuridine staining, flow cytometric, cell adhesion and transwell assays. The study of mechanisms was conducted through comet, immunofluorescence and western blot assay, analysis of The Cancer Genome Atlas and molecular docking. Results: The results revealed that both compounds exhibited stronger cytotoxicity to CRPC cells along with significant inhibition of colony formation, especially compound 1. Further experimental evidence indicated that 1 significantly inhibited DNA replication, induced cell-cycle arrest and cell apoptosis. Additionally, treatment with 1 inhibited cell-matrix adhesion and migration of CRPC cells. Mechanistically, the results suggest that 1 inhibited FAK expression and phosphorylation, as well as affected its distribution, resulting in intense DNA damage and strong DNA damage response. Conclusion: We discovered two chalcone derivatives and collective results indicated that 1 inhibited CRPC cell proliferation and migration through FAK-mediated DNA damage and may be a potential therapeutic drug against CRPC.
- Castration-resistant prostate cancer
- focal adhesion kinase
- chalcone derivative
- DNA damage
- proliferation
- migration
Prostate cancer (PCa) is among the most common malignancies in men in both developed and developing countries, and its morbidity and mortality have been increasing in recent years (1-3). Since androgens and androgen receptors play predominant roles in the progression of PCa, androgen-deprivation therapy (ADT) is a basic element of PCa treatment (4). However, patients receiving long-term ADT eventually develop castration-resistant prostate cancer (CRPC), which is the major cause of death from PCa (5). The progress of CRPC is characterized by the rapid proliferation and migration of CRPC cells despite ADT (6, 7). Among the major treatments for CRPC, chemotherapy is the preferred choice for oncologists (8, 9). However, several drawbacks such as development of resistance to chemotherapy and severe side-effects have limited its efficiency in clinical therapy (9, 10). Therefore, researchers continue to explore factors that affect the progress of CRPC, aiming to find more efficient strategies for curing it.
Focal adhesion kinase (FAK) is a key regulatory molecule in cell proliferation and migration signal transduction processes (11-18), and is commonly overexpressed in cancer, including CRPC. Furthermore, FAK expression has been closely correlated with the survival and prognosis of patients with cancer clinically (19, 20). Moreover, experimental evidence suggests that FAK inhibition in cancer cells would induce persistent DNA damage and provoke a strong DNA-damage response (DDR) (21-23). These data indicate that FAK might play an important role in the pathological progress of cancer and could be further explored as a valuable anticancer drug target, particularly in CRPC treatment. Indeed, FAK has been the attention of research (19, 24). However, few studies were focused on natural products, FAK and CRPC.
Chalcone, a common simple chemical scaffold in the flavonoid family, widely exists in many natural plant products, including spices, vegetables, fruits, and teas (25). Chalcone and its derivatives have been shown to have anticancer activity mainly through cell-cycle arrest, cell apoptosis, and immunomodulatory and inflammatory mediators (26, 27). Further molecular investigation indicated that the chalcone skeleton interacts with DNA via Van der Waal and π-stacking, as well as electrostatic interaction (28-30). Since there is a lack of previous studies investigating the anticancer activity of chalcone derivatives in FAK-mediated signaling pathways in CRPC, we synthesized two chalcone derivatives and conducted a series of experiments to evaluate their anti-proliferative and anti-migratory activities against DU145 and PC3 CRPC cells and explored the underlying mechanism.
Materials and Methods
Cell and culture. CRPC cells (DU145 and PC3) and the human normal prostatic stromal myofibroblast cell line (WPMY-1) were purchased from the Cell Resource Center of Peking Union Medical College (Beijing, PR China). The cells were cultured at 37°C with 5% CO2 in Dulbecco’s modified Eagle’s medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (Gibco) and 100 U/ml of penicillin and streptomycin (Gibco).
