Flavokavain C Suppresses Breast Cancer Cell Viability and Induces Cell Apoptosis by Triggering DNA Damage

Abstract

Breast cancer, presented by multiple breast cancer subtypes that coexist within a diagnosed tumor in clinical, has ranked as the most common malignancy in women in recent years. Evidence suggested that limited effective drugs caused the unsatisfactory therapeutic efficacy of breast cancer. Flavokavain C exhibited anticancer activity on colon cancer cells HCT116. It is yet unknown if it can be used to treat breast cancer. This study aims to believe the mechanisms by which Flavokavain C suppresses cell proliferation and the pathways that impact on this effect in breast cancer. 3-(4,5-Dimethythiazol)-2,5-diphenyltetrazolium bromide assay was chosen to evaluate cell cytotoxicity. Colony formation and cell proliferation assays using 5-ethynyl-2′-deoxyuridine staining were performed. Cell cycle progression and apoptosis were examined via flow cytometry and Western blotting, respectively. Five methods (comet assay, immunofluorescence, Western blotting, agarose gel electrophoresis and molecular docking) were used to quantify DNA damage and its cellular response. Compared to cisplatin, Flavokavain C possessed a comparable or more substantial inhibitory effect on breast cancer cell viability while having lower cytotoxicity on human mammary cells. Breast cancer cells treated with Flavokavain C had their colony formation suppressed, DNA replication blocked, the G2/M phase cell cycle arrested, and apoptosis. Furthermore, the results indicated that Flavokavain C would directly interact with DNA and induce DNA cleavage, demonstrating that DNA is an attractive substrate for Flavokavain C. These results suggested that Flavokavain C had strong anticancer activity against multiple subtypes of breast cancer cells.

INTRODUCTION

The number of new breast cancer cases each year is 2.26 million (24.5 percent of all female tumor cases), and the number of deaths from cancer each year is 685000 (15.5 percent of all female cancer-related deaths). This makes breast cancer the most common tumor in women worldwide, overtaking lung tumors.^1–3) It is well established that the treatment outcomes, prognosis, and survival in breast tumor patients are tightly associated with the hormonal status of mammary tissues.^4–6) In addition, the HER2 oncogene status and the estrogen receptor (ER) and progesterone receptor (PR) expressions all act as critical effects in choosing the best course of clinical action.^5,7) Breast cancer was classified into ER-positive, HER2/ERBB2 amplified, and triple-negative (TNBC) subtypes relying on the expression of ER, PR, and HER2.⁸⁾ Clinical studies showed that breast cancer frequently consists of multiple breast cancer subtypes,⁹⁾ significantly increasing the difficulty of treatment. Therefore, it is pressing to excavate new drugs with broad-spectrum anticancer activity for breast cancer.

Chalcones, the top flavonoid compounds in fruits, vegetables, and tea, possess many biological activities, especially their anticancer activity.¹⁰⁾ Mechanically, previous research suggested that the antitumor activity of chalcones could be mainly involved in enhancive superoxide formation, cellular glutathione exhaustion, and phenoxide radical production.^11,12) Besides those, reported studies also indicated that the interaction among chalcones and plentiful kinases, microtubules, polytherapy-resistant proteins, and various signaling pathways relevant to cell survival and death contributed to their excellent anticancer activity.¹¹⁾ Hence, natural chalcone compounds gained the great interest of scientists in the anticancer drug development field. Flavokavain C is one of the naturally occurring chalcones that extract from the root of the kava plant.¹³⁾ Cell cycle arrest and apoptosis were previously linked to Flavokavain C’s anticancer activity in colorectal cancer HCT116 cells.¹⁴⁾ Flavokavain C may have anticancer effects against breast cancer cells, however, it still needs to be determined whether this is the case or how.

We conducted this research to ascertain whether Flavokavain C has any anticancer properties and, if so, how it does so against various subtypes of breast cancer. We showed that apoptosis and cell cycle halt induced by DNA damage was caused by Flavokavain C, which reduced breast cancer cell’s ability to divide and form colonies.

