Naringenin induces different proliferative effects in estrogen receptor-alpha-66 negative breast cancer cells

Naringenin induces different proliferative effects in estrogen receptor-alpha-66 negative breast cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Naringenin induces different proliferative effects in estrogen receptor-alpha-66 negative breast cancer cells Zhixiang Xu, Siyuan Hu, Nao Luo, Jun Liu, Xiaomin Ren, Xuejun Pan This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3266516/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Naringenin is a flavanone able to suppress the growth of various cancer cells including estrogen receptor-alpha-66 (ERα66)-positive breast cancer cells, but the anti-tumorigenic roles of naringenin in ERα66-negative breast cancer remain unclear. This study aims to determine the potential effects of naringenin on two ERα66-negative breast cancer cells (SKBR3 and MDA-MB-231) and to define their mechanisms of action. Herein, several cellular biological behaviors, including cell viability, migration, cycle, apoptosis, reactive oxygen species (ROS) generation, and oxidative stress (e.g., LDH, SOD and GSH), were evaluated in the present study. Then the gene expression levels involved in cell migration, cycle and apoptosis, as well as two membrane estrogen receptors (ERα36 and GPR30) were analyzed with real-time quantitative polymerase chain reaction (RT-qPCR) assay and western blotting. Results showed that naringenin exerted a concentration-dependent response on the growth of both ERα66-negative cell lines. The cell growth regulation was accompanied by modulation of cell migration, cycle and apoptosis, which were dependent on the ROS-regulated p53 signaling cascade. Importantly, ERα36 and GPR30 were involved in the (anti)-proliferative effects. These findings indicate naringenin possesses contrasting effects on ERα66-negative breast cancer cell growth depending on its dose Naringenin Bidirectional dose effects In vitro ERα66-negative Reactive oxygen species Membrane estrogen receptors Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Highlights Naringenin exerts different effects on the growth of SKBR3 and MDA-MB-231 cells. Cell growth is accompanied by the modulation of cell migration, cycle and apoptosis. Cell (anti)-proliferation depends on the ROS-regulated p53 signaling cascade. ERα36 and GPR30 participate in the naringenin-regulated growth of ERα66-negative breast cancer. 1. Introduction Flavonoids ( e.g. , flavones, isoflavones, flavanones, and flavonols) represent a group of polyphenolic phytochemical naturally-occurring compounds that are commonly abundant in consumed fruits, vegetables, and herbs (Liu et al. 2019, Wang et al. 2012). Numerous studies have reported that flavonoids are associated with reduced risks of many chronic diseases including cancer (Liu et al. 2019, Pan et al. 2014, Wang et al. 2012). Due to the benefits of their medicinal and pharmacological properties in the prevention and treatment of several diseases, phytochemical flavonoids have been extensively investigated over the past decades (Kanno et al. 2006, Zhang and Zuo 2004). Structurally, various flavonoids resemble estrogen and therefore are commonly considered phytoestrogen (Ye and Shaw 2019). Naringenin, a natural antioxidant that is primarily present in grapefruit, oranges and the skin of tomatoes showed low antioxidant properties among other flavonoids due to its structural characteristics (Islas et al. 2015). Previous studies have shown that naringenin possessed pleiotropic health benefits, such as anti-inflammatory, anti-oxidant, and anti-tumor (Pereira et al. 2007, Totta et al. 2004). Indeed, MCF-7 proliferation assay and recombinant yeast screen have shown that naringenin exerts weak estrogenic activity relative to 17β-estradiol (7.7~7.8 × 10 -3 ).(Breinholt and Larsen 1998) Recently, the in silico molecular modeling further demonstrated that naringenin can bind to the ligand-binding cleft of estrogen receptor alpha (ERα), and then mediate the estrogenic effects (Ye and Shaw 2019). However, not all flavonoids and their actions are necessarily beneficial. Due to its large-scale consumption, naringenin should be consequently ubiquitous in the aquatic environment as well as discharged from sewage treatment plants although few studies have been conducted to investigate the environmental levels, thus potentially increasing the health risks of wildlife (Sakalli et al. 2018). As an example, naringenin at 10 mg/L was reportedly teratogenic on amphibians (Pérez-Coll and Herkovits 2004). Naringenin at high doses (≥ 50 μM) has been shown to exhibit antiproliferative activities and was able to cause death in various cancer cell lines (Ahamad et al. 2014, Arul and Subramanian 2013, Shi et al. 2015). Moreover, naringenin in combination with anticancer agents like tamoxifen has been found to enhance cancer chemotherapy for ERα-positive breast cancer in our previous study (Xu et al. 2018). Above pieces of research indicate that the dual role of naringenin by producing either beneficial or toxic effects seems to depend on doses and the experimental model. Although no evidence has been raised that humans can be exposed to such higher concentrations of naringenin from environmental media like water, there are still several potential risks originating from environmental exposure directly or indirectly. Moreover, humans could also expose to these phytoestrogens more or