Damnacanthal Suppresses Breast Cancer Cells by Inducing Apoptosis and Cell Cycle Arrest via NF-ĸB Signaling.

1
Introduction
Breast cancer is a commonly diagnosed cancer that causes a high death rate in women all over the world [1]. Several lines of evidence have been reported on various signaling pathways associated with breast cancer progression. Nuclear factor kappa B (NF‐ĸB) is a superfamily of proinflammatory transcription factors that are often activated in breast cancer. Inhibition of NF‐ĸB leads to an increase in the sensitivity to apoptosis mediated by chemotherapeutic agents and restores hormone sensitivity in cancer cells [2]. NF‐ĸB comprises five subunits, including NF‐ĸB1 (p50), NF‐ĸB2 (p52), RelA protein (p65), Rel B protein, and C‐Rel [3]. These homologous and/or heterologous dimers can bind to specific sequences of target genes to regulate gene transcription, including genes involved in inhibiting apoptosis, interacting with cell cycle regulation, promoting cell invasion, thereby contributing to tumorigenesis and inflammation, and supporting metastatic growth and chemoresistance [4, 5]. In the canonical pathway, NF‐ĸB heterodimer typically consists of p65 and p50 proteins. Once activated, NF‐ĸB translocates to the nucleus, where it binds to specific sequences and regulates gene transcription [6, 7].
Epidermal growth factor receptor (EGFR) is a tyrosine kinase receptor. Overexpression of EGFR in breast cancer is associated with poor clinical outcomes and increased tumor progression [8, 9]. Activation of EGFR can trigger several signaling cascades, including the PI3K/AKT, Ras–Raf‐MAPK, and JNK pathways. The PI3K/AKT pathway is one of the major signaling pathways downstream of EGFR and plays an important role in the cell cycle and cell proliferation, which leads to breast cancer progression [9, 10, 11].
Phytochemicals have received considerable attention as natural alternative substances for cancer treatment and prevention.
Morinda citrifolia
L., commonly known as noni, belongs to the Rubiaceae family and has traditionally been used for various medicinal purposes in Thailand and other Southeast Asian countries.
Damnacanthal (3‐hydroxy‐1‐methoxy‐anthraquinone‐2‐aldehyde), a natural anthraquinone compound initially isolated from the roots of M. citrifolia (Figure 1), has been reported to target various molecular targets associated with tumor inhibition. The capability of damnacanthal to decrease cell growth has been reported among various cancer cells, including human adenocarcinoma cells (HT‐29), prostate cancer (PC‐3), oral squamous cell carcinoma (H400), hepatocarcinoma cells (Hep G2), and breast cancer cell line (MCF‐7) [12, 13, 14, 15, 16]. Damcanthal exhibited anti‐cancer properties via different mechanisms, including inducing apoptosis, inhibiting cell migration, reducing inflammation, acting as a tyrosine kinase inhibitor and Ras inhibitor, and showing promising immunomodulatory potential [12, 13, 14, 15, 16, 17, 18, 19, 20].
Damnacanthal treatment exhibited as an inducer of the tumor suppressor protein, non‐steroidal anti‐inflammatory drug‐activated gene (NAG‐1) [12]. It also increased apoptosis and caspase 3/7 activity, reduced cyclin D1 expression [13], upregulated Bax, p53, and p21 protein expression levels, and induced caspase‐3/9 activity [16], decreased the phosphorylation level of AKT, and targeted matrix metalloproteinase‐2 secretion [15]. Moreover, damnacanthal demonstrated strong inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn, and EGFRs [17], suppressing cell migration and invasion primarily through the inhibition of LIM kinase activity [18]. The inhibitory effect of damnacanthal on the NF‐ĸB signaling pathway in melanoma and mast cells has been reported [19, 20]. However, its impact on the NF‐ĸB signaling pathway in breast cancer remains unexplored. This study examined the molecular mechanisms underlying damnacanthal's antitumorigenic effect, including mRNA expression of RELA (NF‐ĸB subunit 65), apoptosis‐related protein expression, and the EGFR/PI3K/AKT signaling pathway. The antitumorigenic effect of damnacanthal on cell proliferation, apoptosis, and cell cycle was also investigated.