MATERIALS AND METHODS

Cells and Agents

Originating from the Peking Union Medical College’s Cell Resource Center, we got the normal human mammary epithelial cell (MCF-10A), and three subtypes of breast cancer: MDA-MB-231 (triple-negative breast cancer, TNBC cells), MDA-MB-453 (HER2/ERBB2 amplified breast cancer cells), and MCF-7 (ER-positive breast cancer cells). Cells were grown in RPMI1640 (Gibco) with streptomycin (HyClone-GE, Marlborough, MA, U.S.A.), penicillin (100 U/mL), and fetal calf serum (10%, PPA-GE, HyClone-GE) (37 °C, 5% CO₂). Flavokavain C was purchased from Sigma (St. Louis, MO, U.S.A.) with a purity of no less than 96% (No. PHL83854).

Viability of Cells

The cell viability was evaluated by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Cells (5 × 10³ cells/well) were cultivated in 96-well plates (48 h, 37 °C) with or without Flavokavain C treatment before MTT (20 µL/well, 0.5 mg/mL) was added. After disposing of the growing medium, dimethyl sulfoxide (DMSO) (100 µL) was chosen to dissolve the formazan. The final step was performed with a DTX880 spectrophotometer (λ = 490 nm, Beckman Coulter, CA, U.S.A.). The viability of the cell was evaluated via absorbance (three measurements ± standard deviation).

Colony Formation

Twelve-well plates were planted with 800 breast cancer cells/well. Following attachment, the cells were incubated for 2 weeks in normal growth media (0.01% DMSO) or co-incubated with the relevant Flavokavain C solution before colony growth was evaluated. After being rinsed with phosphate-buffered saline (PBS) (×3), the colonies were fixed in formaldehyde (4%, 15 min) and stained crystal violet (0.04%, 1 h). The colonies on each plate were examined under a microscope after being washed twice with ddH₂O. Three separate experiments tracked the number of colonies.

5-Ethynyl-2′-deoxyuridine (EdU) Staining

Cell proliferation was discovered by the Edu Staining Proliferation Kit (Beyotime, Shanghai, China). Twelve-well plates with coverslips were used to transfect and cultivate breast cancer cells 3 × 10⁴ cells/well in terms of cell density. Flavokavain C was added to the coverslip in varying quantities and then added to the cells. At last, a Nikon fluorescence microscope was used to examine the cells.

Cell Cycle Analysis Using Flow Cytometry

After trypsinized and rinsed in PBS at 4 °C for 12 h, 70% ethanol was used to fix the cells, and PBS was used for two washes. The cells were left in a water bath containing ribonuclease (RNase) (20 µg/mL, 37 °C, 30 min). At last, the cells were stained for 10–15 min with PI (50 °C staining solution). A flow cytometer (BD Biosciences, Franklin Lakes, NJ, U.S.A.) was chosen to analyze the cells.

Apoptosis

3 × 10⁵ cells per well of six-well culture trays were used to spawn breast cancer cells and left overnight to be tested for apoptosis. Following 48 h exposure to Flavokavain C, cells were stained by a flow cytometer (BD Biosciences) to examine results from a fluorescein isothiocyanate (FITC)-conjugated Annexin V/propidium iodide (PI) apoptosis Kit (Sigma).