Reagents and antibodies. All the reagents and solvents were respectively purchased from Sigma–Aldrich (St. Louis, MO, USA) and Alfa Aesar (Haverhill, MA, USA). Other chemicals were obtained from local suppliers and used directly. 5-ethynyl-2′-deoxyuridine (EdU) staining kit was purchased from Beyotime Biotech (Shanghai, PR China). Human fibronectin was obtained from Millipore (Billerica, MA, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) powder, glyceraldehyde 3-phosphate dehydrogenase, phalloidin and antibodies to p53-binding protein 1 (53BP1), cleaved poly (ADP-ribose) polymerase (c-PARP), BCL2 apoptosis regulator (BCL2), BCL2-associated X, apoptosis regulator (BAX), FAK and phospho (p)-FAK were obtained from Cell Signaling Technology (Danvers, MA, USA). 4′,6-Diamidino-2-phenylindole was obtained from Vectashield (Vector Laboratories, Burlingame, CA, USA). Doxorubicin was obtained from Sigma–Aldrich. Transwell plates were purchased from Corning Costar (Corning, NY, USA). Propidium iodide (PI) and flow cytometry apoptosis kits were obtained from Sigma-Aldrich.
Synthesis of chalcone derivatives 1 and 2. ((E)-3-(5-Bromo-4-hydroxy-2-methoxyphenyl)-1-(4-(piperidin-1-yl)phenyl)prop-2-en-1-one) (compound 1) and ((E)-4-(3-(5-bromo-4-hydroxy-2-methoxyphenyl) acryloyl)phenyl 4-nitrobenzoate) (compound 2) (Figure 1A) were synthesized according to the procedure reported in literature (37). 1H nuclear magnetic resonance (NMR) spectra were recorded using a Bruker instrument (Bruker AVANCE DRX-500, Bruker Corporation, Billerica, MA, USA), with all chemical shifts being presented in parts per million relatives to tetramethylsilane.
Bromo-retrochalcone derivatives 1 and 2 inhibit the cell viability of and colony formation of DU145 and PC3 castration-resistant prostate cancer (CRPC) cells. A: Chemical structure of bromo-retrochalcone derivatives 1 and 2. B: The half maximal-inhibitory concentration (IC50) values of compounds 1 and 2 against normal prostatic stromal myofibroblast cells (WPMY-1) and CRPC cells (PC3 and DU145) were measured by MTT assay after treatment with 1 and 2 for 48 h. 0.01% dimethyl sulfoxide (DMSO)-treated cells were used as control and the default cell survival rate was 100%. C: Representative images of colonies of compound 1- or 2-treated and untreated DU145 and PC3 cells. Cells were treated with 0.01% dimethyl sulfoxide (DMSO; control) or the indicated concentrations of 1 or 2 for 48 h before the colony formation assay was performed. D: Quantification of data shown in C. The values represent the average±standard deviation of at least three independent experiments. Unpaired Student’s two-tailed t-test was used to determine the statistical significance. Significantly different at: *p<0.05, **p<0.01 and ***p<0.01 compared with the control; #p<0.05 and ##p<0.01, compared with doxorubicin (Dox).
MTT cell proliferation assay. MTT assay was performed as described previously (31). Briefly, PC3, DU145, and WPMY-1 cells were seeded on 96-well plates (3×103 cells/well) after overnight pre-incubation, following which 1.5625, 3.125, 6.25, 12.5, 25, 50, 100, or 200 μM of bromo-retrochalcone derivatives 1 or 2 or doxorubicin were added for another 48 h, and then 20 μl of 0.5 mg/ml MTT was added to each well for a further 4 h. The formazan reaction product was dissolved in 100 μl of dimethyl sulfoxide and detected by reading the absorbance at 490 nm using a Multiskan FC automatic microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).
Colony-formation assay. PC3 and DU145 cells were plated onto 12-well plates at a density of 1×103 cells/well and incubated overnight until the cells were attached to the dish. The cells were then exposed to 5, 10 or 20 μM of compounds 1 or 2 or 0.01% DMSO (control). The medium was changed every 2 days until the density of colonies reached ~80%. The colonies were then washed, fixed with 4% paraformaldehyde, and stained with crystal violet. The colonies in each plate were photographed under a light microscope and then the crystals were dissolved in 500 μl acetic acid (33%) and the absorbance was detected at 560 nm by a spectrophotometer.