less through diet or medicines (Weng and Yen 2012, Zamora-Ros et al. 2016). Unfortunately, little is known about their impact on environmental safety as well as human health risks. Some phytoestrogens are known sometimes to have estrogenic potency at very low concentrations even though no toxic activity is evident at environmental concentrations (Breinholt and Larsen 1998). In contrast, higher concentrations of phytoestrogens exposure would also induce oxidative or toxic effects (Pérez-Coll and Herkovits 2004, Sakalli et al. 2018). There are several assessment methods, covering in vitro , in vivo and epidemiological studies, that were used to assess the potential health risks of those phytoestrogens (Xu et al. 2017b). Among these methods, the in vitro model systems using cultured cells, especially the immortalized tumor cells, treated with various compounds may be more suitable for evaluating any adverse effects on humans (Xu et al. 2017b). Moreover, the biological significance of gene expression patterns including estrogen receptors (ERs) can be systematically assessed by the in vitro models (Xu et al. 2017a, Xu et al. 2017b). The in vitro methods, therefore, are promising in assessing the potentially toxic effects along with exploring the underlying mechanism induced by phytoestrogens. Generally, anti-estrogenic drugs and phytoestrogens bind to the ERs and then participate in the regulation of cell growth via modulating the 17β-estradiol (E2)-induced gene transcription (Liu et al. 2008, Möller et al. 2010). Estrogen receptor alpha 66 (ERα66) and estrogen receptor beta (ERβ), the two types of nuclear estrogen receptors (nERs), are firstly recognized for their ability to influence the development of female characteristics including breast cancer mediated by genomic estrogen pathways (Xu et al. 2017a). And naringenin has been shown to exert its antiproliferative and pro-apoptotic effects only in the presence of either ERα66 or ERβ (Totta et al. 2004). Interestingly, our recent studies have demonstrated that naringenin inhibited cell growth at higher doses (≥ 200 µM) also involved in membrane estrogen receptors (mERs)-mediated pathways in ERα66-positive breast cancer MCF-7 cells (Xu et al. 2018). However, whether naringenin showed similar effects in ERα66-negative like ERα66-positive breast cancer cells, and whether mERs are associated with such growth inhibition remains to be investigated. As the name implies, mERs differ from nERs in that they are primarily localized to the cytoplasm and plasma membrane and mediate membrane-initiated non-genomic signaling pathways (Xu et al. 2017a). Estrogen receptor alpha 36 (ERα36) and G protein-coupled estrogen receptor 30 (GPR30) are two typical mERs. These two mERs could mediate the activation of phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase (Akt) and mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathways, resulting in cell proliferation.(Chimento et al. 2014, Lin et al. 2010) In the present study, naringenin was screened at different doses and incubation times in two kinds of ERα66-negative breast cancer cells, SKBR3 and MDA-MB-231. Our results suggest naringenin could induce cell proliferation at lower doses but inhibit cell growth at higher doses in both SKBR3 and MDA-MB-231 cells. These bidirectional dose effects are associated with the regulation of cell migration, cell cycle arrest, cell apoptosis, and ROS generation that is mediated by GPR30 and ERα36. 2. Materials and methods 2.1. Chemicals and reagents 17β-estradiol (E2) and naringenin standards were purchased from Sigma-Aldrich (St Louis, MO, USA). Stock solutions at 100 μM and 200 mM, respectively, were prepared in dimethyl sulfoxide (DMSO) and stored at −20 °C. The working solutions were freshly diluted in the basal medium. Dulbecco's modified Eagle's medium (DMEM), phosphate-buffered saline (PBS), heat-inactivated fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Thermo Fisher Scientific (USA). Charcoal-dextran stripped fetal bovine serum (CS-FBS) was ordered from Gemini Bio-Products (West Sacramento, CA, USA). 2.2. Cell lines and cell culture Breast adenocarcinoma cell lines SKBR3 and MDA-MB-231 were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA) and preserved by our laboratory. These cell lines were selected based on different phenotypes for signaling pathways to elucidate if certain pathways were required. Cells were routinely maintained in phenol red DMEM, supplemented with 10% FBS and 1% penicillin-streptomycin at 37 °C in a water-saturated atmosphere of 5% CO 2 incubator. Before starting the exposure experiments, cell media were changed to phenol red-free DMEM containing 10% CS-FBS 48 h to deplete the influence of phenol and endogenous steroids (Berthois et al. 1986). Cells in the logarithmic growth phase were used to determine the proliferative or antiproliferative effects of naringenin. For all in vitro assays, cells were treated with increasing doses of naringenin dissolved in phenol red-free DMEM containing 5% CS-FBS for specific times. 2.3. Cell proliferation and cytotoxicity assessment Cell proliferation and cytotoxicity were evaluated using both a CCK-8 assay and a real-time cell impedance analyzer (RTCA) according to our previous studies (Xu et al. 2018). Briefly, SKBR3 and MDA-MB-231 cells were seeded into 96-well microplates and 16-well E-plates at a cell density of 1×10 4 and 6×10 3 cells/well, respectively. Following incubation to adhere for 24 h, cells were treated with various doses of naringenin ranging from 1 to 1000 μM or its vehicle in phenol red-free DMEM containing 5% CS-FBS for 12−72 h (up to 96 h). 