Comet

During the previous day, a 12-well plate with breast cancer cells was planted, and the cells were allowed to develop in the incubator. The cells were harvested after 48 h of exposure to 5, 10, or 20 µM Flavokavain C. Then, 100 µL of preheated, low-melting agarose at 0.65% was added to 2 × 10⁴ to 1 × 10⁵ single cells, and the resulting mixture was stacked on 1.5% agarose with a normal melting point applied onto microscope slides. Slides were lysed at 4 °C overnight. DNA unwinding was performed by incubating them in the cold, freshly prepared electrophoresis buffer (1 mM Tris–HCl, 0.3 M NaOH, pH 13) at the same temperature for 30 min. A 20 min run at 4 °C and 30 V at 300 mA (mA) electrophoresis were used. The Tris–HCl buffer (pH 7.5, 0.4 M) neutralized the slides. PI dye was used to stain the DNA (20 mg/mL). The results were analyzed by a fluorescence microscope (Nikon Ti, Nikon, Japan) and CASP software at a magnification of 40×. One hundred cells were counted for each concentration, and traits such as Tail DNA % and tail strength were assessed.

Immunofluorescence (IF)

The IF assay was conducted the same way as previously described.¹⁵⁾ To summarize, paraformaldehyde (4%) was chosen to fix the cells. Triton X-100 (0.5% in 1 × PBS) was chosen to permeabilize them on the coverslip. The cells were exposed to primary antibodies against 53BP1 (CST) for a whole night at 4 °C, washed six times, and then subjected to secondary antibody incubation (DyLight 488-conjugated anti-rabbit). Six cycles of PBST washing were used on the coverslip. 4′-6-Diamidino-2-phenylindole (DAPI) was then used to stain the cells and mount them. A Nikon Ti microscope was utilized for fluorescence detection and imaging. To determine the frequency of 53BP1 foci, we randomly sampled over 200 cells from each group across three assays.

Western Blotting

Various concentrations of Flavokavain C were added to cell cultures on a 6-well plate (48 h). Cells were diluted in cell lysis buffer and lysed after being cleaned with PBS (×2). The Bradford assay (Bio-Rad, CA, U.S.A.) was chosen to calculate the concentrations. A sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) gel (12%) was used to transfer the proteins to polyvinylidene difluoride (PVDF) membranes. Nonfat milk (5%) in TBST was chosen to block the blots (2.5 h, room temperature). Primary antibodies were incubated at 4 °C for 12 h. Once the membranes had been washed with TBST (×3), they were cultured with secondary antibodies conjugated to peroxidase (1 h, room temperature). The immunoreactive bands could be seen using an ECL detection kit (Bio-Rad).

Agarose Gel Electrophoresis

Plasmid pBR322 (0.5 µg/µL, Thermo Fisher Scientific (Waltham, MA, U.S.A.)) was mixed with different concentrations of Flavokavain C (5.0, 10.0, 20.0 µM) at 37 °C for 2 h. Before loading, add loading buffer (10 × loading buffer), then add the sample containing Gel Red (Beyotime) 1% agarose gel electrophoresis was carried out in the gel hole, the prepared gel was put into the electrophoresis tank, then added TAE (l×) electrode buffer and adjusted the voltage to 100 V. After electrophoresis for 35 min, the electrophoresis completed gel was removed, and the experimental results were observed in the gel imaging system. Finally, quantitative analysis was performed by ImageJ software.

Molecular Docking

The crystal structure of the B-DNA dodecamer was obtained from Protein Data Bank (PDB ID: 355D). Water molecules were manually removed by using PyMOL software. The predicted binding poses of Flavokavain C were carried out using AutoDock (version 4.2.6). Prepar_eligand.py and prepare_recptor.py scripts from AutoDock-Tools were used to prepare the initial files including adding hydrogen atoms and charges. Next, a grid box of 60 × 60 × 60 with a spacing of 0.40 Å enclosed the binding site. The Lamarckian genetic (LGA) was adopted to search for the best binding poses. The specific docking settings were as follows: trials of 100 dockings, 300 individuals per population with a crossover rate of 0.8, and the local search rate set to 0.06. Other parameters were set as default during the docking.

Statistical Analysis

All p-values are displayed in the figures; Two-tailed t-tests on unpaired students was chosen to establish statistically evident (ns = not significant represents p > 0.05; *, **, and *** represents p < 0.05, p < 0.01, and p < 0.001).

