Chalcone-3 Inhibits the Proliferation of Human Breast Cancer MDA-MB-231 Cell Line

Objective: Chalcone-3 has been shown to be cytotoxic and selective against luminal subtype breast cancer cell lines, which are suspected to occur through the mechanism of epidermal growth factor receptors (EGFR) inhibition. However, the cytotoxic effect has never been tested on cell strains from patients with triple negative breast cancer (TNBC), where EGFR expression is known to increase. This study aimed to identify the role of chalcone-3 in one of the downstream targets of EGFR as an antiproliferative agent. Methods: Chalcone-3 was examined for its effect on proliferation in human breast cancer MDA-MB-231 cell lines. The percentage of proliferation inhibition was analyzed using methyl-thiazol tetrazolium assay. Flow cytometry was used to analyze the population of cell cycle distribution and the expression of cyclin-D1 and pEGFR. Results: Chalcone-3 inhibited the proliferation of MDA-MB-231 cells in a dose and time-dependent manner with an IC50 value of 17.98±6.36 µg/mL by inducing cell cycle arrest at the G2/M phase. Flow cytometry assays showed that chalcone-3 significantly reduced the expression of pEGFR and cyclin-D1, contributing to cell cycle arrest. Conclusion: Chalcone-3 might have potential as an anti-proliferative drug to treat TNBC.


Chalcone-3 Inhibits the Proliferation of Human Breast Cancer MDA-MB-231 Cell Line
targeted therapy, resistance to chemotherapy is also challenging in TNBC treatment (Sharom, 2008;Yamada et al., 2013). Accordingly, new effective treatment strategies are urgently needed. In terms of anticancer pharmacological activity, one of the compounds that is often studied today is the chalcone derivative (Bonakdar et al., 2017;Wang et al., 2016;Dong et al., 2018;Xu et al., 2015;Harmastuti et al., 2012;Zhang et al., 2012;Maioral, et al., 2013;Wankhede et al., 2017). Suma et al. (2019) have synthesized a chalcone derivative with chloro substituent, chalcone 3 or (E) -1-(4-chlorophenyl) -3-(3,4-dimethoxyphenyl) prop-2-en-1-one with high yields (96.69%) and in a short time (65 min.) using the sonication method. The study showed a good anticancer effect on T47D and MCF7 cell lines, with IC 50 of 0.34 and 0.80 µg / mL, respectively. In vitro and in vivo studies on these compounds have not been conducted yet. However, a docking simulation study involving epidermal growth factor receptors (EGFR) showed an interaction between chalcone-3 and EGFR. The interaction occurs through two hydrogen bonds between the carbonyl oxygen atom and the MET769 residue, and between the p-OCH3 oxygen atom and the LYS721 residue. This interaction is thought to cause inactivation of EGFR and inhibit the growth of tumor cells.
EGFR has a very important role in the vital process of tumor cells, including proliferation. EGF activation of the EGFR stimulates many complex intracellular signaling pathways, including RAS/RAF/MEK/ERK and the PI3K/AKT axes (Batzer et al., 1994;Prenzel et al., 2000;Vivanco & Sawyers, 2002). These protein kinases have various cytoplasmic and nuclear targets, which aid in the transcription and translation of cyclin-D1, leading to G1/S cell cycle progression and proliferation (Wee & Wang, 2017;Onodera et al., 2005;Lo et al., 2005). Based on the above research, chalcone-3 is cytotoxic and selective against luminal subtype breast cancer cell lines, which are suspected to occur through the mechanism of EGFR inhibition. However, the cytotoxic effect has never been tested on cell strains from patients with TNBC, where EGFR is frequently overexpressed (Manupati et al., 2017). Therefore, this study aimed to determine the antiproliferative activity of chalcone-3 in MDAMB-231 TNBC cell lines.

