Gingerenone A Induces Antiproliferation and Senescence of Breast Cancer Cells

Ginger is a popular spice and consists of several bioactive antioxidant compounds. Gingerenone A (Gin A), a novel compound isolated from Zingiber officinale, is rarely investigated for its anti-breast-cancer properties. Some ginger extracts have been reported to initiate senescence, an anticancer strategy. However, the anticancer effects of Gin A on breast cancer cells remain unclear. The present study aims to assess the modulating impact of Gin A acting on proliferation and senescence to breast cancer cells. Gin A diminished the cellular ATP content and decreased the cell viability of the MTS assay in several breast cancer cell lines. It also showed a delayed G2/M response to breast cancer cells (MCF7 and MDA-MB-231). N-acetylcysteine (NAC), an oxidative stress inhibitor, can revert these responses of antiproliferation and G2/M delay. The oxidative stress and senescence responses of Gin A were further validated by increasing reactive oxygen species, mitochondrial superoxide, and β-galactosidase activity, which were reverted by NAC. Gin A also upregulated senescence-associated gene expressions. In addition to oxidative stress, Gin A also induced DNA damage responses by increasing γH2AX level and foci and generating 8-hydroxyl-2′-deoxyguanosine in breast cancer cells, which were reverted by NAC. Therefore, Gin A promotes antiproliferation and senescence of breast cancer cells induced by oxidative stress.


Introduction
Breast cancer is the most commonly diagnosed cancer type and the leading cause of cancer death in women, according to 2020 global cancer statistics [1]. Breast cancer accounts for 11.7% of all cancer cases, and several valuable markers have been advocated, such as estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), and Ki67 [2]. For the purposes of therapy, breast cancer is often classified into four subtypes: luminal type, pure HER2, triple-positive, and triplenegative. The triple-negative breast cancer (TNBC) is the most challenging category to (10 mM, 1 h), and Gin A was post-treated with cells to evaluate the contribution of oxidative stress in each experiment.

Cell Viability
Viability was assessed using an ATP kit (PerkinElmer Life Sciences, Boston, MA, USA) [22], MTS cell proliferation assay [23], and trypan blue reagent [24] based on the user manual's instruction.

Senescence Detection
Senescence was determined by monitoring the expression of β-galactosidase using flow cytometry and fluorescence microscopy. For flow cytometry, senescent cells were mixed using a Senescence Detection Kit (Fluorometric) (ab228562; Abcam) [26] with the requirements of 37 • C and 60 min, measured by flow cytometer (Guava easyCyte), and analyzed by FlowJo software.
For fluorescence monitoring, cells were fixed with 4% paraformaldehyde for 10 min. After washing, cells were incubated with senescence fluorescent dye (Abcam) at 1:1000 and bisBenzimide H 33342 trihydrochloride (Sigma-Aldrich) 1:1000 for 2 h at 37 • C without CO 2 . Cells were washed three times with PBS and mounted with Dako Fluorescence Mounting Medium (Invitrogen, Grand Island, NY, USA). Slides were photographed using a DMi8 microscope (Leica Microsystems, Wetzlar, Germany).

Statistics
ANOVA after Tukey's HSD Post hoc Tests (JMP ® 12 software, SAS Institute Inc., Cary, NC, USA) was applied for statistical analysis to assess multicomparison. Different treatments were labeled with connecting letters. When the connecting letters were overlapping, the difference between them was non-significant. In contrast, their difference was significant when the connecting letters were overlapping.

Gin A Diminished Proliferation of Breast Cancer Cells
Gin A (0, 20, 40, 60, and 80 µM) treatment for 48 h inhibited cell viability (ATP and MTS assays) of several kinds of cell lines of breast cancer in a dose-dependent manner ( Figure 1A). In  Normal breast cells (M10) show higher cell viability than breast cancer cells. By adding the ROS scavenger NAC, we assessed the impact of oxidative stress in antiproliferation of Gin A on breast cancer cells. After NAC pretreatment, Gin A-induced antiproliferation effects were suppressed, and they were recovered to 100% viability-like control ( Figure 1B).

Gin A Delayed G2/M Progression of Breast Cancer Cells
The impact of Gin A on the cell cycle of breast cancer cells (MCF7 and MDA-MB-231) was assessed using flow cytometric analysis ( Figure 2A). Gin A (80 μM) treatment for 48 h showed a minor increase in subG1, a moderate decrease in G1, and an increase in G2/M populations compared to control ( Figure 2B).
By adding the ROS scavenger NAC, we assessed the impact of oxidative stress on cell cycle regulation of Gin A in breast cancer cells. After NAC pretreatment, Gin A-induced subG1 accumulation, G1 decrement, and G2/M increment were reverted ( Figure  2B).