Comet assay. The comet assay was conducted as described previously (32). Briefly, cells treated with 5, 10, 20 μM of compounds 1 or 2 or 0.01% dimethyl sulfoxide (DMSO) (control) were first mixed with 0.5% low-melting-temperature agarose before being loaded onto slides coated with 1.5% normal agarose. These slides were then lysed in a lysis buffer containing 2.5 M NaCl, 10 mM Tris (pH 8.0), 100 mM ethylene diamine tetraacetic acid (EDTA), 0.5% Triton X-100, 1% N-lauroylsarcosine, and 3% DMSO. Electrophoresis was carried out in a buffer containing 300 mM sodium acetate, 100 mM Tris-HCl, and 1% DMSO. The slides were then treated with PI solution (20 μg/ml), visualized under a Nikon fluorescence microscope (Tokyo, Japan), and analyzed by CASP (Trevigen Inc., Gaithersburg, MD, USA).
Immunofluorescence (IF) assay. IF assay was performed as previously described (33). Briefly, cells treated with 5, 10 or 20 μM of compounds 1 or 2 or 0.01% DMSO (control) were seeded over a coverslip and fixed with 4% paraformaldehyde. The fixed cells were then permeabilized using 0.5% Triton X-100. Subsequently, the cells were incubated with primary antibodies against 53BP1 or FAK and then with secondary antibodies (DyLight 488-conjugated anti-Rabbit). The cells were mounted with DAPI (Vectashield), and fluorescent images were captured using a Nikon Ti microscope. Quantification of focal adhesion surface was determined with ImageJ (NIH, Rockville Pike, MD, USA). Briefly, the cell cytoskeleton area (red) and nucleus’ area (blue) were firstly calculated with the measurement function of ImageJ then focal adhesion surface area was calculated by the difference between the two. To quantify the degree of 53BP1 foci, over 200 cells from each group were randomly chosen from three independent experiments.
EdU staining assay. The DNA replication rate of PC3 and DU145 cells treated with bromo-retrochalcone derivative 1 or 0.01% DMSO (control) was examined using the EdU staining kit according to the manufacturer’s instructions. A coverslip was loaded onto 12-well plates, and cells were seeded on the coverslip at a density of 3.0×104 cells/well. The cells on the coverslip were treated with 5, 10 or 20 μM of 1 or 0.01% DMSO (control) for 48 h. The images were observed under a Nikon fluorescence microscope (Tokyo, Japan) and the percentage of EdU-positive cells were compared from the proportion of EdU-stainined cells (red) out of total cells (blue); over 200 cells from each group were randomly chosen from three independent experiments for these measurements.
Flow cytometric cell-cycle assay. PC3 or DU145 cells pretreated with compound 1 or 0.01% DMSO (control) for 48 h were trypsinized, washed once with phosphate-buffered saline (PBS), and fixed with 70% ethanol overnight at 4°C. After fixation, the cells were washed twice with PBS were then incubated with 20 μg/ml RNAse in a 37°C water bath for 30 min. Finally, the cells were stained with PI (50 μg/ml) staining solution for 15 min. Flow-cytometric analysis was performed using a flow cytometer (BD Biosciences, Sunnyvale, CA, USA).
Western blot assay. Western blot assay was performed as previously described (34). Cells treated with 5, 10 or 20 μM of compound 1 or 0.01% DMSO (control) were lysed using cell lysis buffer. The concentrations of protein were determined by using the Bradford assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The signals were captured using a Bio-Rad ChemiDoc XRS+ system (Bio-Rad).
Flow cytometric apoptosis assay. Annexin V/PI apoptotic assays (Sigma) were applied using flow cytometry according to the manufacturer’s instructions. PC3 and DU145 cells were seeded in 6 cm2 dishes at a density of 2.0×105 cells/dish. After the attachment of cells to the dish, 5, 10 or 20 μM of compound 1 or 0.01% DMSO (control) were added to the medium for another 48 h. Then the cells were harvested for annexin V/PI apoptotic assays and assessed using a flow cytometer (BD FACS Calibur; BD Biosciences).
Cell adhesion assay. The cell adhesion assay was carried out as described previously (35). A 96-well plate was pre-coated with 50 μl of human fibronectin (2.5 μg/ml) in 1× PBS at 4°C overnight. PC3 and DU145 cells pretreated with 5, 10 or 20 μM of compound 1 or 0.01% DMSO (control) for 48 h were trypsinized, washed once with PBS, and re-seeded onto a 96-well plate (4.0×104 cells/well). The cells were cultured for another 0.3-0.5 h at 37°C in an incubator with 5% CO2. The cells were then rinsed thrice with 10% formalin and stained with crystal violet for 5 min at room temperature, followed by washing with double-distilled water thrice, and the stained cells were dissolved in 100 μl of acetic acid (33%). Absorbance at 560 nm was detected using a Synergy H1 Multi-Mode Reader (BioTek, Winooski, VT, USA). The relative number of cells attached to the extracellular matrix was calculated using the equation: Mean optical density of treated cells/mean optical density of control cells.