2
Materials and Methods
2.1
Chemicals and Reagents
Damnacanthal or 3‐hydroxy‐1‐methoxy‐anthraquinone‐2‐aldehyde was purchased from Calbiochem (Merck, Darmstadt, Germany). The compound was dissolved in 0.1% dimethylsulfoxide (DMSO, Sigma, St. Louis, MO, USA). Minimum Essential Medium (MEM), phosphate buffer saline (PBS), 0.25% trypsin–EDTA solution, fetal bovine serum (FBS), penicillin/streptomycin solution, and other tissue culture reagents were purchased from Gibco (Grand Island, NY).

2.2
Cell Culture
Human breast carcinoma MCF‐7 (ATCC, Cat# HTB‐22, RRID: CVCL_0031) and MDA‐MB‐231 (ATCC, Cat# HTB‐26, RRID: CVCL_0062) were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). The cell lines were contamination‐free before use (microscopic observation and mycoplasma screening) and were maintained in MEM supplemented with 10% FBS, 2 mM l‐glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin in a humidified incubator at 37°C and 5% CO2.

2.3
Cell Viability
The effect of damnacanthal on the cell viability of cell lines was determined using a 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay. The cell lines were seeded on a 96‐well microtiter plate with a density of 9 × 103 cells/mL. Various concentration dilutions of compounds were 6.25, 12.5, 25, 50, and 100 μM, and the vehicle control contains 0.1% DMSO. After exposure to damnacanthal for 24, 48, and 72 h, respectively, MTT solutions (5 mg/mL) were added to each well, and cells were incubated for 4 h. The formazan crystals were dissolved in DMSO, and the absorbance of the samples was measured at 562 nm by a microplate reader (Thermo Fisher Scientific, Marietta, OH, USA). The cell viability of treatment groups was calculated as a percentage compared to the control group (calculated by the formula: Light absorption value in experimental samples/light absorption value in experimental samples ×100%). IC50 was calculated using the SPSS program.

2.4
qRT‐PCR Analysis
Cells were treated with 25, 50, and 75 μM of damnacanthal for 48 h. Total RNA was isolated from cells using TRIzol. The amount of isolated RNA was quantified by NanoDrop. The RNA was converted to cDNA by using the iScript cDNA Synthesis Kit (Bio‐Rad, Hercules, CA, USA). The amplification was performed utilizing the ABI PRISM7500 Sequence Detection System and analytical software (Applied Biosystems, Carlsbad, CA, USA). Melting curve analysis was performed to detect the amplicon by using SYBR green dye. The iScript reverse transcription supermix and iTaq universal SYBR green supermix were obtained from Bio‐Rad (Hercules, CA, USA). To provide quantification, the amplification plot was investigated at a point during the log phase of product accumulation by assigning a fluorescence threshold above the background, defined as the threshold cycle (Ct) number. The relative mRNA expression ratio was normalized with GAPDH, the internal control, and was calculated by using the equation: 2(ΔCt sample‐ ΔCt control). All primer sequences were designed using NCBI/Primer‐Blast.

2.5
Apoptosis Detection
Cells were treated with 25, 50, and 75 μM of damnacanthal for 48 h. After washing and harvesting, the cells were mixed with the binding buffer. Then, annexin V and PI from the Annexin V‐FITC apoptosis detection kit (BD Biosciences, San Jose, CA, USA) were added to the cells and incubated for 15 min. Afterward, stained cells were added and mixed with 500 μL of 1× binding buffer, and the apoptosis of the cells was analyzed by BD FACSCanto (BD Bioscience, Becton and Dickinson) with BD FACSDiva software (BD Bioscience, San Jose, CA). Results were revealed as the percentage of apoptotic cells from total cells.

2.6
Cell Cycle Analysis
The breast cancer cell lines were treated with control, 25, 50, and 75 μM of damnacanthal concentration for 48 h. After that, cells were washed with PBS and then fixed with 70% ethanol at −20°C overnight. Fixed cells were washed with PBS before being stained with 500 μL PI/RNase staining buffer in the dark for 15 min at room temperature. Cellular DNA content was quantified by the fluorescence of propidium iodide‐stained DNA with BD FACSCanto and using BD FACSDiva software, which was used for the analysis of the percentage of cells in the different phases of the cell cycle. PI/RNase staining buffer was purchased from BD Pharmingen (Indianapolis, IN).