RESULTS

Breast Cancer Cells’ Cell Viability and Proliferation Are Inhibited by Flavokavain C

For starters, the anticancer activity of Flavokavain C (Fig. 1A) was tested in an MTT assay versus the three cells representing three distinct subtypes of breast cancer (Fig. 1B). The results exhibited that compared with normal mammary cells MCF-10A, Flavokavain C exhibited stronger cytotoxicity on the three breast cancer cells, with the values of IC₅₀, were 66.9 ± 1.5 µM (MCF-10A), 30.8 ± 2.2 µM (MCF-7), 34.7 ± 1.4 µM (MDA-MB-453), 27.5 ± 1.1 µM (MDA-MB-231), respectively (Fig. 1B). Moreover, the data also indicated that as compared with cisplatin, Flavokavain C is more cytotoxic to MDA-MB-231, but is less cytotoxic to human normal MCF-10A cells (Fig. 1B). Additionally, Flavokavain C’s capacity to impede the development of the cells (MCF-7, MDA-MB-231 and MDA-MB-453) was tested by colony formation assay. Flavokavain C significantly suppressed the formation of the three cell colonies, and its effects appeared to be concentration-dependent (Figs. 1C–F). The above results suggested that the breast cancer cells’ viability and growth were inhibited considerably by Flavokavain C.

Fig. 1. Breast Cancer Cells’ Ability to Reproduce and Form Colonies Was Hindered by Flavokavain C

(A) The molecular structure of Flavokavain C. (B) Flavokavain C’s IC₅₀ values in MCF-10A, MCF-7, MDA-MB-453, and MDA-MB-231. The IC₅₀ values were obtained by MTT testing. Cells were treated with cisplatin or Flavokavain C for 48 h. (C) Representative pictures of the cells (MCF-7, MDA-MB-231 and MDA-MB-453) used in the colony formation assay. (D–F) Quantification of C. The values represented the mean of at least three different investigations, or mean standard ± deviation (S.D.). Two-tailed t-tests on unpaired students was chosen to establish statistical obvious (ns = not significant is p > 0.05; ** p < 0.01, *** p < 0.001 vs. the control group).

Flavokavain C Slows Down the DNA Replication Ratio and Causes Arrest of the Cell Cycle in the G2/M Phase

There is no doubt that the DNA replication ratio positively correlates with cell proliferation.^16,17) Using an EdU staining technique, we looked more closely at DNA replication in treating cells (MCF-7, MDA-MB-231 and MDA-MB-453) with Flavokavain C. Compared with the untreated (control) group, the results exhibited that Flavokavain C administration dramatically decreased the DNA replication proportion of the three cells (Figs. 2A, C, E). Quantitative studies showed that Flavokavain C inhibited DNA replication in a dose-dependent manner (Figs. 2B, D, F).

Fig. 2. Breast Cancer Cells (MCF-7, MDA-MB-231, and MDA-MB-453)’ DNA Replication Was Slowed by the Flavokavain C

(A) Typical pictures of the EdU staining experiment in MCF-7 cells. When Flavokavain C or 0.01% DMSO (Control) was used at the recommended amounts for 48 h, cells were employed in an EdU staining test. (B) Quantification of A. (C) Similar to A. However, MDA-MB-231 cells were used instead. (D) Quantification of C. (E) Similar to A. However, MDA-MB-453 cells were used instead. (F) Quantification of E. The values represented the mean of at least three different investigations, or mean ± S.D. Two-tailed t-tests on unpaired students was chosen to establish statistical obvious (ns = not significant is p > 0.05; * p < 0.05; ** p < 0.01 vs. the control group).

DNA replication would affect cell cycle progression.¹⁸⁾ Flow cytometry analysis confirmed that 20.0 µM Flavokavain C administration strongly triggered the three cells to undergo G2/M phase arrest with the proportion of G0/G1 cells reduction. (Figs. 3A, B). Treatment with 20.0 µM Flavokavain C consistently resulted in a considerable reduction in the amount of CDC2 and cyclin B1, which proteins link to the G2/M phase, according to a Western blotting test (Figs. 3C–I).