Transwell assay. Transwell assay was conducted using a transwell kit (Corning Costar) according to the manufacturer’s instructions. Briefly, cells were pretreated with 5, 10 or 20 μM of compound 1 or 0.01% DMSO (control) for 48 h and then re-seeded into a transwell permeable support (insert) that was pre-equilibrated with 100 μl of serum-free DMEM at a density of 1.0×105 cells/insert. The insert was then placed in a 24-well plate containing 600 μl of DMEM with 10% fetal bovine serum for another 24 h. The cells on the upper surface of the insert were removed using cotton-tipped swabs; the cells on the backside of the insert were fixed with 10% formalin, stained with crystal violet, and washed thrice with double-distilled water. The stained cells were dissolved in 500 μl of acetic acid (33%), and the absorbance were measured at 560 nm using a spectrophotometer (DTX880; Beckman Coulter, CA, USA).
The Cancer Genome Atlas (TGCA) data analysis. The selected samples of 1,239 patients were obtained from the Xenabrowser (https://xenabrowser.net) TCGA Prostate Cancer (PRAD) and GTEx datasets, which contained 498 normal samples and 741 tumor samples. Survival data were stratified at the median by protein tyrosine kinase 2 (PTK2) expression and subsequently analyzed. Data analysis was carried out by using R language (version 4.0.2) and GraphPad Prism 5.0 (San Diego, CA, USA).
Molecular modeling analysis. Molecular docking analysis of compound 1 and the crystallographic structure of FAK (PDB ID 6YT6) were performed using Autodock vina (36). Hydrogen atoms were neglected in compound 1, and water molecules were removed from the PDB file. Autodock vina docked compound 1 into FAK using an empirical scoring function and a patented search engine to identify the most stable and favorable orientations. The image in this article was created from SYBYL 7.3 with the intercalation site.
Statistical analysis. GraphPad Prism version 5 (San Diego, CA, USA) was used for the statistical analysis. In histograms, all data were presented as the mean±standard deviation of at least three independent replicates for each experiment. The statistical significance of the data was assessed by Student’s two-tailed unpaired t-test or two-way analysis of variance; p<0.05 was considered statistically significant.
Results
Chalcone derivatives. Both compound 1 and 2 (Figure 1A) were obtained by column chromatography using Merck silica gel 60 (200-300 mesh ASTM) (Merck KGaA, Darmstadt, Germany) as a light-yellow solid. 1H NMR of compound 1 (400 MHz, DMSO-d6) δ (ppm) (Supplementary Figure S1): 10.90 (s, 1H), 8.18 (s, 1H), 8.04 (d, J=9.0 Hz, 2H), 7.82 (d, J=15.0 Hz, 2H), 6.99 (d, J=9.0 Hz, 2H), 6.67 (s, 1H), 3.86 (s, 3H), 3.41 (s, 4H), 1.62 (s, 6H). 1H NMR of compound 2 (400 MHz, DMSO-d6) δ (ppm) (Supplementary Figure S2): 10.99 (s, 1H), 8.39 (d, J=6.7 Hz, 4H), 8.26 (d, J=8.7 Hz, 2H), 8.21 (s, 1H), 7.94 (d, J=15.6 Hz, 1H), 7.83 (d, J=15.6 Hz, 1H), 7.52 (d, J=8.7 Hz, 2H), 6.63 (s, 1H), 3.83 (s, 3H).
Derivatives 1 and 2 inhibit cell viability and colony formation by CRPC cells. The effect of compounds 1 and 2 on the viability of CRPC cells (DU145 and PC3) and the normal prostatic stromal myofibroblast cell line (WPMY-1) were evaluated by MTT assay. Doxorubicin was selected as a positive control drug in this study as it was the most widely used chemotherapeutic drug against CRPC in a clinical setting. The results indicated that both 1 and 2 possessed comparable or stronger anticancer activity with less cytotoxicity in normal cells compared to Dox (Figure 1B). To further explore the anti-CRPC activity of compounds 1 and 2, a colony-formation assay was carried out. The results indicated that the derivatives had a concentration-dependent effect on colony formation. Compared to compound 2, 1 had a stronger inhibitory effect on colony formation of DU145 and PC3 cells (Figure 1C and D). The results were consistent with MTT data, suggesting compound 1 might have better anti-CRPC activity than compound 2. Thus, compound 1 was selected in the subsequent experiments in this study.