Gin A Delayed G2/M Progression of Breast Cancer Cells
The impact of Gin A on the cell cycle of breast cancer cells (MCF7 and MDA-MB-231) was assessed using flow cytometric analysis ( Figure 2A). Gin A (80 µM) treatment for 48 h showed a minor increase in subG1, a moderate decrease in G1, and an increase in G2/M populations compared to control ( Figure 2B).
By adding the ROS scavenger NAC, we assessed the impact of oxidative stress on cell cycle regulation of Gin A in breast cancer cells. After NAC pretreatment, Gin A-induced subG1 accumulation, G1 decrement, and G2/M increment were reverted ( Figure 2B).

Gin A Provoked ROS Increment in Breast Cancer Cells
The impact of Gin A on the ROS level of breast cancer cells (MCF7 and MDA-MB-231) was assessed using flow cytometric analysis ( Figure 3A,C). Gin A dose-responsively promoted ROS levels in breast cancer cells ( Figure 3B). Moreover, Gin A (80 µM) treatment at 1.5 and 3 h showed higher ROS levels in breast cancer cells than in the control (0 h) ( Figure 3D).
By adding NAC, we assessed the impact of oxidative stress on the ROS level of Gin A in breast cancer cells. After NAC pretreatment, Gin A-induced ROS increments were partly reverted ( Figure 3D).

Gin A Provoked ROS Increment in Breast Cancer Cells
The impact of Gin A on the ROS level of breast cancer cells (MCF7 a 231) was assessed using flow cytometric analysis ( Figure 3A,C). Gin A dos promoted ROS levels in breast cancer cells ( Figure 3B). Moreover, Gin A ment at 1.5 and 3 h showed higher ROS levels in breast cancer cells than in h) ( Figure 3D).
By adding NAC, we assessed the impact of oxidative stress on the RO A in breast cancer cells. After NAC pretreatment, Gin A-induced ROS inc partly reverted ( Figure 3D). . When the connecting letters are overlapping, their difference was significant for multiple comparisons (p < 0.05 to 0.0001). For example, the G1 phase for MCF7 cells (B), control, -NAC, and NAC/Gin A showing a differ non-significantly because they overlap with the same letter. These three treatments marked with a differ significantly from Gin A marked with b. For the example of the G2/M phase for MCF7 cells (B), Gin A, control, and NAC/Gin A showing a, c, and b differ significantly because they do not overlap with the same letter.

Gin A Provoked MitoSOX Increment of Breast Cancer Cells
The impact of Gin A on the MitoSOX level of breast cancer cells (MCF7 and MDA-MB-231) was assessed by flow cytometric analysis ( Figure 4A,C). Gin A dose-responsively promoted MitoSOX level in breast cancer cells ( Figure 4B). Moreover, Gin A (80 µM) treatment at 24 and 48 h promoted MitoSOX level in breast cancer cells in a time-dependent manner ( Figure 4D).

Gin A Provoked MitoSOX Increment of Breast Cancer Cells
The impact of Gin A on the MitoSOX level of breast cancer cells (MCF7 and MDA-MB-231) was assessed by flow cytometric analysis ( Figure 4A,C). Gin A dose-responsively promoted MitoSOX level in breast cancer cells ( Figure 4B). Moreover, Gin A (80 μM) treatment at 24 and 48 h promoted MitoSOX level in breast cancer cells in a time-dependent manner ( Figure 4D).
By adding NAC, we assessed the impact of oxidative stress on the MitoSOX level of Gin A in breast cancer cells. After NAC pretreatment, Gin A-induced MitoSOX increments were partly reverted ( Figure 4D).

Gin A Provoked MitoSOX Increment of Breast Cancer Cells
The impact of Gin A on the MitoSOX level of breast cancer cells (MCF7 and MDA-MB-231) was assessed by flow cytometric analysis ( Figure 4A,C). Gin A dose-responsively promoted MitoSOX level in breast cancer cells ( Figure 4B). Moreover, Gin A (80 μM) treatment at 24 and 48 h promoted MitoSOX level in breast cancer cells in a time-dependent manner ( Figure 4D).
By adding NAC, we assessed the impact of oxidative stress on the MitoSOX level of Gin A in breast cancer cells. After NAC pretreatment, Gin A-induced MitoSOX increments were partly reverted ( Figure 4D). By adding NAC, we assessed the impact of oxidative stress on the MitoSOX level of Gin A in breast cancer cells. After NAC pretreatment, Gin A-induced MitoSOX increments were partly reverted ( Figure 4D).

Gin A Provoked the Appearance of Senescence in Breast Cancer Cells
Since fluorescence is more sensitive than visual light, the fluorescence-based β-galactosidase activity was measured using flow cytometry and fluorescence microscopy ( Figure 5).