10 nM E2 was used as the positive control, and DMSO (0.5%) was used as the untreated control. For the CCK-8 assay, after being incubated with naringenin for a specific time, cells were then incubated with 20 μL CCK-8 solution per well for another 3 h at 37 °C. In the end, the absorption values were measured at 450 nm using a microplate reader (SpectraMax M5, Molecular Devices, CA, USA). To verify that ERα36 and GPR30 signaling pathways are involved in naringenin-regulated antiproliferative effects, cells were treated with Broussoflavonol B and G36, respectively, for 1 h before naringenin addition. The results are reported as a percentage compared with the controls calculated from their relative absorbance, so 100% indicates no cytotoxicity. For RTCA analysis, the impedance was recorded every 5 minutes for up to 96 h, and these data were normalized as the last time point (approximately at 24 h) before objectives addition through the integrated software previously reported (Xu et al. 2018). The cytotoxicity of naringenin at higher doses to SKBR3 and MDA-MB-231 cells was also assessed using a Lactate dehydrogenase cytotoxicity kit (LDH, Beyotime, China). Following treatment with naringenin for 48 h, the supernatant was collected to assess the LDH activity following the manufacturer’s instructions. Morphological changes at 48 h were also observed using an inverted fluorescence microscope (IX73, Olympus) at a magnification of 200 ×. 2.4. Colony formation assay The SKBR3 (50 cells/well) and MDA-MB-231 (25 cells/well) were seeded in 6-well plates in triplicate. Following incubation to adhere for 24 h, cells were treated with various low concentrations of naringenin (1, 5 and 10 μM). Then, cells were allowed to grow at 37 °C in 5% CO 2 for 14 days, and the culture medium was changed every 2 days. In the end, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet, and the colonies were photographed. 2.5. Cell migration assessment Cell migration was evaluated using the wound healing assay as described previously with some specific modifications (Xu et al. 2018). Briefly, SKBR3 and MDA-MB-231 cells were seeded in a 12-well plate at a density of 4×10 5 and 2×10 5 cells/well, respectively. After forming a confluent monolayer, it was scratched with a sterile pipette tip (10 μL) and washed with a serum-free medium to remove the floating and detached cells. Afterward, cells were treated with lower doses (1 and 10 μM) and a higher dose (200 μM) of naringenin according to the results from the cell viability assay. The digitized images at 0, 12, 24, 36, and 48 h were monitored using an inverted fluorescence microscope (IX73, Olympus Corporation, Tokyo, Japan) at a magnification of 100 ×, and then analyzed with Image-Pro Plus software (Media Cybernetics, L.P., Silver Spring, MA, USA). 2.6. Cell cycle and apoptosis analysis Cell cycle and apoptosis were analyzed using the Cell cycle and apoptosis analysis kit (Beyotime, China) and Annexin V-FITC apoptosis detection kit (BD Biosciences, San Jose, CA, USA), respectively, according to the manufacturer's instructions. Briefly, SKBR3 and MDA-MB-231 cells were seeded in 6-well plates at a density of 6×10 5 and 3×10 5 cells/well, respectively. Following incubation to adhere for 24 h, cells were treated with naringenin the same as cell migration for 48 h in the condition of 5% CS-FBS. At the end of the time point, both adherent and floating cells were collected into a flow cytometry tube and centrifuged at 1000 g for 5 min to obtain cell pellets. Thereafter, cells were washed with precooling PBS. For cell cycle analysis, cells were firstly fixed in 70% ethanol overnight at 4 °C. And then, fixed cells were washed twice with precooling PBS and incubated with propidium iodide (PI) staining solution for 30 min at 37 °C. For cell apoptosis, cells were incubated with Annexin V-FITC/PI double staining solution. Both cell cycle and cell apoptosis were measured with NovoCyte™ Flow Cytometer (ACEA Biosciences, California, USA) within 30 min. In each analysis, over 10, 000 events were recorded. The percentages of cell distribution for cell cycle (Sub-G0, G0/G1, S-phase, and G2/M DNA content) and cell apoptosis (early apoptosis and late apoptosis) were calculated using the built-in analysis software. 2.7. Measurement of ROS generation, SOD activity and GSH content Intracellular reactive oxygen species (ROS) were examined with 2,7-dichloride-hydro fluorescein diacetate (DCFH-DA)-fluorescence probe (10 μM, Beyotime) as previously reported (Xu et al. 2018). Briefly, cells were treated with different doses of naringenin for 48 h. And then, cells were harvested, washed, and incubated with a DCFH-DA probe that was freshly prepared with the serum-free medium at 37 ℃ for 30 min in the dark. Subsequently, cells were washed with serum-free medium thrice to remove the unloaded probes, and the fluorescence was analyzed with the NovoCyte™ Flow Cytometer (ACEA Biosciences). Superoxide dismutase (SOD) activity and glutathione reductase (GR) activity were determined with a total superoxide dismutase assay kit with NBT (Beyotime, China) and glutathione reductase assay kit (Nanjing JianCheng Bio Institute, Nanjing, China), respectively, according to previous studies.