Fig. 3. Cells (MCF-7, MDA-MB-231 and MDA-MB-453) Had Cell Cycle G2/M Phase Arrest Due to Flavokavain C

(A) Studying the cell cycle of the cells (MCF-7, MDA-MB-231 and MDA-MB-453) treatment of Flavokavain C. Following a 48-h exposure to Flavokavain C or 0.01% DMSO (Control), all cells were examined using flow cytometry to track the various cell cycle phases. (B) Quantification of A. (C) Cyclin B1 and CDC2 proteins of MCF-7, MDA-MB-231 and MDA-MB-453 cells were evaluated by Western blot after treatment of Flavokavain C or 0.01% DMSO (Control) (48 h), respectively. The loading control used was GAPDH. (D–I) Quantification of C. The values represented the mean of at least three different investigations, or mean ± S.D. Two-tailed t-tests on unpaired students was chosen to establish statistical obvious (ns = not significant is p > 0.05; * p < 0.05; ** p < 0.01 vs. the control group).

Apoptosis Is Induced by Flavokavain C

Cell apoptosis could occur due to cell cycle arrest.¹⁹⁾ Following Flavokavain C therapy, a flow cytometric apoptosis assay was used to learn more about what happened to cells (MCF-7, MDA-MB-231 and MDA-MB-453). Flavokavain C treatment caused the three cells to exhibit a considerable improvement in apoptotic cells, particularly at 20.0 µM (Figs. 4A–D). Finally, we used a Western blot assay with antibodies against cleaved-poly(ADP-ribose)polymerase (PARP) and cleaved-caspase 3 to evaluate these proteins levels in the three cells that were treated with Flavokavain C. Apoptosis in cells is often tracked by looking for signatures such as cleaved-PARP or cleaved-caspase 3.^20,21) Using 20.0 µM Flavokavain C, we discovered that the three cells had higher cleaved-PARP and cleaved-caspase 3 protein levels. In contrast, the caspase 3 protein expression was unaffected (Figs. 4E–G). Quantitative research revealed that Flavokavain C administration induced a concentration-dependent rise in cleaved-PARP and cleaved-caspase 3/caspase 3 protein levels (Figs. 4H, I). Our findings suggest that Flavokavain C caused apoptosis in breast cancer cells by elevating the apoptosis-related protein expressions such as cleaved-PARP and cleaved-caspase 3 protein.

Fig. 4. Cells (MCF-7, MDA-MB-231 and MDA-MB-453) Underwent Cell Death Due to Flavokavain C

(A) Using the Annexin V/PI staining assay, Flavokavain C caused the cell death of the cells. (B–D) Quantification of A. (E–G) To ascertain the expression of Cleaved-Caspase 3, Cleaved-PARP, and Caspase 3 proteins in the cells after treatment with Flavokavain C at the appropriate doses (48 h), a Western blot was chosen. The loading control utilized was GAPDH. (H) and (I) Quantification of E–G. The values represented the mean of at least three different investigations, or mean ± S.D. Two-tailed t-tests on unpaired students was chosen to establish statistical obvious (ns = not significant is p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001 vs. the control group).

Flavokavain C Induces Severe DNA Damage and Prompts a Robust DNA Damage Response

When cells experience DNA damage, the cell cycle checkpoints are triggered, and in some cases, apoptosis is triggered.²²⁾ As a result, DNA damages in cells (MCF-7, MDA-MB-231 and MDA-MB-453) were measured by a comet test to determine the effect of Flavokavain C. Flavokavain C caused extensive DNA damage in the three cells, as predicted, with DNA fragments exhibiting a tail pattern during electrophoresis (Fig. 5A). Quantitative research revealed that 20% or more of cells, most notably Flavokavain C-treated MDA-MB-231 cells, had increased DSB levels compared to the control (5% tail DNA signal) (Figs. 5B, C). The results also demonstrated a concentration-dependent relationship between Flavokavain C and DNA damage (Fig. 5).