2.7
Western Blot Analysis
MCF‐7 and MDA‐MB‐231 cells were treated with control, 25, 50, and 75 μM of damnacanthal concentration for 48 h. Cell pellets were lysed with RIPA buffer containing protein inhibitors. The concentrations of proteins were determined by the BCA Protein Assay Kit (Bio‐Rad, USA). Samples were separated by 10% Sodium Dodecyl Sulfate‐PolyAcrylamide Gel Electrophoresis (SDS‐PAGE) and transferred to the PVDF membrane (Bio‐Rad, USA). The membranes were incubated with primary antibodies (Bio‐Rad, USA), including EGFR (1:500), p‐EGFR (1:200), PI3K (1:1000), PTEN (1:1000), AKT (1:1000), p‐AKT (1:200), mTOR (1:1000), p‐mTOR (1:1000), Bax (1:1000), Bcl‐2 (1:1000), and β‐actin antiserum (1:5000) and incubated with the secondary antibody horseradish peroxidase (HRP). The membranes were added with an enhanced chemiluminescent reagent (ECL). Protein bands were quantified using Quantity One software (Bio‐Rad Laboratories Inc.) relative to β‐actin, the control for the loading and transfer.
BCA‐protein assay kit, protease inhibitor, polyvinylidene difluoride (PVDF) membrane, nonfat dry milk, and precision plus protein dual color standard were purchased from Bio‐Rad (Hercules, CA, USA). Bovine serum albumin (BSA) was obtained from Sigma‐Aldrich (St. Louis, MO, USA). A phosphatase inhibitor was purchased from Roche Diagnostics (Mannheim, Germany). Antibody including PI3 Kinase p100α rabbit mAb, p‐AKT (S473) rabbit pAb, AKT rabbit pAb, p‐EGFR, p‐mTOR rabbit pAb, mTOR rabbit pAb, β‐actin, and secondary antibody horseradish peroxidase (HRP), were purchased from Cell Signaling Technology (Danvers, MA, USA). EGFR mouse mAb was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Enhanced chemiluminescent reagent (ECL) was purchased from Millipore Corporation (Waltham, MA, US).

2.8
Statistical Analysis
All experimental data were presented as mean ± standard error of the mean (SEM). Statistical analyses were performed by using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA, USA), followed by ANOVA, and p < 0.05 was significant.

2.9
Nomenclature of Targets and Ligands
Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY, and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20.

3
Results
3.1
Damnacanthal Inhibited the Cell Viability of MCF‐7 and MDA‐MB‐231 Cells
After treatment with damnacanthal for 48 and 72 h, the cell viability was reduced (Figure 2A–C). The IC50 of damnacanthal on MCF‐7 cell viability was 100.7 μM and 60.15 μM at 48 and 72 h, respectively. In MDA‐MB‐231 cells, cell viability decreased following treatment with damnacanthal, with IC50 values of 53.67 μM at 48 h and 41.69 μM at 72 h (Figure 2D–F). Therefore, the suppressive effects of damnacanthal on cell viability were time‐ and concentration‐dependent.

3.2
Damnacanthal Downregulates RelA Gene Expression in MCF‐7 and MDA‐MB‐231 Cells
The activation of the RelA gene that encodes NF‐ĸB subunit 65 contributes to tumorigenesis [21, 22, 23]. To determine the effect of damnacanthal on the NF‐ĸB pathway in its transcription level, the mRNA expressions of RELA in MCF‐7 and MDA‐MB‐231 cells were determined by qRT‐PCR. In MCF‐7, damnacanthal significantly reduced the expression of the RELA gene at 50 and 75 μM (Figure 3A). In MDA‐MB‐231, results revealed that damnacanthal reduced the expression of the RELA gene at 75 μM (Figure 3B), indicating that damnacanthal exerts an inhibitory effect on RelA by blocking its transcription.
Since damnacanthal more effectively reduced the RelA gene in MCF‐7 compared to MDA‐MB‐231, NF‐ĸB signaling pathway molecules were also investigated. To determine the effect of damnacanthal on TNFα, tumor necrosis factor‐alpha, and IĸBα, nuclear factor of kappa light polypeptide gene enhancer in B‐cells inhibitor alpha in their transcription level, the mRNA expression of TNF‐α and IĸBα in MCF‐7 cells was determined by qRT‐PCR. At 48 h, damnacanthal upregulated TNF‐α gene expression at concentrations of 50 and 75 μM in MCF‐7 cells (Figure 3C). However, the expression of IĸBα was not changed by the effect of damnacanthal when compared with the control (Figure 3D).