Derivative 1 slows down the DNA replication rate and induces cell-cycle arrest and apoptosis of CRPC cells. It is well established that DNA replication is a central process in cell proliferation regulation (38, 39). Thus, an EdU assay was performed to evaluate the inhibitory effect of compound 1 on the DNA replication rate of DU145 and PC3 CRPC cells. The results indicated that DU145 and PC3 cells treated with compound 1 had a lower DNA replication rate compared to the untreated cells (Figure 2A), and this inhibitory effect was concentration-dependent (Figure 2B). A change in DNA replication rate might lead to cell-cycle arrest (39). Indeed, the results from the flow cytometric assay suggested that treatment with compound 1 induced G0/G1 phase arrest of DU145 cells, accompanied by a reduction of the percentage of cells in both the S and the G2/M phase in a concentration-dependent manner (Figure 2C and D). On the other hand, treatment with 1 arrested PC3 cells at the G2/M phase, reducing the percentage of cells in the G0/G1 phase and the S phase in a concentration-dependent manner (Figure 2C and D).
Bromo-retrochalcone derivative 1 inhibits DNA replication and induces cell-cycle arrest of DU145 and PC3 cells. A: Representative images of 1-treated or 0.01% dimethyl sulfoxide (DMSO)-treated (control) DU145 and PC3 cells in 5-ethynyl-2′-deoxyuridine staining (EdU) staining assays. Before the EdU staining assay, cells were treated with the indicated concentrations of 1 for 48 h. B: Quantification of EdU staining data. C: Representative images of the cell-cycle distribution of 1-treated DU145 and PC3 cells using flow cytometry. Cells were treated with the indicated concentrations of 1 or 0.01% DMSO (control) for 48 h. D: Quantification of cell-cycle distribution data. Values represent the average±standard deviation of at least three independent experiments. Unpaired Student’s two-tailed t-test was used to determine the statistical significance. Significantly different at: *p<0.05 and **p<0.01 compared with the control.
To further explore the fate of compound 1-treated DU145 and PC3 CRPC cells, we examined the expression of apoptosis-related proteins and the production of apoptotic cells in treated DU145 and PC3 cells by western blot assay and flow cytometry, respectively. The expression of cleaved-PARP, BAX and BCL2 was determined by western blot assay under the consideration that all of them play a key regulatory role in cell apoptosis (40-42). The results indicated that treatment with compound 1 significantly increased the expression of cleaved PARP and BAX protein but reduced the expression of BCL2 protein in DU145 and PC3 cells (Figure 3A). The quantitative analysis data indicated that 1 treatment changed the expression of cleaved PARP, BCL2 and BAX protein in a concentration-dependent manner (Figure 3B). Moreover, the quantitative analysis also indicated that treatment with compound 1 significantly reduced the BCL2/BAX ratio of both DU145 and PC3 cells in a concentration-dependent manner (Figure 3C). Additionally, the results from flow cytometry further demonstrated that treatment with compound 1 significantly elevated the proportion of apoptotic DU145 cells and PC3 cells in a concentration-dependent manner (Figure 4). Together, above results demonstrated that the chalcone derivative 1 induced CRPC cell apoptosis by reducing the BCL2/BAX ratio.
Bromo-retrochalcone derivative 1 regulates the expression of apoptosis-related proteins in DU145 and PC3 cells. A: Western blot determination of apoptosis-related proteins, cleaved poly (ADP-ribose) polymerase (c-PARP), BCL2 apoptosis regulator (BCL2) and BCL2-associated X, apoptosis regulator (BAX) in DU145 and PC3 cells was conducted. Cells were treated with the indicated concentrations of 1 for 48 h before cell lysis and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Representative blots are shown. B: Densiometric quantification of western blot data. C: Quantification of the relative BCL2/BAX ratio. Values represent the average±standard deviation of at least three independent experiments. Unpaired Student’s two-tailed t-test was used to determine the statistical significance. Significantly different at: *p<0.05, **p<0.01 and ***p<0.001 compared with the control.