(Kapoor and Kakkar 2014) Briefly, following treatment with various doses of naringenin for 48 h, cells were collected and resuspended in PBS, and the lysates were prepared under ultrasonic and centrifuged. The supernatants were quantified using a BCA protein assay (Beyotime, China). Then, the lysates were used to determine the SOD and GR activities, and all results were normalized to protein content. 2.8. RNA purification and RT-qPCR To determine gene expression changes involved in cell proliferation and anti-proliferation, a real-time quantitative polymerase chain reaction (RT-qPCR) was performed to examine the gene expression in mRNA transcription levels. Briefly, SKBR3 and MDA-MB-231 cells were seeded in 6-well plates at a density of 6×10 5 and 3×10 5 cells/well, respectively. Following tread with naringenin, cells were washed with PBS and the total RNA was isolated from cell samples using the RNAiso Plus (Takara, Dalian, China) according to our previous studies (Xu et al. 2018). And then the first strand of cDNA was reverse-transcribed using the PrimeScript-RT reagent kit (Takara, Dalian, China). The mRNA transcription levels were analyzed using validated primers and SYBR Premix Ex Taq TM Ⅱ (Tli RNaseH Plus) (TakaRa, Dalian, China) in the StepOnePlus Real-time PCR System (Applied Biosystems, CA, USA). Specific primers are shown in Table S1 . The cycler was programmed with the following conditions: (i) initial denaturation at 95 °C for 30 sec, followed by 40 cycles, (ii) 95 °C for 5 sec, (iii) annealing of the primer-template at 60 °C for 30 sec. β-actin in the same incubations was used for internal normalization, and the mRNA levels were determined using the 2 −ΔΔCt method as described in our previous studies (Xu et al. 2018). 2.9. Western blotting Western blotting was performed to analyze the protein expression levels of mERs (ERα36 and GPR30) following treatment with different concentrations of naringenin (1, 10, and 200 μM) for 48 h. The details have been described previously (Xu et al. 2018). The information on antibodies used is shown in Table S2 . Protein bands were quantified with the Image-Pro Plus software (Media Cybernetics, L.P., Silver Spring, MA, USA). 2.10. Statistical analysis All experiments were conducted thrice independently. Data were analyzed with SPSS 17.0 (Chicago, IL), and results were presented as the mean ± standard deviation (SD). Statistical significance was assessed by one-way analysis of variance (ANOVA), followed by the post-hoc test analysis. Differences at p < 0.05 were considered statistically significant. 3. Results 3.1. Lower-dose naringenin induces cell proliferation but higher doses inhibit cell growth To investigate the influence of naringenin on the growth of ERα66-negative breast cancer cells, SKBR3 and MDA-MB-231 cells were treated with increasing doses of naringenin ranging from 0.1 to 1000 μM for 48 h, and the cell viability was first analyzed with CCK-8 assay, as shown in Fig. 1A . Interestingly, different effects were observed on cell viability. Naringenin at low doses (0.1−20 μM) promoted cell proliferation in comparison with the control group. And the maximal proliferative effects of naringenin were achieved at 10 μM for SKBR3 cells (112.59±1.71%) and 1 μM for MDA-MB-231 cells (111.69±1.67%), respectively, which were almost equivalent to the effect of 10 nM E2 (Fig. 1A) . However, naringenin significantly inhibited cell growth in both cell lines when the exposure doses were high than 50 μM. Similar results were also observed with the RTCA assessment (Fig. 1B –C) . Particularly, naringenin at low concentrations significantly stimulated cell growth in both SKBR3 and MDA-MB-231 cells. The colony-forming assay was also conducted to testify that low concentrations of naringenin can increase the proliferation of ERα66-negative breast cancer cells, results are shown in Fig. 1E . Since higher doses of naringenin could significantly inhibit cell proliferation, cells were then treated with higher doses of naringenin at 100, 200 and 500 μM for 12−72 h to investigate their timely responses, and results showed naringenin decreased cell viability in both time- and dose-dependent manners of both SKBR3 and MDA-MB-231 cell lines (Fig. 1D) . Similar to other results (Islas et al. 2015), the half-maximal inhibitory concentration (IC50) of naringenin for SKBR3 and MDA-MB-231 cells was more than 200 μM, accounting for 362±15 μM and 410±26 μM, respectively. LDH is located predominantly in the cytoplasm and its presence in the extracellular medium serves to detect disruption of cell membrane integrity (Dong et al. 2017). To further confirm the cytotoxic effects of higher doses of naringenin on cells, the LDH assay was also performed as another indicator of cytotoxicity. Results demonstrated that higher-doses naringenin induced a marked dose-dependent increase in LDH leakage of both SKBR3 and MDA-MB-21 cells for 72 h (Fig. 1F) . Thus, higher-doses naringenin enhanced against cytotoxicity by increasing LDH release and this activation was higher in the SKBR3 cell line. From Fig. 1G , an inverse linear correlation was observed between LDH release and cell viability in SKBR3 (R 2 = 0.9956) and in MDA-MB-231 (R 2 = 0.9764) cells following treatment with higher doses of naringenin. Furthermore, the microscopic evaluation also revealed that cells exposed to 200 μM naringenin reduced significantly of the cell attachment number, normal morphology and adhesion capacity compared to those of the experimental