Fig. 5. In Cells (MCF-7, MDA-MB-231 and MDA-MB-453), Flavokavain C Caused DNA Damage

(A) The DNA damage level was evaluated using the alkaline comet assay after Flavokavain C treatment, or no treatment was applied to the cells. The cells were treated with either flavokavain C or 0.01% DMSO (Control) and grown for 48 h. (B, C) Measurements were made of the percentages of tail DNA in the cells treated with Flavokavain C (B) and the quantity of the cells treated with Flavokavain C that contained >10% tail DNA (C). Five hundred or more cells from each group were analyzed following 48 h of treatment with the cited Flavokavain C concentrations (5.0, 10.0, or 20.0 µM) or 0.01% DMSO (Control). The values represented the mean of at least three different investigations, or mean ± S.D. Two-tailed t-tests on unpaired students was chosen to establish statistical obvious (*** p < 0.001 vs. the control group).

Therefore, an IF test was carried out using an antibody against p53-binding protein 1 (53BP1), a key regulator in response to DNA damage.^23,24) Using an IF test, researchers discovered that treating the three cells (MCF-7, MDA-MB-231 and MDA-MB-453) with Flavokavain C dramatically enhanced the frequency of 53BP1 foci (Fig. 6A). A quantitative study indicated that Flavokavain C administration led to a concentration-dependent rise in the number of 53BP1 foci (Figs. 6B–D). Results were in agreement with those from a Western blot experiment, which indicated a dose-dependent improvement in the γ-H2AX protein amount (a DNA double-strand break indicator) in cells treated with Flavokavain C²⁵⁾ (Figs. 6E–H).

Fig. 6. In Cells (MCF-7, MDA-MB-231 and MDA-MB-453), Flavokavain C Induced a DNA Damage Response

(A) An immunofluorescence test was used to assess the buildup of nuclear foci that are colored green by 53BP1 and are a marker of the DNA damage response. With DAPI, the nuclei were stained (blue). (B–D) Quantification of A. About 200 cells from each group were evaluated after 48 h of treatment with the appropriate concentration of Flavokavain C (5.0, 10.0, or 20.0 µM) or 0.01% DMSO (Control). (E) The expression of γ-H2AX protein levels was evaluated using a Western blot. Before being employed, cells underwent a 48-h treatment with either 0.01% DMSO (Control) or Flavokavain C at the stated concentrations (5.0, 10.0, or 20.0 µM). (F–H) Quantification of E. The values represented the mean of at least three different investigations, or mean ± S.D. Two-tailed t-tests on unpaired students was chosen to establish statistical obvious (ns = not significant is p > 0.05; * p < 0.05; ** p < 0.01; *** p < 0.001 vs. the control group).

Moreover, agarose gel electrophoresis was performed further to determine the interaction between Flavokavain C and DNA. The results indicated that Flavokavain C treatment increased the amount of Form II of DNA in a concentration-dependent manner (Figs. 7A, B), demonstrating Flavokavain C might induce DNA damage by cleaving DNA directly. Additionally, the results from molecular docking study suggested that the entire Flavokavain C molecule lodges inside the major groove of double-stranded DNA with several hydrogen bonds predicted between Flavokavain C and the surrounding nucleotides in the drug binding pocket (Fig. 7C). The carbonyl oxygen and oxygen atom on the phosphodiester bond of DT-8 (thymine) form two hydrogen bonds with the two hydrogen atoms on the methoxy group of Flavokavain C, respectively. And the carbonyl oxygen on the phosphodiester bond in DC-9 (cytosine) was engaged in a hydrogen bond with the hydrogen atom on methoxy of Flavokavain C. In addition, the carbonyl oxygen atom in DG-10 (guanine) also interacted, by hydrogen bonding, with the phenolic group of Flavokavain C.