Bromo-retrochalcone derivative 1 induces apoptosis of DU145 and PC3 cells. A: Cells were treated with the indicated concentrations of 1 or 0.01% dimethyl sulfoxide (DMSO) (control) for 48 h. Apoptotic cells were assayed by annexin V/propidium iodide (PI) staining and fluorescence-activated sorting. B: Quantification of the apoptotic cells shown in A. The values represent the average±SD of at least three independent experiments. Unpaired Student’s two-tailed t-test was used to determine the statistical significance. Significantly different at: *p<0.05 and **p<0.01 compared with the control.
Compound 1 inhibits the adhesion and migration of CRPC cells. About 80-90% of patients with CRPC develop distant metastasis, which is mainly responsible for patient death (43). Therefore, we investigated the effects of the derivative 1 on the adhesion and migration of DU145 and PC3 CRPC cells. Firstly, a cell adhesion assay was carried out between treated and untreated DU145 and PC3 CRPC cells to confirm the effect of compound 1 on cell adhesion. The results showed that treatment with derivative 1 significantly reduced the adhesion of DU145 and PC3 cells to the matrix in a concentration-dependent manner (Figure 5A).
Bromo-retrochalcone derivative 1 inhibits the adhesion and migration of DU145 and PC3 cells. A: Compound 1 reduces cell adhesion to the extracellular matrix in a concentration-dependent manner. Cells were treated with 1 or 0.01% dimethyl sulfoxide (DMSO) at the indicated concentrations for 48 h before undergoing this assay. B: Representative images showing compound 1 inhibition of the migration of DU145 and PC3 cells in a transwell assay. Representative images are shown. C: Quantification of migration data. The values represent the average±standard deviation of at least three independent experiments. Unpaired Student’s two-tailed t-test was used to determine the statistical significance. Significantly different at: *p<0.05 and **p<0.01 compared with the control.
Cell adhesion is often positively correlated with cell migration (44, 45). To confirm that treatment with compound 1 inhibited the migratory ability of DU145 and PC3 CRPC cells, the trans-well assay was performed. The results indicated that compound 1-treated DU145 and PC3 cells had weaker ability to penetrate membranes and migrate compared to the untreated cells (representative images are shown Figure 5B). The quantitative data demonstrated that the migratory ability of 1-treated DU145 and PC3 CRPC cells was significantly inhibited compared to untreated cells (Figure 5C).
Derivative 1 suppresses the expression and phosphorylation of FAK, along with reducing its distribution at the cell margins of CRPC cells. FAK has been considered as a key regulation molecular in cell proliferation and migration (46-48). To test the idea that FAK might regulate the proliferation and migration of CRPC cells, transcriptomic data from 1,239 CRPC tissue biopsies were obtained from TCGA database and analyzed. The results revealed that compared to normal tissues, mRNA for PTK2 (FAK-encoding gene) was overexpressed in CRPC tissues (Figure 6A), and that FAK expression level was negatively correlated with patient survival (Figure 6B). Thus, we speculated that compound 1 inhibited CRPC cell proliferation and migration might be mediated by FAK. The results from the western blot assay showed that treatment with 1 suppressed the expression and phosphorylation of FAK in a concentration-dependent manner in DU145 and PC3 cells (Figure 6C and D). Moreover, we also noticed that treatment with 1 reduced the distribution of FAK at the leading edge of cells in a concentration-dependent manner (Figure 7A) accompanied with reduced FA surface area (Figure 7B) through immunofluorescence assay. Additionally, molecular docking studies suggested that compound 1 might interact with FAK through two hydrogen bonds with CYS-502 (Figure 7C).