control cells (Fig. 1H) . The above results demonstrated that cells treatment with lower doses of naringenin increased cell viability in both SKBR3 and MDA-MB-231 cells, and that exposure to higher doses of naringenin inhibit cell growth. Fig. 1 Naringenin regulated cell proliferation and cytotoxicity in SKBR3 and MDA-MB-231 cells 3.2. Lower-dose naringenin promotes cell migration but higher doses inhibit it in SKBR3 and MDA-MB-231 cells To investigate whether naringenin regulates cell migration, both SKBR3 and MDA-MB-231 cells were treated with naringenin at 1, 10 and 200 μM based on the results of cell viability for 12−48 h, and then the relative migration rates were evaluated with a wound-healing assay. In general, naringenin regulated cell migration in a cell type-, dose-, and time-dependent manner, as shown in Fig. 2A –C . Herein, the wound healing percentage of untreated SKBR3 cells increased to (17.63 ± 1.21) % at 48 h, but higher healing ratios were observed for MDA-MB-231 cells in which the healing percentages increased to (59.90 ± 1.25) %, (70.07 ± 1.37) % and (99.79 ± 1.24) % for 12, 24 and 36 h, respectively. Lower doses of naringenin (1 and 10 μM) marginally stimulated the cell migration of SKBR3 cells at 24 and 48 h, whereas no significant changes in cell migration were observed in MDA-MB-231 cells in all periods except for 24 h. Different from the potential stimulatory effects of naringenin at low-dose exposure, higher-dose naringenin significantly inhibited cell migration in both SKBR3 and MDA-MB-231 cells. However, naringenin exerted more relative inhibitory effects on cell migration in MDA-MB-231 cells than in SKBR3 cells. In detail, the percentage of migrated cells at the ending point increased from (1.30 ± 0.44) % to (11.10 ± 1.04) % for SKBR3 cells but only increased from (9.72 ± 0.99) % to (29.40 ± 1.19) % for MDA-MB-231 cells, respectively. Then the migration-linked protein E-cadherin, N-cadherin, MMP-2 and MMP-9 were further assessed, as illustrated in Fig. 2D . In line with the migration inhibition, the mRNA transcription levels of E-cadherin were significantly increased when cells were treated with higher-dose naringenin (200 μM), whereas the N-cadherin, MMP-9 and MMP-2 were decreased in both SKBR3 and MDA-MB-231 cells. As for low-dose exposure, SKBR3 cells treated with 1 μM naringenin significantly down-regulated the mRNA transcription of E-cadherin, but it markedly up-regulated the transcription levels of N-cadherin, MMP-9 and MMP-2 at 10 μM. Different regulatory manners have been found in MDA-MB-231 cells. The mRNA transcription levels of E-cadherin, N-cadherin and MMP-2 were markedly decreased, but no significance was found in the expression of MMP-9. As a whole, these results indicate that cell migration regulation is one of the ways through which naringenin regulated cell viability on ERα66-negative breast cancer cells. Fig. 2 Effects of naringenin on cell migration in SKBR3 and MDA-MB-231 cells 3.3. Naringenin shows dual effects on cell cycle in SKBR3 and MDA-MB-231 cells To evaluate the impacts of naringenin on the cell cycle in SKBR3 and MDA-MB-231 cells, flow cytometry was applied after cells were treated with increasing doses of naringenin ranging from 1 to 200 μM for 24 and 48 h. As shown in Fig. 3A & Fig. S1 , despite no significant differences were observed in cell cycle distribution for SKBR3 cells after being treated with lower doses of naringenin (1 and 10 μM) for 24 h and 48 h compared with the control group, higher doses of naringenin (100 and 200 μM) caused a significant increase of cell population in the S and G2/M phases. Similar results have been observed in MDA-MB-231 cells, namely naringenin (1−200 μM) increased the percentage of cells in the S and G2/M phases in comparison to that of the control group, concomitant with this increase was a decrease in the percentage of cells in the G0/G1 phase (Fig. 3B & Fig. S1) . However, it seemed that MDA-MB-231 cells were more sensitive to naringenin than SKBR3 cells since 10 μM naringenin began to arrest cells in the S phase. These results are following the data obtained from the cell proliferation analysis. These results demonstrated that naringenin possibly exerted its effect on cell anti-proliferation at S phase entry. Based on the results of cell cycle distribution, several cell cycle-regulatory genes, including cyclin A, cyclin B, cyclin D and cyclin D, were analyzed in mRNA transcription levels. As shown in Fig. 3C , a higher-dose naringenin (200 μM) exposure significantly increased the mRNA transcription levels of cyclin A and cyclin E, but it markedly decreased the mRNA transcription levels of cyclin B and cyclin D, which was consistent with the accumulation of cells in the S-phase. However, no significant difference has been found in cell cycle-regulated genes when cells were treated with lower doses of naringenin (1 and 10 μM). Fig. 3 Effects of naringenin on cell cycle distribution in SKBR3 and MDA-MB-231 cells 3.4. Dose-dependent effects of naringenin on cell apoptosis in SKBR3 and MDA-MB-231 cells We also investigated the influence of naringenin on cell apoptosis in SKBR3 and MDA-MB-231 cells. From Fig. 4A –B , lower doses of naringenin (1 and 10 μM) inhibited cell apoptosis both in SKBR3 (48 h) and MDA-MB-231 cells (24 and 48 h), whereas naringenin at higher doses (≥ 100 μM) promoted cell apoptosis in a dose-dependent manner. Moreover, the promotion of those inhibitive effects showed