3.3
Damnacanthal Induced Cell Apoptosis in MCF‐7 Cells
NF‐ĸB homologous and/or heterologous dimers can inhibit apoptosis and the cell cycle regulation process [4, 5]. To investigate the ability of damnacanthal to regulate cell apoptosis in MCF‐7 cells, cells were treated with 25, 50, and 75 μM of damnacanthal for 48 h and analyzed by flow cytometer with Annexin V/PI staining (Figure 4A–D). Upon exposure to 50 and 75 μM damnacanthal, the percentage of live cells was significantly reduced compared to the control group. The proportion of late apoptotic cells increased following treatment with 50 and 100 μM damnacanthal. Furthermore, damnacanthal at a concentration of 75 μM significantly increased the percentage of necrotic cells (Figure 4E).

3.4
Cell Cycle Arrest at G0/G1 and S Phase in MCF‐7 Cells Was Induced by Damnacanthal
The stimulation of NF‐ĸB in breast cancer has been reported to upregulate the expression of cyclin D1 and cyclin‐dependent kinase 2 (CDK2), leading to activation of tumor growth, cell cycle progression, and cell proliferation [24, 25, 26, 27, 28]. To investigate the ability of damnacanthal to regulate the cell cycle in MCF‐7 cells, the cells were treated with damnacanthal and analyzed by flow cytometry with PI staining (Figure 5A–D). The percentage of the G0/G1 phase was significantly increased at 50 and 75 μM compared to the control group. The percentage of the S phase was also significantly increased at 25, 50, and 75 μM (Figure 5E).

3.5
The Protein Expression of the PI3K/AKT/mTOR Signaling Pathway in MCF‐7 Was Not Suppressed by Damnacanthal
The activation of PI3K‐110α leads to the phosphorylation of the downstream NF‐ĸB subunit p65 (RelA), thereby allowing NF‐ĸB translocation into the nucleus [28, 29]. Therefore, NF‐ĸB and PI3K/AKT/mTOR signaling pathways can be linked to increased tumorigenic processes [29, 30]. To investigate the ability of damnacanthal on the PI3K/AKT/mTOR pathway in MCF‐7 cells, the protein expressions of PI3K (p110α), p‐AKT (Ser473), AKT, PTEN, p‐mTOR, and mTOR were examined by western blotting. The cells were treated with 25, 50, and 75 μM of damnacanthal for 48 h. The results showed that damnacanthal did not change the protein expression of PI3K and PTEN compared to the control group. Moreover, damnacanthal unchanged AKT and mTOR in both phosphorylated forms and total protein levels (Figure 6A,B).

3.6
Damnacanthal Unaltered Cell Cycle Arrest in MDA‐MB‐231 Cells
To investigate the ability of damnacanthal to regulate the cell cycle in MDA‐MB‐231 cells, cells were treated with 25, 50, and 75 μM of damnacanthal for 48 h compared with the control. The cells were stained with propidium iodide (PI) and analyzed by flow cytometry (Figure 7A–D). After treatment for 48 h with increasing concentrations of damnacanthal, the percentage of the cell cycle phase was not changed when compared with the control group (Figure 7E).

3.7
The Protein Expression of the EGFR/PI3K/AKT Pathway Was Not Suppressed by Damnacanthal in MDA‐MB‐231 Cells
To investigate the ability of damnacanthal on the EGFR/PI3K/AKT pathway in MDA‐MB‐231 cells, the protein expression of EGFR, p‐EGFR (y1045), PI3K (p110α), p‐AKT (Ser473), and AKT was examined by western blotting. The cells were treated with 25, 50, and 75 μM of damnacanthal for 48 h. The results showed that damnacanthal did not change the protein expression of EGFR in both phosphorylated forms and total protein compared to the control group (Figure 8A–F). Moreover, damnacanthal also did not affect the protein expression of PI3K and AKT in both phosphorylated forms and total protein levels.