Measurement of cell cytotoxicity
The methyl-thiazol tetrazolium (MTT) assay was used to determine the cytotoxicity of chalcone-3 toward MDA-MB-231 cell lines. Cells were seeded in a 96-well plate to a final concentration of 10,000 cells/well and incubated for 24 h (37ᵒC, 5% CO2). The cells were then treated with serial concentrations of chalcone-3 (6.25, 12.5, 25, 50, 100 and 200 μg/mL) and control drugs (doxorubicin and gefitinib) prepared in growth media (0.781, 1.562, 3.125, 6.25, 12.5, 25, and 50 μg/mL), and incubated for an additional 24 h. Next, the medium was removed and fresh medium was added to each well along with 50 µl of MTT reagent (5 mg/mL). Subsequent to incubation at 37°C for 3 h, 150 μl MTT solvent (ab211091) was added to solubilize the MTT-formazan product and mixed thoroughly. The plates were wrapped in foil and kept overnight at room temperature. The spectrometric absorbance at 590 nm was measured using a microplate absorbance reader iMark (Bio-Rad, USA) and converted into the percentage of viable cells by using the following formula: Dose-response curves were generated, and the concentrations of drugs required to inhibit cell viability by 50% (IC 50 ) were calculated using probit regression analysis, with SPSS (IBM Corp., Armonk, NY, USA). Three reduplicative wells were used for each treatment. The IC 50 was calculated for each experiment, and the mean IC 50 s were calculated and presented as ± standard error (SE).

Determination of cell proliferation
The IC 50 evaluation determined from the cytotoxicity assay for each treatment, was used in the cell proliferation assay. Cells were seeded at a density of 10,000 cells/ well in 96-well plates and treated with chalcone-3 for the desired concentrations (1/2xIC 50 , IC 50 , 2xIC 50 , 4xIC 50 ) and time intervals (24, 48, and 72 h). Following incubation, the cell proliferation was analyzed using MTT assay as described above. The percentages of cell proliferation and proliferation inhibition were calculated using the following equations: Three reduplicative wells were used for each treatment and the cell proliferations were calculated and presented as mean ± SE.

Analysis of cell cycle distribution
MDA-MB-231 cells were plated at 5 x 10 5 cells/mL in a six-well plate. After a 24 h incubation (37ᵒC, 5% CO 2 ), the cells were treated with chalcone-3 (1/2xIC 50 , IC 50 , 2xIC 50 , 4xIC 50 ), doxorubicin, and gefitinib, and then incubated for an additional 24 and 48 h. The cell cycle phase evaluation was performed as described by Huang et al., (2017). Following trypsinization, cells were centrifuged at 3,000 x g for 5 min, at room temperature, and the pellet was resuspended in 0.5 mL of phosphate buffered solution (PBS). Cells were fixed in 70% ethanol overnight at 4°C and then treated with a staining PBS containing 1 mg/mL PI and 10 mg/mL RNase A at 37°C in the dark for 30 min. Cells were analyzed to determine the cell cycle stage using flow cytometry (BD FACSCantoTM II, San Jose, CA, USA) with an excitation wavelength of 488 nm and an emission at 670 nm. Percentages of cell populations distributed in the various phases of the cell cycle (sub-G1, G1, S and G2/M) were calculated. subsequent experiments at serial concentration of 1/2xIC 50 , IC 50 , 2xIC 50 , 4xIC 50 and time interval of 24, 48, and 72 h. Thus, the cells were exposed to 9, 18, 36, or 72 μg/mL of chalcone-3 with the desired time intervals. As shown in Figures 2 and 3, chalcone-3 affects cell proliferation even at a half concentration of IC 50 (9 μg/mL). The percentage of inhibition increased significantly to reach about 45.4% at a concentration of 18 μg/mL after 24 h of exposure. By augmenting chalcone-3 concentration to 72 μg/mL, inhibition percentage doubled to reach almost 83% compared to control cells. Chalcone-3 markedly inhibited cell proliferation at 9-72 μg/mL after 24 h of treatment in a dose-dependent manner. Similarly, a marked increase in the percentage of inhibition was detected at 48 h. The percentage of inhibition was about 11% at a half concentration of IC 50 , and enhanced to a five-to eightfold increase by doubling the concentration to 18-72 μg/ mL, respectively. A significant increase in the percentage of inhibition was also observed as time of exposure progressively increases to 72 hours. Chalcone-3 inhibited cell proliferation about 61% at a concentration of 18 μg/ mL, and further increased to 93% inhibition at 72 μg/mL compared to control cells. This inhibitory action was also similar to doxorubicin and gefitinib, where potentiation of proliferation inhibitory was noticeable after 24 h of treatment and exerted the highest effect at 72 h. Therefore, chalcone-3 significantly inhibited cell proliferation in a time and concentration-dependent manner, and appeared to be capable of exerting a proliferation inhibition on MDA-MB-231 cells under the present experimental conditions.