Fig. 7. Flavokavain C Interacted with DNA and Induced pBR322 DNA Cleavage

(A) Agarose gel electrophoresis image of pBR322 plasmid DNA incubated with Flavokavain C at the indicated concentrations (5.0, 10.0, 20.0 µM) at 37 °C for 2 h. DNA incubated with 0.01% DMSO as control. (B) Quantification of A. (C) The binding modes of Flavokavain C with the major groove of double-strand DNA. The values represented the mean of at least three different investigations, or mean ± S.D. Two-tailed t-tests on unpaired students was chosen to establish statistical obvious (** p < 0.01; *** p < 0.001; ^## p < 0.01; ^### p < 0.001 vs. the control group).

These results provided more evidence in favor of the hypothesis that Flavokavain C treatment caused extensive DNA damage and induced a strong DNA damage response by directly interacting with DNA.

DISCUSSION

Tumor cells tend to multiply uncontrolled and avoid the cell death program.²⁶⁾ Consequently, a substance with the ability to stop the growth of tumor cells while also causing them to die has the potential to be utilized to cure cancer. A natural chalcone called Flavokavain C, which is generated from the root of the kava plant, has been demonstrated to suppress the development of cancer cells and cause apoptosis in colorectal cancer.^14,27) Flavokavain C’s anticancer effects on several human tumor cells, notably breast cancer cells, remain unknown. This study was carried out to investigate Flavokavain C’s anticancer properties on breast cancer cells. Firstly, our data demonstrated that compared to cisplatin, Flavokavain C exhibited a comparable or stronger inhibitory effect on breast cancer cell viability while having lower cytotoxicity on human mammary cells MCF-10A (Fig. 1B), demonstrating that Flavokavain C might be a potential drug in breast cancer treatment.

It is well established that the classification of breast cancer primarily determines clinical treatment strategies, therapeutic drugs, and outcomes.^28,29) While the efficient and specific diagnosis of breast cancer has been desperately lacking.^30,31) and clinical breast cancer is always composed of multiple types.^7,9) Consequently, the diversification of breast cancer classification is responsible for breast cancer treatment challenges.²⁾ Thus, developing a drug possessing a pan-anticancer activity of all types of breast cancer cells would be extremely valuable. Our findings demonstrated that Flavokavain C suppressed breast cancer cell’s ability to divide and create clones, regardless of their hormonal condition (Figs. 1C–F), further demonstrating that Flavokavain C had the potential in clinical breast cancer treatment. Our results suggested that Flavokavain C treatment slowed down cells (MDA-MB-231, MDA-MB-453 and MCF-7)’s DNA replication rates (Fig. 2), down-regulated the cell check-point protein expression, causing the G2/M phase of the cell cycle (Fig. 3), ultimately led to cell apoptosis (Fig. 4). Together, our findings indicated that DNA might have a part in the anticancer impact of Flavokavain C on breast cancer cells. Previous research has shown that chalcones frequently interact with DNA to cause DNA damage and elicit a reaction, which is how they typically exercise their anticancer impact.^32,33) Our findings also exhibited that the cells (MCF-7, MDA-MB-231 and MDA-MB-453) experienced significant DNA damage (Figs. 5, 6), indicating that Flavokavain C likely targeted DNA to have its anticancer effects on these cells. Mechanically, Flavokavain C would directly interact with the major groove of double-stranded DNA via several hydrogen bonds and then cleaved DNA efficiently (Fig. 7). These findings showed that Flavokavain C hindered breast cancer cells’ capacity to form colonies and replicate their DNA, which caused the arrest of the cell cycle and apoptosis by inflicting severe DNA damage. These results prove that Flavokavain C has potential as a future treatment for breast cancer and improves understanding of the substance’s biological functions.

Acknowledgments

The Natural Science Foundation of Zhejiang Province (LY20H160010) provided funding for this project.

Conflict of Interest

The authors declare no conflict of interest.

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