3.8
The Protein Expression of Anti‐Apoptotic Bcl‐2 and Pro‐Apoptotic Bax Was Not Regulated by Damnacanthal
The expression of Bax and Bcl‐2 is associated with the regulation of cell apoptosis in cancer cells [31, 32]. To investigate the ability of damnacanthal on Bax and Bcl‐2 in MCF‐7 and MDA‐MB‐231 cells, the protein expression of Bax and Bcl‐2 was examined by western blotting. The cells were treated with 25, 50, and 75 μM of damnacanthal for 48 h. The results showed that damnacanthal did not change the protein expression of Bax and Bcl‐2 compared to the control group in both MCF‐7 (Figure 9A–C) and MDA‐MB‐231 cells (Figure 9D–F).

4
Discussion
Anthracyclines and other anthraquinone chemicals have long been utilized as potent anticancer medications [33, 34]. Anthraquinones, which include groove binders, alkylating, and intercalator chemicals, have been shown as DNA‐recognizing molecules. Anthraquinone's tricyclic structure has been extensively studied for tumorigenic therapy and is crucial to its cytotoxic properties [35, 36, 37]. An anthraquinone chemical called damnacanthal has the potential to decrease the viability of numerous cancer cell lines [12, 13, 14, 15, 16]. Our findings verified that damnacanthal can stop MCF‐7 and MDA‐MB‐231 cells from growing. The MTT assay results from a previous study showed that damnacanthal, which was obtained from the Faculty of Applied Sciences, Mara University of Technology (Shah Alam, Malaysia), could inhibit the growth of MCF‐7 cells at a concentration of 8.2 μg/mL for 72 h [16] and that it significantly decreased after three days at 50 μM [13]. Damnacanthal, which was purchased from Calbiochem and partially dissolved with DMSO, has an IC50 of 60.15 μM on MCF‐7 after 72 h in this experiment. Additionally, the damnacanthal demonstrated the capacity to inhibit the MDA‐MB‐231 cell line's proliferation. As a result, damnacanthal could reduce cell viability in the breast cancer cell lines in a time‐ and concentration‐dependent manner.
Treatment with damnacanthal has been shown to suppress NF‐ĸB protein expression in melanoma and mast cells [19, 20]. However, little is known about how damnacanthal affects the NF‐ĸB signaling pathway in the breast cancer cell line. According to our findings, damnacanthal significantly reduced the expression of the RELA gene, NF‐ĸB (subunit 65). Nevertheless, our findings suggested that damnacanthal may suppress breast cancer cells at the mRNA level through NF‐ĸB (subunit 65). Research has been done on the effects of NF‐ĸB (subunit 65) on breast cancer. In the instance of human breast cancer, 89.3% of tumors more than 5 cm in size and 86.9% of high‐grade (Grade III) tumors activated p65 [38]. Prior studies have demonstrated that NF‐ĸB plays a role in both initiating and sustaining epithelial‐to‐mesenchymal transition (EMT) in the process of breast cancer progression [39, 40]. As a transcriptional regulator of EMT transcription factors, including SLUG, SIP1, and TWIST1, NF‐ĸB/p65 is crucial [41]. In human breast cancer cell lines, MDA‐MB‐231 and HCC‐1954, inhibition of NF‐ĸB/p65 reduced migration and invasion [42].
One mechanism associated with chemoresistance in breast cancer cells is the activation of NF‐ĸB. Resistance to endocrine and chemotherapies is linked to NF‐ĸB activation in cancers that express estrogen receptor [43, 44]. A cohort study reported that patients with negative NF‐ĸB expression were more likely to respond to chemotherapy compared to those with positive NF‐ĸB expression [45]. To overcome tumorigenesis and chemoresistance, NF‐ĸB is a crucial therapeutic target in the setting of breast cancer.