a time-dependent manner. For instance, 1−10 μM naringenin inhibited cell apoptosis in both SKBR3 and MDA-MB-231 cells compared to that of the control group. On the contrary, following treatment with 200 μM naringenin for 48 h, the percentage of viable cells was significantly decreased, whereas the apoptotic cells were increased from (16.16 ± 1.22) % to (65.56 ± 1.37) % for SKBR3 cells and from (16.92 ± 0.16) % to (37.05 ± 0.94) % for MDA-MB-231 cells, respectively. We then examined the expression changes of four important apoptosis-related genes ( i.e. , p21, p53, Bcl-2 and Bax) to explore whether this apoptosis regulation was regulated by mitochondrial pathways. As shown in Fig. 4C , when cells were treated with lower-dose naringenin (1 and 10 μM), the mRNA transcription levels of proapoptotic genes ( i.e ., p21, p53 and Bax) were significantly decreased, whereas the Bcl-2 was increased. Opposite results were observed when cells were treated with higher-dose naringenin (200 μM). Moreover, Bcl-2/Bax ratios were significantly increased following treatment with 1 and 10 μM naringenin but decreased following treatment with 200 μM naringenin. These results are consistent with the apoptosis status determined by flow cytometry. Therefore, apoptosis regulation is one of the ways through which naringenin regulated cell survival or death. Fig. 4 Effects of naringenin on cell apoptosis in SKBR3 and MDA-MB-231 cells 3.5. Naringenin regulates ROS generation and oxidative stress in SKBR3 and MDA-MB-231 cells To investigate whether naringenin induces oxidative stress, the effects of naringenin on ROS production were studied by employing DCFH-DA fluorescent dye. As shown in Fig. 5A , the ROS was significantly increased after being treated with higher doses of naringenin for 48 h compared to that of the control group, whereas lower doses of naringenin decreased the ROS levels. In line with the results of ROS levels, although no significant changes were found when cells were treated with lower doses of naringenin, higher doses of naringenin treatment resulted in a significant depletion of SOD (Fig. 5B) and intracellular GR activity (Fig. 5C) in both SKBR3 and MDA-MB-231 cells. These results indicate that naringenin especially at higher doses could affect the cellular redox status. Fig. 5 Effects of naringenin on ROS generation, SOD and GSH in SKBR3 and MDA-MB-231 cells 3.6. Naringenin modulates mERs expression in SKBR3 and MDA-MB-231 cells It has been reported that naringenin can bind to nERs (ERα66 and ERβ) and acts as a mimetic of E2 to activate the pro-apoptotic cascades in the presence of either ERα66 or ERβ (Totta et al. 2004). Unlike the ERα66-positive breast cancer cells, SKBR3 and MDA-MB-231 belong to ERα66-negative breast cancer cells but they still expressed two membrane estrogen receptors (mERs), ERα36 and GPR30. To further characterize the molecular mechanisms, we examined the influence of naringenin on mERs expression at both mRNA and protein levels in SKBR3 and MDA-MB-231 cells. As shown in Fig. 6A–B , naringenin regulated the mRNA transcription of ERα36 and GPR30 in a dose-dependent manner. In detail, naringenin at 1 and 10 μM significantly down-regulated the mRNA transcription of ERα36 and GPR30 in both SKBR3 and MDA-MB-231 cells compared with the control group. In contrast, naringenin at 200 μM markedly up-regulated the mRNA transcription of ERα36 by up to 15.25 ± 2.49% in SKBR3 cells but significantly decreased it in MDA-MB-231 cells. Opposite regulatory effects were observed on GPR30 transcription in SKBR3 and MDA-MB-231 cells, namely 200 μM naringenin treatments significantly up-regulated the mRNA transcription of GPR30 in MDA-MB-231 cells but slightly increased in SKBR3 cells ( p ＞0.05). Therefore, naringenin mediates ERα66-negative breast cancer cell growth via mERs signaling pathways. Fig. 6C–D showed that naringenin regulated the protein expression of ERα36 and GPR30 similar to mRNA transcription levels. It is noteworthy that the participation levels of ERα36 and GPR30 in SKBR3 and MDA-MB-231cells have more or fewer differences. Generally, the downregulation of ERα36 and GPR30 contributed to cell proliferation in both SKBR3 and MDA-MB-231 cells when they were exposed to low concentrations of naringenin. However, an upregulation of either ERα36 or GPR30 would result in cell antiproliferative effects induced by higher concentrations of naringenin in SKBR3 and MDA-MB-231 cells, respectively. To verify such antiproliferative effects of naringenin are mediated by ERα36 and GPR30, we treated cells with Broussoflavonol B (ERα36 antagonist) and G36 (GRP30 antagonist), respectively, for 1 h before the addition of 200 μM naringenin. And then the cell viability was analyzed with CCK-8 assays (Fig. 6E–F) . As expected, SKBR3 cells pretreated with Broussoflavonol B inhibited cell antiproliferation induced by high-concentration naringenin to some extent. Similar results were found in MDA-MB-231 cells when pretreated with G36. These results indicate that naringenin-induced cell apoptosis depends on high expression levels of both ERα36 and GPR30 in SKBR3 cells, and high GPR30 expression in MDA-MB-231 cells, respectively. Fig. 6 Naringenin regulated mERs expression in SKBR3 and MDA-MB-231 cells 4. Discussion As a heterogeneous disease with distinct clinical behavior and molecular properties, breast cancer can be divided into two categories based on the presence or absence of ERα, namely