Gin A Provoked Senescence-Associated Gene Expressions of Breast Cancer Cells
The impact of Gin A on senescence-inducing effects of breast cancer cells (MCF7 and MDA-MB-231) was also assessed using qRT-PCR analysis ( Figure 6). Senescence-related genes [28,29] were detected, including EDN1, ANKRD1, CDKN1A, SERPINE, and TAGLN. Gin A at 80 μM showed higher mRNA levels of these senescence-related genes on breast cancer cells than control.

The impact of Gin A on senescence-inducing effects of breast cancer cells (MCF7 and
MDA-MB-231) was assessed using flow cytometric analysis ( Figure 5A,C). Gin A doseresponsively promoted senescence levels in breast cancer cells ( Figure 5B). Moreover, Gin A (80 µM) treatment at 24 and 48 h showed higher senescence levels in breast cancer cells than control (0 h) ( Figure 5D).
By adding NAC, we assessed the impact of oxidative stress on senescence level of Gin A in breast cancer cells. After NAC pretreatment, Gin A-induced senescence increments were partly reverted ( Figure 5D).
Moreover, the impact of Gin A on senescence-inducing effects in breast cancer cells was also assessed by fluorescence microscopy ( Figure 5E). β-galactosidase-detected senescence phenotype appeared in Gin A-treated breast cancer cells showing green fluorescence rather than in control. This Gin A-induced senescence phenotype was reduced by NAC pretreatment.

Gin A Provoked Senescence-Associated Gene Expressions of Breast Cancer Cells
The impact of Gin A on senescence-inducing effects of breast cancer cells (MCF7 and MDA-MB-231) was also assessed using qRT-PCR analysis ( Figure 6). Senescence-related genes [28,29] were detected, including EDN1, ANKRD1, CDKN1A, SERPINE, and TAGLN. Gin A at 80 µM showed higher mRNA levels of these senescence-related genes on breast cancer cells than control.

Gin A Provoked γH2AX Appearance of Breast Cancer Cells
The impact of Gin A on the γH2AX level in breast cancer cells (MCF7 and MDA-MB-231) was assessed by flow cytometric analysis ( Figure 7A,C). Gin A dose-responsively promoted γH2AX levels in breast cancer cells ( Figure 7B). Moreover, in a time-dependent manner, Gin A (80 μM) treatment at 24 and 48 h elevated γH2AX levels in breast cancer cells ( Figure 7D).
By adding NAC, we assessed the impact of oxidative stress on the γH2AX level of Gin A in breast cancer cells. After NAC pretreatment, Gin A-induced γH2AX increments were partly reverted ( Figure 7D).
Moreover, the impact of Gin A on γH2AX foci in breast cancer cells was assessed using fluorescence microscopy ( Figure 7E). γH2AX foci appeared in Gin A-treated breast cancer cells showing green fluorescence spots rather than in control ( Figure 7F). These Gin

Gin A Provoked γH2AX Appearance of Breast Cancer Cells
The impact of Gin A on the γH2AX level in breast cancer cells (MCF7 and MDA-MB-231) was assessed by flow cytometric analysis ( Figure 7A,C). Gin A dose-responsively promoted γH2AX levels in breast cancer cells ( Figure 7B). Moreover, in a time-dependent manner, Gin A (80 µM) treatment at 24 and 48 h elevated γH2AX levels in breast cancer cells ( Figure 7D).

Gin A Provoked 8-OHdG Increment of Breast Cancer Cells
The impact of Gin A on the 8-OHdG level of breast cancer cells (MCF7 and MDA-MB-231) was assessed using flow cytometric analysis ( Figure 8A,C). Gin A dose-responsively promoted 8-OHdG level in breast cancer cells ( Figure 8B). Moreover, Gin A (80 μM) treatment at 24 and 48 h elevated 8-OHdG levels in breast cancer cells more than in the control (0 h) ( Figure 8D).
Using adding NAC, we assessed the impact of oxidative stress on the 8-OHdG level of Gin A in breast cancer cells. After NAC pretreatment, Gin A-induced 8-OHdG increments were partly reverted ( Figure 8D). By adding NAC, we assessed the impact of oxidative stress on the γH2AX level of Gin A in breast cancer cells. After NAC pretreatment, Gin A-induced γH2AX increments were partly reverted ( Figure 7D).
Moreover, the impact of Gin A on γH2AX foci in breast cancer cells was assessed using fluorescence microscopy ( Figure 7E). γH2AX foci appeared in Gin A-treated breast cancer cells showing green fluorescence spots rather than in control ( Figure 7F). These Gin A-induced γH2AX foci increments were reduced by NAC pretreatment.

Gin A Provoked 8-OHdG Increment of Breast Cancer Cells
The impact of Gin A on the 8-OHdG level of breast cancer cells (MCF7 and MDA-MB-231) was assessed using flow cytometric analysis ( Figure 8A,C). Gin A dose-responsively promoted 8-OHdG level in breast cancer cells ( Figure 8B). Moreover, Gin A (80 µM) treatment at 24 and 48 h elevated 8-OHdG levels in breast cancer cells more than in the control (0 h) ( Figure 8D).