NF‐ĸB signaling pathway molecules were also examined since damnacanthal more efficiently decreased the RelA gene in MCF‐7 than MDA‐MB‐231. After cytokines like TNFα and interleukin‐1α/β bind to their receptors, an inflammatory stimulation can activate the NF‐ĸB pathway [46]. TNF‐α recruits the death domain (DD) that can lead to the activation of signal transduction pathways that induce apoptosis. The recruitment of TNF‐RI‐associated factors (TRAFs) can lead to the activation of multiple cell survival intracellular signals such as NF‐ĸB, JNK, p38, and Erk [47, 48]. The NF‐κB signaling pathway can be activated by TNFα. However, our findings indicated that damnacanthal increased TNF‐α mRNA expression while decreasing NF‐ĸB mRNA expression. In some situations, increased levels of TNF‐alpha (a pro‐inflammatory cytokine) can lead to a reduced expression of the RelA gene, which is a key component of the NF‐κB signaling pathway. This occurs through various mechanisms. The increase in TNF‐alpha might lead to a feedback mechanism that downregulates RELA expression, potentially as a way to limit excessive NF‐kB activity and prevent overactivation of inflammatory pathways. NF‐ĸB expression may be influenced by other variables such as reactive oxygen species (ROS), interleukin 1‐beta (IL‐1β), growth factors, ultraviolet radiation, and bacterial lipopolysaccharides [49]. No research has investigated the interaction of damnacanthal with NF‐κB and TNF‐α in the same environment. The mechanism of damnacanthal's disruption of the TNF‐α‐enhanced NF‐κB pathway remains unknown.
TNF‐alpha can influence DNA methylation patterns at RelA target genes. While some studies show DNA demethylation at RelA binding sites, other studies suggest that the extent of demethylation can be affected by RelA itself, and thus, a reduced RelA expression could lead to changes in DNA methylation patterns. RELA expression can be controlled by other molecules or signaling pathways [50, 51, 52] while TNF‐α's action can also be modulated by many signaling pathways [53, 54]. To prevent overactivation of inflammatory pathways, a prolonged elevation in TNF‐α can trigger a negative feedback loop that reduces NF‐κB expression [55, 56]. Increased TNF‐α can cause cancer cells to undergo apoptosis in certain situations, but it can also encourage survival and growth in others [57, 58, 59, 60]. TNF‐α can initiate cell death pathways, but it also frequently stimulates NF‐κB. It has the ability to trigger programmed cell death or apoptosis, and this apoptotic signaling can reverse the NF‐κB activation. The cleavage of the death domain kinase resulted in the blockage of TNF‐induced NF‐κB activation, leading to TNF‐induced apoptosis [61]. Inactivation of NF‐κB (p65) in NB4 and MCF7 cells induces autophagy in response to TNFα, and autophagy is one of the mechanisms involved in the control of death in cancer cells [62]. The NF‐κB activation can be reversed by apoptotic signaling [61, 63, 64]. Even when TNFα is present, these conflicting signals may cause NF‐κB activity to decline.
TNF‐α signaling pathways have the ability to initiate apoptosis (programmed cell death). TNF‐α acts as a mediator of the apoptotic process and has selective cytotoxicity against malignant breast tumor cells, promoting an apoptotic type of cell death in MCF‐7 cells [65]. p38/MNK/PML network regulates TNFα‐induced apoptosis in breast cancer cells [54]. Damnacanthal was reported to increase TNF‐α levels by inducing apoptosis in SKHep1 liver cancer cells through the activation of the p38 mitogen‐activated protein kinase (MAPK) pathway. This activation involves the death receptor (DR) pathways, specifically DR5/TRAIL and TNFR1/TNF‐α, ultimately leading to caspase‐8 activation and cell apoptosis [66]. According to our findings, damnacanthal caused MCF‐7 cells to express more TNF‐α gene. Furthermore, our Annexin V‐FITC apoptosis detection assay also demonstrated that damnacanthal administration caused cell apoptosis in MCF‐7. Consequently, it is possible that the elevated TNF‐α in MCF‐7 may have a more significant impact on the apoptotic pathway than it would as an activator of the NF‐ĸB pathway.
Because apoptosis contributes to cell death, it is essential for preventing cancer. According to earlier research findings, damnacanthal caused MCF‐7 cells to undergo apoptosis via increasing proapoptotic gene expression, including p21, caspase‐7, and increased the protein level of p53 (tumor suppressor protein) [16]. Our study's findings were consistent with previous studies and demonstrated that damnacanthal could increase cell apoptosis at late apoptotic and necrosis in a concentration‐dependent pattern. Bax is a pro‐apoptotic protein, whereas Bcl‐2 belongs to the Bcl‐2 family members, which are anti‐apoptotic proteins. Nevertheless, our findings indicated that damnacanthal did not alter the protein expression of Bax and Bcl‐2, representing that damnacanthal induces apoptosis in breast cancer cell lines independently of Bax and Bcl‐2 proteins.