Bromo-retrochalcone derivative 1 inhibits the expression and phosphorylation of focal adhesion kinase (FAK) in DU145 and PC3 castration-resistant prostate cancer (CRPC) cells. A: Expression of FAK-encoding gene (protein tyrosine kinase 2, PTK2) in CRPC samples (n=741) compared to normal tissues (n=498). The data were acquired from The Cancer Genome Atlas (TCGA). Boxes represent the sets of PTK2 expression in normal (black) and tumor (red) patient samples; bars represent the minimum to maximum PTK2 expression values; lines in boxes represent the average PTK2 expression. B: Kaplan–Meier analysis showing the association between PTK2 expression and the overall survival of patients with CRPC. The data were acquired from TCGA. C: Western blot determination of FAK and phospho (p)-FAK levels in control (0.01% dimethyl sulfoxide) and 1-treated DU145 and PC3 cells. Cells were treated with the indicated concentrations of 1 for 48 h before cell lysis and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. Representative blots are shown. D: Quantification of western blot data in C. The values represent the average±standard deviation of at least three independent experiments. Unpaired Student’s two-tailed t-test was used to determine the statistical significance. Significantly different at: *p<0.05, **p<0.01 and ***p<0.001 compared with the control.
Bromo-retrochalcone derivative 1 reduces the distribution of focal adhesion kinase (FAK) at the front edge of cells. A: Representative images of FAK in compound 1-treated and untreated DU145 and PC3 cells in immunofluorescence assays. Anti-FAK (green) and phalloidin (red) were used to visualize FAK and F-actin, respectively. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Images were obtained using a Nikon microscope. B: Quantification of FAK distribution in DU145 and PC3 cells. Boxes represent the minimum to maximum FA surface area; bars represent the error of each group; lines represent the average FA surface area. C: Representation of the mode of binding between 1 and FAK as determined by molecular docking. The values in charts represent the average±standard deviation of at least three independent experiments. Unpaired Student’s two-tailed t-test was used to determine the statistical significance. Significantly different at: *p<0.05 and ***p<0.001 compared with the control.
Treatment with compound 1 triggers intense DNA damage and provokes a strong DDR in CRPC cells. FAK inhibition would induce DNA damage (21-23). Chalcone and its derivatives commonly achieve anticancer biological activity by triggering DNA damage (28, 30, 34). We hypothesized that the anti-proliferative activity of 1 might be mediated by inducing DNA damage. To confirm this hypothesis, the comet assay was first performed to evaluate the level of DNA damage of compound 1-treated or untreated DU145 and PC3 CRPC cells (Figure 8A). As expected, compound 1-treated DU145 and PC3 cells showed a higher level of DNA damage than untreated cells, leading to DNA fragments that left the genome and formed ‘tails’ during the comet assay. The extent of DNA damage induced by treatment with compound 1 was measured by the percentage of DNA tails, which indicates the abundance of DNA fragments induced. The quantitative analysis indicated that more than 20% of the cells, especially compound 1-treated DU145 cells, displayed higher levels of double-strand breaks than in the control (5% tail DNA signal) (Figure 8B and C).
Bromo-retrochalcone derivative 1 triggers intense DNA damage in DU145 and PC3 castration-resistant prostate cancer (CRPC) cells. DU145 and PC3 cells were treated with compound 1 at the indicated concentrations or 0.01% dimethyl sulfoxide (control) for 48 h before being subjected to assays. A: Representative results of the alkaline comet assay. B: The percentage of DNA in the tails of compound 1-treated and control DU145 and PC3 cells. C: The percentage of 1-treated and control DU145 and PC3 cells with over 10% tail DNA was measured. The values represent the average±standard deviation of at least three independent experiments; lines represent the average tail DNA or olive tail moment of each group. Unpaired Student’s two-tailed t-test was used to determine the statistical significance). Significantly different at: **p<0.01 and ***p<0.001 compared with the control.
Considering that intense DNA damage would provoke strong DDR, an IF with antibodies against 53BP1, an important regulator in response to DNA damage (49, 50), was performed. The amount of 53BP1 indicates the level of DDR. The number of 53BP1 foci per nucleus was calculated in both compound 1-treated and untreated DU145 and PC3 CRPC cells (Figure 9A). Unsurprisingly, the number of 53BP1 foci in DU145 and PC3 cells increased to an average of ~61 and ~20 foci per nucleus, respectively, after treatment with 20.0 μM of 1 for 48 h (Figure 9B). Moreover, the number of 53BP1 foci per cell in 1-treated CRPC cells was observed to be concentration-dependent (Figure 9). These results suggested that treatment with compound 1 triggered intense DNA damage and provoked strong DDR in CRPC cells.