ERα66-positive and ERα66-negative cancers (Rouzier et al. 2005). While the majority of breast cancers belong to ERα66-positive, approximately 25–30% of cases are ERα66-negative (Chimento et al. 2014). Compared with the ERα66-positive breast cancer patients, the ERα66-negative classes have shorter disease-free intervals and worse overall survival (Pan et al. 2014). Several previous studies have shown that naringenin exerted antiproliferative effects or even caused cell death in various cancer cell lines including the ERα66-positive MCF-7 cells (Ahamad et al. 2014, Arul and Subramanian 2013, Shi et al. 2015). Herein, SKBR3 and MDA-MB-231 cells were selected as the in vitro models to investigate the potential regulatory effects of naringenin on ERα66-negative breast cancer progression in our present study. CCK-8 and RTCA results showed naringenin induced different effects on the cell growth of the above two kinds of ERα66-negative cells. In detail, naringenin at lower doses (≤ 10 μM) induced cell proliferation, but high-doses naringenin (≥ 50 μM) significantly inhibited it in a dose- and time-dependent manner. These results suggest that naringenin at higher doses possesses chemotherapeutic potential against human ERα66-negative breast cancer but lower doses would promote cancer development. Several specific cellular biological behaviors and molecular signaling pathways involved in cell growth promotion or inhibition were further studied. In agreement with the cell viability results, morphology and cell migration assessment showed lower doses of naringenin (1 and 10 μM) induced cell migration to some extent as well as increased cell numbers but without any changes in cell morphology. Conversely, cell attachment number and morphology, and migration capacity of both SKBR3 and MDA-MB-231 cells significantly declined following treatment with higher doses of naringenin (200 μM). It is worth mentioning that the migration capacity of MDA-MB-231 cells was greater than that of SKBR3 cells due to their different growth characteristics (Alamer and Darbre 2018). Epithelial to mesenchymal transition (EMT) plays a crucial role in cell migration and metastasis of breast cancer, and the characteristic feature of EMT is the switch of the epithelial E-cadherin to mesenchymal N-cadherin (Wei et al. 2018, Weng and Yen 2012). Consistent with this theory, higher-dose naringenin treatment resulted in an increase of E-cadherin but a decrease of N-cadherin in ERα66-negative cells, whereas contrary results were obtained when cells were treated with lower doses of naringenin. The activation of matrix metalloproteinases (MMPs), including MMP-2 and MMP-9, could also facilitate cells to invade the extracellular matrix, resulting in the promotion of tumor metastasis and deterioration (Kessenbrock et al. 2010, Weng and Yen 2012). As expected, lower-doses naringenin up-regulated the expression of MMP-2 and MMP-9 in SKBR3 cells but not in MDA-MB-231 cells, whereas higher-doses naringenin significantly down-regulated their expression. Similar results have been found by Magee and co-workers in MDA-MB-231 cells treated with genistein (Magee et al. 2004). In addition, the p21/p53 tumor transcription factor was also considered to suppress cell migration and invasion (Kim et al. 2017), which is consistent with the cell migration results of our present study. Dysregulation of the cell cycle is a hallmark of tumorigenesis and development (Evan and Vousden 2001). Naringenin has been found to induce cell cycle arrest in various cells ( e.g. , THP-1, HepG2 and A431 cells) but in different manners (Ahamad et al. 2014, Arul and Subramanian 2013, Shi et al. 2015), resulting in different cell antiproliferative effects. Herein, higher doses of naringenin (100 and 200 μM) caused a significant accumulation of cell population in the S and G2/M phases along with a decrease in the G0/G1 phase in both SKBR3 and MDA-MB-231 cells. Moreover, no significant difference was observed in cell cycle distribution for SKBR3 cells following treatment with lower doses of naringenin (1 and 10 μM) for 24 h and 48 h compared with the control group, whereas it still gently increased the percentage of cells in the S and G2/M phases in MDA-MB-231cells. The cell cycle, of course, is controlled at multiple checkpoints which are regulated by various kinds of cell cycle-regulating proteins, including cyclin A, cyclin B, cyclin D and cyclin E (Evan and Vousden 2001, Xie et al. 2016, Zhao et al. 2017). Consistent with the cell cycle distribution results, cyclin A and cyclin E were significantly increased following treatment with 200 μM naringenin, whereas the expression of cyclin D and cyclin B were markedly decreased. Additionally, no significant changes in mRNA transcription of cycle-regulatory genes were observed. Other than cell cycle phase arrest, cell apoptosis is also involved in cell growth regulation (Evan and Vousden 2001). Apoptosis is a common form of cell death morphologically characterized by cellular shrinkage, chromatin condensation, DNA fragmentation, and cell disruption into apoptotic bodies (Kapoor and Kakkar 2014). The death receptor pathway and the mitochondria pathway are two well-established mechanisms involving apoptotic cell death (Dong et al. 2017). Among these, the death receptor pathway is mediated by caspases, and the mitochondrial-dependent apoptosis is regulated by the Bcl-2 family proteins, particularly Bax and Bcl-2 (Adams and Cory 