Extrinsic and intrinsic pathways are two major routes of apoptotic processes. The extrinsic signaling pathway is involved with transmembrane death receptor‐mediated interactions [67, 68]. Death receptors such as TNF (tumor necrosis factor), TRAIL (TNF‐related apoptosis‐inducing ligand), and Fas‐L have a death domain that plays a significant role in transmitting the death signal from the cell surface to the intracellular signaling pathways [69, 70]. Meanwhile, the intrinsic pathway is a mitochondrial‐mediated pathway. Other regulatory mechanisms within the cell's apoptosis pathway are probably engaged when apoptosis happens despite no discernible change in Bcl‐2 and Bax levels. While Bcl‐2 and Bax are key players in regulating the mitochondrial pathway of apoptosis, other variables can also cause apoptosis, and their activity is influenced by other routes. The extrinsic signaling pathways are involved. Death receptors such as TNF (tumor necrosis factor). According to experimental studies, treatment with damnacanthal increased TNF‐α and did not alter Bcl‐2 and Bax levels. Therefore, cells may undergo the apoptosis process via the extrinsic signaling pathways rather than the mitochondrial system.
Cyclin D1 is a cell cycle regulator that controls the G1 to S phase of the cell cycle. Damnacanthal was found to induce cell cycle arrest at the G1 checkpoint in MCF‐7 cells and decrease cyclin D1 protein expression [13]. Similarly, our results revealed that the percentage of the G0/G1 phase in MCF‐7 was significantly increased. In the MDA‐MB‐231 cell line, the percentage of the cell cycle phase did not change when compared with the control group. This result was similar to a previous study, which showed that damnacanthal had no significant effect on cell cycle distribution in human breast cancer carcinoma (MDA‐MB‐231) [15].
Damancanthal acts as a multi‐kinase inhibitor, effectively targeting tyrosine kinases (PDGFR, erbB2, EGFR, and insulin receptor) with IC50 values in the micromolar range [17]. Damnacanthal (10 mM) inhibited more than 50% of the in vitro activity of several kinases, including related tyrosine kinases: VEGFR1‐3, FGFR1, 2, and 4, c‐Met, and EGFR [71]. However, the results of protein expression from western blot analysis in this study exhibited that damnacanthal did not change EGFR level and its phosphorylated forms on MDA‐MB‐231 cells. The limitations of computational modeling in docking and in vitro kinase assays, which use purified enzymes and substrates in a controlled environment that may not accurately reflect physiological conditions, This. may cause discrepancies in results because in vitro kinase assay and protein‐ligand docking were used in earlier references to study the inhibition activity of damnacanthal with EGFR [17, 71]. Western blots detect the presence and amounts of proteins, including post‐translational alterations like phosphorylation, in complicated biological materials, while in vitro kinase assay and protein‐ligand docking are useful for initial inhibitor screening. Several factors, including transcription factors, post‐translational modifications, epigenetic modifications, and others, may influence the results produced using the difference technique. These changes may be seen in cellular environments but not in vitro assay.
Pro‐inflammatory cytokines production initiates the PI3K/AKT/mTOR pathway, which is then activated by phosphorylation of the PI3K regulatory subunit p85. This subsequently increases the catalytic activity of PI3K‐110α, phosphorylates the downstream NF‐ĸB subunit p65 (RelA), thus allowing NF‐ĸB translocation into the nucleus [28, 29]. Autophosphorylation of tyrosine kinase receptors, such as EGFR, can lead to triggering the PI3K/AKT signaling pathway, which plays a critical role in breast tumorigenesis. A previous study reported the inhibitory effect of some anthraquinones on the PI3K/AKT pathways in breast cancer cell lines [72, 73, 74]. Damnacanthal was found to act against hepatocellular