Bromo-retrochalcone derivative 1 provokes a strong DNA damage response in DU145 and PC3 castration-resistant prostate cancer cells. A: DU145 and PC3 cells were treated with the indicated concentrations of 1 or 0.01% dimethyl sulfoxide (control) for 48 h. The number of p53-binding protein 1 (53BP1) foci was determined by immunofluorescence with 53BP1 antibodies. Cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) as shown in the ‘merge’ images. Representative results are shown. B: Quantification of the number of 53BP1 foci per cell in A. The values represent the average±standard deviation of at least three independent experiments. Unpaired Student’s two-tailed t-test was used to determine the statistical significance. Significantly different at: *p<0.05 and **p<0.01 compared with the control.
Discussion
Chalcone, which widely naturally exists, is a common simple scaffold and useful template in anticancer agent development (51). To date, a plethora of chalcone derivatives have been synthesized and some of them had been demonstrated to have good anticancer activity both in vitro and in vivo (26, 27). However, to our knowledge, none of them had been reported to inhibit CRPC cell proliferation and migration through FAK-mediated DNA damage pathway. Therefore, this study was conducted to provide a theoretical basis for the development of chalcone derivatives as potential chemotherapeutic agents against CRPC via FAK-mediated DNA damage.
In this study, we first identified and synthesized two bromo-retrochalcone derivatives (compounds 1 and 2, Figure 1A). Then we demonstrated that both compounds had significant anti-proliferative effect on CRPC cells by effectively suppressing the cell viability and colony formation (Figure 1B-D), as well as slowing down DNA replication (Figure 2A and B), inducing cell-cycle arrest (Figure 2C and D) and promoting cell apoptosis (Figure 3 and Figure 4). Both 1 compound and 2 strongly inhibited extracellular matrix adhesion and migration of CRPC cells (Figure 5). We demonstrated that FAK, which is a key regulatory molecular in cell proliferation and migration (20, 24, 46, 52), was overexpressed in CRPC tissues and was negatively correlated with patient survival (Figure 6A and B). Strikingly, the results presented here showed that treatment with 1 not only inhibited the expression and phosphorylation of FAK (Figure 6C and D), but also reduced its distribution at the margin of cells (Figure 7A and B). Furthermore, the molecular docking analysis suggested that compound 1 interacts with FAK by forming hydrogen bond with CYS-502 (Figure 7C).
Previous studies suggested that FAK inhibition would induce DNA damage and provoked strong DNA damage response (21-23). Meanwhile, DNA was generally thought to be the preferred target of chalcone derivatives (26-28). Our results demonstrate that treatment with 1 triggered intense DNA damage (Figure 8) and provoked strong DDR (Figure 9) by FAK inhibition, demonstrating the anticancer effect of 1 might be a consequence of FAK-mediated DNA damage or DDR in treated DU145 and PC3 CRPC cells. Additionally, taking the function of FAK in regulation of cell migration into consideration, our results suggested that 1 might achieve its anti-migratory effect by inhibiting the expression and phosphorylation of FAK protein and reducing its distribution at the edge of cells.
In conclusion, this study demonstrated two bromo-retrochalcone derivatives, especially compound 1, to be potential anticancer agents for treatment of FAK-overexpressing CRPC and determined that its anticancer activities were due to FAK-mediated DNA damage. Our results provide a theoretical basis for future development and application of chalcone derivatives in CRPC treatment.
Acknowledgements
This study was sponsored by Wenzhou Science and Technology Plan Project, China (No. Y20210944) and the Zhejiang Medicine and Health Science and Technology Project (no. 2020RC082). We thank Elsevier (http://webshop.elsevier.com/languageediting/) for its linguistic assistance during the preparation of this article.
Footnotes
Authors’ Contributions
HX, FL and YX conceived the idea. YX, HX, ZW, QW and CZ designed, conducted the experiments, and analyzed data. HX, FL and YX wrote the article. All Authors were involved in the discussion of the project. All Authors took part in drafting the final version for submission and accept overall accountability for the accuracy and integrity of the article.
↵Supplementary Material
Available at:<https://1drv.ms/u/s!Amd7Gy5fxhnqbwtWxK7F6wVaqGU?e=9JG4lf>
Conflicts of Interest
The Authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
- Received July 31, 2022.
- Revision received September 8, 2022.
- Accepted September 12, 2022.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
References
In this issue
Jump to section
Related Articles
Cited By...
- No citing articles found.