Discussion
The present study reported that Gin A showed antiproliferation and senescence properties in breast cancer cells. The relationship between senescence-associated changes and DNA damages connecting to oxidative stress was discussed.
Several bioactive compounds, such as gingerols, shogaols, gingediols, zingerone, dehydrozingerone, gingerinone, and diarylheptanoids, have been isolated from ginger extracts [8][9][10]. Some of these ginger derivatives were reported to exhibit oxidative stressinducing ability. For example, (6)-Gingerol can induce ROS generation and apoptosis in Using adding NAC, we assessed the impact of oxidative stress on the 8-OHdG level of Gin A in breast cancer cells. After NAC pretreatment, Gin A-induced 8-OHdG increments were partly reverted ( Figure 8D).

Discussion
The present study reported that Gin A showed antiproliferation and senescence properties in breast cancer cells. The relationship between senescence-associated changes and DNA damages connecting to oxidative stress was discussed.
Several bioactive compounds, such as gingerols, shogaols, gingediols, zingerone, dehydrozingerone, gingerinone, and diarylheptanoids, have been isolated from ginger extracts [8][9][10]. Some of these ginger derivatives were reported to exhibit oxidative stressinducing ability. For example, (6)-Gingerol can induce ROS generation and apoptosis in gastric cancer cells [45]. 6-Shogaol promoted ROS and endoplasmic reticulum stress so as to trigger apoptosis in endometrial cancer cells [46]. Gingediols caused ROS-dependent apoptosis in colon cancer cells [47]. Similarly, Gin A caused oxidative stress by inducing ROS (Figure 3) and MitoSOX ( Figure 4) in a dose-and time-dependent manner. These reports suggest that ginger derivatives have a potential ROS-modulating function.
Oxidative stress causes oxidative DNA damage [48,49]. Moreover, oxidative stress can cause DNA damage to initiate senescence [50]. DNA damage exhibits senescence-inducing effects on cancer cells in vitro and in vivo [51]. For example, oncogenic RAS upregulating mitochondrial mass and ROS can induce DNA damage and senescence [52]. Similarly, Gin A upregulated γH2AX level and foci as detected by flow cytometry and fluorescence microscopy ( Figure 7). Gin A also provoked oxidative DNA damage as detected by 8-OHdG flow cytometry (Figure 8).
Ginger extract can induce senescence in lung cancer cells [53] or protect the senescence of myoblasts [54]. Accordingly, the senescence responses of ginger extract still need further investigation. Although ginger derivative 6-Shogaol promoted apoptosis in endometrial cancer cells [46], it suppressed apoptosis and senescence in human dermal fibroblasts [55]. Conversely, Gin A induced β-galactosidase activity in breast cancer cells as detected by flow cytometry and fluorescence microscopy ( Figure 5), suggesting that Gin A induced senescence in breast cancer cells. Therefore, different bioactive compounds of ginger may exhibit different responses to senescence. The nature of cell types also needs to be considered for the senescence response to ginger derivatives.
Several senescence genes (EDN1, ANKRD1, CDKN1A, SERPINE, and TAGLN) were reported [28,29]. These genes were upregulated at 48 h Gin A treatment for breast cancer cells (MCF7 and MDA-MB-231) ( Figure 6). The expression status for these senescence genes was different for these cell lines. For example, EDN1 mRNA was highly expressed among these senescence-associated genes in MCF7 cells but only slightly expressed in MDA-MB-231 cells. In contrast, TAGLN mRNA was highly expressed among these senescenceassociated genes in MDA-MB-231 cells but expressed only somewhat in MCF7 cells. These results indicate that Gin A differentially regulates different senescence-associated genes in different breast cancer cell lines.
NAC pretreatment could revert the changes for proliferation, cell cycle progression, oxidative stress-associated modulations, senescence phenotypes, and DNA damages in breast cancer cells (Figures 1-5, 7 and 8). These results indicate that oxidative stress plays a central role in the antiproliferation and senescence effects of Gin A in breast cancer cells.

Conclusions
Ginger is a popular spice that is famous for several of its bioactive compounds. Gin A is a novel compound isolated from ginger. We report for the first time that Gin A induced antiproliferation and senescence responses acting on breast cancer cells. Senescence-associated genes were also upregulated by Gin A. Several experiments confirmed that Gin A generated oxidative stress and DNA damages to breast cancer cells. These responses to Gin A in breast cancer cells were confirmed to be ROS-dependent using NAC pretreatment. Therefore, Gin A is an antiproliferation-and senescence-inducing natural product against breast cancer cells involving oxidative-stress-associated responses.