carcinoma cells by decreasing the phosphorylation level of AKT [15]. On the other hand, this study discovered that damnacanthal had no effect on the expression of PI3K (p110α), AKT, and its phosphorylated form. Consequently, the EGFR/PI3K/AKT signaling pathway in MDA‐MB‐231 cells had no bearing on the reduced cell viability caused by damnacanthal. In the MCF‐7 cells, damnacanthal had no effect on the protein expression of PI3K and PTEN, nor did it alter AKT and mTOR in both phosphorylated and total protein levels. Therefore, the decreased cell viability by damnacanthal was independent of the PI3K/AKT signaling pathway in MCF‐7 cells. Differences in protein expression between cell types are an important feature of cellular biology. Although all cells in an organism have the same DNA, they have different protein profiles due to selective gene expression. Breast cancer cell lines may respond differently from other types of cells. distinct cell lines (hepatocellular vs. breast cancer cells) may express distinct sets of genes, or the same genes at varying levels, depending on their tissue of origin, mutation status, or epigenetic landscape.
In conclusion, damnacanthal exerts anticancer activity on MCF‐7 and MDA‐MB‐231 cell lines via blocking NF‐ĸB (subunit 65) expression at mRNA levels. MCF‐7 is an ideal model to study molecular mechanisms in the estrogen receptor‐positive breast cancer cell line. In MCF‐7 cells, damnacanthal induces late apoptosis and increases cell cycle arrest in the G0/G1 and S phase, decreases RELA gene expression, and enhances TNF‐α gene expression.
MDA‐MB‐231 is a triple‐negative breast cancer cell line that does not present estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2). It is commonly used as a model of triple‐negative breast cancer (TNBC) in medical research laboratories. In MDA‐MB‐231, after treatment for 48 h with increasing concentrations of damnacanthal, the percentage of the cell cycle phase was not changed. The RELA gene expression was decreased; however, damnacanthal more effectively reduced the RelA gene in MCF‐7 compared to MDA‐MB‐231. Therefore, TNFα and IĸBα were mainly determined in MCF‐7 cells.
The inhibition effect of damnacanthal at 48 h on both breast cancer cell lines was PI3K/AKT independent signaling pathway. Nevertheless, additional time points for PCR and proteomics analysis are equally intriguing. Variations in gene expression might happen at different times.
Further experiments can confirm the effects of damnacanthal on post‐transcriptional, post‐translational, and nuclear translocation mechanisms of NF‐ĸB (subunit 65) by using a western blot to detect the protein expression and using automated fluorescent microscopy computer‐assisted image analysis to detect nuclear translocation of NF‐ĸB. Kinase activity assays can be used to further establish the effects of damnacanthal on the activity of phosphorylated protein forms. To better understand the mechanisms, more research may be done on molecules implicated in apoptosis, such as PARP cleavage and caspase 3/7 activation.
An intriguing compound that may be a substitute for NF‐ĸB inhibitor in human breast cancer is damnacanthal. This study provided more information about the mechanism of this phytochemical compound in breast cancer Figure 10.

Author Contributions

Onnichar Jongcharoen: conceptualization, data curation, formal analysis, investigation, methodology, validation, visualization, writing – original draft. Somrudee Reabroi: investigation, resources, validation, writing – review and editing. Pimtip Sanvarinda: formal analysis, investigation, resources. Duangjai Tungmunnithum: formal analysis, methodology, resources, writing – review and editing. Warisara Parichatikanond: conceptualization, formal analysis, methodology, writing – review and editing. Darawan Pinthong: conceptualization, data curation, formal analysis, funding acquisition, methodology, project administration, resources, supervision, writing – review and editing.

Funding
This work was supported by the Science Achievement Scholarship of Thailand (SAST) and the CIF and CNI Grant, Faculty of Science, Mahidol University.

Conflicts of Interest
The authors declare no conflicts of interest.