Analysis of cyclin-D1 and pEGFR expression
Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 5 min and then blocked with 1% BSA/10% normal goat serum/0.3 M glycine in 0.1% PBS-Tween for 1 h. The cells were then labeled with Cyclin-D1 (ab16663) at 1/50 dilution or pEGFR (ab223499) at 1:400 dilution in 1%BSA-PBS overnight at 4ᵒC, followed by a further incubation at room temperature for 1 h with a goat secondary antibody to Rabbit IgG (Alexa Fluor 488, ab150077) at 1/2,000 dilution in 23% glycerol, PBS, 1% BSA. Following a 45 min incubation (4°C, in the dark) and washing with PBS, the cells were centrifuged (5 min, 500 × g) to wash off excess antibodies and resuspended in 200 μl of PBS for flow cytometric analysis (BD FACSAriaTM III Cell Sorter, NJ, USA). Percentages of cyclin-D1 or pEGFR positive cells were calculated and the data presented as representative of those obtained in three independent experiments.

Statistical analysis
Data were presented as mean ± SE of at least three independent experiments. Statistical analysis of data was performed by one-way analysis of variance (ANOVA) followed by Bonferroni's or Games-Howell post-hoc multiple comparison test. A value of P <0.05 was considered statistically significantly different.

Chalcone-3 inhibited MDA-MB-231 cell proliferation
MDA-MB-231 cells were subjected to the MTT assay to evaluate the cytotoxicity of chalcone-3 treatment by measuring cell viability. The half-maximal inhibitory concentration (IC 50 ) after 24 hours of treatment with chalcone-3, doxorubicin, and gefitinib were about 17.98±6.36, 9.48±6.10, and 11.41±3.24 µg/mL, respectively ( Figure 1). These values were used in cycle. Tumor cells treated with 9-72 μg/mL chalcone-3 for 24 h showed higher G2/M population (19.8-20.7%) compared to vehicle control (16.4%). The percentages of sub-G1 phase (apoptotic cells) were significantly increased after cells were treated with 36 and 72 μg/mL chalcone-3, up to 7 and 14.4%, respectively, compared with 2% in control. The results of this experiment suggested that chalcone-3 firstly induces G2/M-phase cell cycle arrest even at low concentrations, whereas, chalcone-3 treatment at higher concentrations (36 and 72 μg/mL) induced cell death in MDA-MB-231 cells. Meanwhile, doxorubicin and gefitinib at 24 h cause significant S-phase arrest by increasing its cell population when compared to controls (12, 17.2, and 5.7%, respectively). The number of cells at the G2/M phase was also increased in doxorubicin and gefitinib treatment at 24 h, indicating cell cycle arrest at the G2/M phase. Moreover, the exposure of tumor cells to all treatment groups for 48 h evidently increased the proportion of cells in the Sub-G1 phases, which was accompanied by a significant decrease in cells in the G2/M phase (Figure 4).

Chalcone-3 downregulates cyclin-D1 expression through the inhibition of EGFR activation
Chalcone-3 induced a pronounced inhibition of proliferation and G2/M cell cycle arrest. Therefore, we investigated the expression of cyclin-D1, a crucial protein in cell cycle and proliferation induction. We found that chalcone-3 induces low levels of cyclin-D1 protein expression, starting significantly at 18 μg/ mL chalcone-3 to almost half expression at 72 μg/mL chalcone-3 exposure ( Figure 5). This protein expression was also reduced in doxorubicin and gefitinib treatment when compared to the control. The downstream signal transduction of EGFR is involved in the initiation of cyclin-D1 translation. Thus, EGFR might be involved in  the chalcone-3 induced inhibition of MDA-MB-231 cell proliferation. The activation of this receptor (pEGFR) at the same concentrations was also confirmed by flow cytometry, which was decreased in a dose-dependent manner ( Figure 6). All the treatment groups except doxorubicin showed inhibition of EGFR activation, where gefitinib had the most significant result.

Discussion
Cell proliferation is the increase in the number of cells, through the process of cell division. It is a physiological process and generally increases in pathological conditions such as malignancy (Dang et al., 2003). Therefore, proliferation has emerged as an important mechanism by which chemical compounds may exhibit chemotherapy  The most common proteins to be altered in chalcone antiproliferative studies are the cell cycle regulating proteins (p21, p27, Rb, Chk1 kinase, cdc2, cylin-B1, D, and E, as well as their complementary CDKs) and tubulins (α, α1c, and β tubulin).
T h e e x p o s u r e o f M D A -M B -2 3 1 c e l l s t o growth-suppressive concentrations of chalcone-3 in this study resulted in a significant increase in the proportion of cells in the G2/M and Sub-G1 phases. These findings are consistent with a recent report that chalcone compounds can induce cell cycle arrest and G2/M blockade in MDAMB-231 cells. As Hsu et al., (2006) reported in their previous study, chalcone (1,3-diphenyl-2-propenone) significantly decreased the expression of cyclin-B1, cyclin-A and Cdc2 protein, contributing to cell cycle arrest in the G2/M phase. Another natural chalcone (cardamonin) reported by Kong et al., (2020) was shown to enhance the expression and nuclear translocation of FOXO3a, inducing the expression of FOXO3a and its target genes, including p21 and p27. Furthermore, this process led to the reduction of cyclin-D1 and G2/M arrest. MDA-MB-231 cells treated with synthetic chalcone (ZK-CH-11d) for 24 h were also shown to cause G2/M cell cycle arrest by significantly downregulating phosphorylated cyclin-B1, cyclin-D1, cdc2, Rb and altering the expression of α, α1c tubulin, and β tubulin with a maximum of 72 h after treatment (Michalkova et al., 2022). The same result was reported by Elkhalifa et al., (2020), when using a nitrogen-based chalcone analog (E)-3-(4-(Bis(2-chloroethyl) amino) phenyl)-1-(3-methoxyphenyl) prop-2-en-1-one to treat MBA-MB-231 cells for 48 h. A chalcone-based small molecule, (E)-3-(2-bromo-3,4,5-trimethoxyphenyl)-1-(2,4-dihydroxyphenyl) prop-2-en-1-one (BDP), efficiently induced the degradation of EGFR, Akt, c-Raf, and Cdk4 in MDAMB-231 cells in a dose-and time-dependent manner. These consequently led to cell cycle arrest at the G2/M phases (Oh and Seo, 2017). Taken together, our study and previous studies suggest that chalcone may blockade the cell cycle progression at the G2/M phase due to several mechanisms, involving cell cycle regulatory proteins such as cyclins and CDKs. Doxorubicin and gefitinib were used in this study as a comparison to chalcone-3. Our results suggested that cell cycle arrest is predominantly induced at the G2/M checkpoint in MDA-MB-231 cells treated with doxorubicin or gefitinib for 24 h, although the effect was more evident in gefitinib. Similar results were also reported in previous studies, where MDA-MB-231 cells accumulated largely in the G2/M phase due to reduced expression of CDK1, CDK2, and cyclin-B1 (Oncul and Ercan, 2017;Wei et al., 2020). A significant increase in a sub-G1 fraction was observed in the cells treated with gefitinib for 24 h compared with control (22.8 and 2%, respectively), and maximum at 48 h (55.1 and 1.7%, respectively). Similar results were reported by a previous study, where gefitinib may increase p21 expression, a potent CDK inhibitor, which is necessary for orderly progression through the S phase and G2/M (Okubo et al., 2004).
Cyclin-D1 forms the cyclin-D1-CDK4/6 complex, and phosphorylates the Rb protein. Inactivation of Rb causes the release of Rb binding to E2F, which is a cyclin-E transcription factor, resulting in cell cycle progression from the G1 phase to the S phase (Cobrinik, 2005). Cyclin-D1 expression also increases in the G2/M phase (Yang et al., 2006). Thus, EGFR inactivation of the four signaling pathways above can suppress the cell proliferation process by inhibiting cyclin-D1 transcription and translation, and further cell cycle progression. Present studies showed that chalcone-3 affects the inhibition of cyclin-D1 expression in a dose and time-dependent manner. These results are consistent with the cell cycle arrest at the G2/M phase. The interaction between chalcone-3 and EGFR occurs through two hydrogen bonds between the carbonyl oxygen atom and residue MET769, and between the oxygen atom of p-OCH3 and residue LYS721, located in the tyrosine kinase domain of this receptor. This interaction is thought to cause EGFR inactivation, thereby inhibiting the various processes above and the growth of breast cancer cells. The results of this study indicated an inactivation of EGFR, which is marked by a decrease in pEGFR expression after treatment with serial concentrations of chalcone-3.
In conclusion, results of the present study provided clear evidence that chalcone-3 inhibits the cell proliferation through cell cycle arrest at the G2/M phase of human MDAMB-231. It may be mediated by the inactivation of EGFR and the subsequent signaling target, cyclin-D1. Thus, our findings provide a mechanistic framework for further exploration of the use of chalcone-3 as a promising chemotherapeutic agent for treatment of human breast cancer.

Author Contribution Statement
All authors conceived and designed the study. The first author conducted the experiment, analyzed the data, and wrote the paper. All authors contributed to manuscript revisions, and approved the final version of the manuscript and agreed to be held accountable for the content therein.