1998). As important tumor suppressor genes, p21, p53 and their p53/p21 complex can induce cell apoptosis by regulating the expression of Bcl-2 and Bax, thus suppressing tumor cell proliferation and preventing tumor development (Fridman and Lowe 2003, Kim et al. 2017). In the present study, when cells were exposed to high doses-induced oxidative stress for 24–48 h, cell apoptosis induction became evident owing to a decline of Bcl-2 protein, but to an elevation of Bax, p21 and p53 proteins. Moreover, changes in Bax/Bcl-2 ratio also result in significant activation of caspases and lead to cell death through the mitochondrial death pathway (Adams and Cory 1998). The Bcl-2/Bax ratio, of course, also markedly declined following treatment with 200 μM naringenin, whereas contrary results were observed when cells were exposed to lower doses of naringenin. ROS acted as an early signal in various biological circumstances, but an excessive generation of ROS that cannot be attenuated by intracellular redox systems would lead to a variety of biochemical and physiological lesions such as lipid peroxidation, protein oxidation, enzyme inactivation and oxidative DNA damage, resulted in cell cycle arrest and apoptosis induction as well as metastatic properties suppression (Islas et al. 2015, Simon et al. 2000). Previously, we found naringenin at a high level of 200 μM caused oxidative stress-induced apoptosis by p53 activation in MCF-7 cells, but pre-treatment with antioxidants blocked cell death induced by naringenin (Xu et al. 2018). An increased generation of ROS would destroy the mitochondrial membrane potential (MMPs), and then induce the release of LDH (Dong et al. 2017, Holder et al. 2012), and cytochrome c from mitochondria into the cytosol (Li et al. 2010). Moreover, the cytochrome c also triggered caspase-9 signaling pathways activation and ultimately caused cell death (Li et al. 2010). Those intracellular ROS production, SOD activity, GSH content and LDH release were then investigated. Results demonstrated that SKBR3 and MDA-MB-231 cells treated with higher doses of naringenin resulted in significant production of ROS and LDH release, but depletion of SOD and intracellular GSH compared to that of the control group. Contrary results were obtained when cells were treated with lower doses of naringenin. In addition, high levels of ROS also induced Bax expression but suppressed Bcl-2 expression through the activation of p38 and JNK signaling pathways, resulting in cell apoptosis (Chen et al. 2010). Herein, high doses of naringenin promoted Bax expression but suppressed Bcl-2 expression. The above results support the assumption that naringenin can induce cell growth inhibition in ERα66-negative breast cancer cells through the increasing rates of apoptosis mediated by ROS accumulation. Phytoestrogens usually act both as agonists and antagonists of estrogens at different concentrations (Totta et al. 2004). Naringenin can mimic natural steroid hormones ( e.g. , E2) and enable it to interact with ERs as an agonist or antagonist. Liu et al. found that nERs (ERα66 and ERβ) played important roles in low-dose naringenin-induced cell growth (Liu et al. 2008). Generally, naringenin induced cell apoptotic cascade in the presence of either ERα66 or ERβ in ER-positive cells (Totta et al. 2004). Nevertheless, whether naringenin exerted the same effects in ERα66-negative cancer as in ERα66-positive cancer cells is unclear. ERα36 as a truncated variant of the classical estrogen receptor—ERα66, is expressed in several breast cancer cells but not in normal mammary epithelial cells MCF10A (Zou et al. 2009). Compared with the ERα66-positive breast cancer cells including MCF-7 and T47D, however, ERα36 was highly expressed in ERα66-negative breast cancer cells such as the SKBR3 and MDA-MB-231 cell lines (Zhang et al. 2012, Zou et al. 2009). The different expression patterns of ERα66 and ERα36 in these cancer cells further indicated that ERα36 is transcriptionally regulated differently from ERα66. Moreover, GPR30 was also highly expressed in ERα66-negative breast cancer and participated in the proliferation/apoptosis processes (Zhou et al. 2016). Naringenin has been said to selectively impair the membrane-initiating signals of the ERα subtype in earlier studies (Galluzzo et al. 2008). In the present study, lower-dose naringenin inhibited the expression of ERα36 and GPR30 in both SKBR3 and MDA-MB-231 cells, but higher-dose naringenin promoted ERα36 expression in SKBR3 cells and GPR30 in MDA-MB-231 cells, resulted in the regulation of cell growth and apoptosis, respectively. Similar results by others also showed that the activation of ERα36 and GPR30 pathways are involved in the anti-proliferation and pro-apoptosis of both ERα66-positive and ERα66-negative breast cancer cells (Chimento et al. 2014, Pan et al. 2014, Weißenborn et al. 2014). Previous studies indicated that mERs, especially ERα36 and GPR30, participated in cellular processes through rapid activation of second messenger pathways, such as an increase in intracellular free calcium or ROS. And then, they triggered functional responses or activated multiple downstream kinase cascades ( i.e. , PI3K/Akt and MAPKs), resulting in cell proliferation, differentiation, apoptosis, or survival (Xu et al. 2017a). The PI3K/Akt and MAPKs, such as ERK1/2, JNK and p38, played critical roles in converting extracellular stimuli into cellular responses (Bulzomi et al. 2010, Chen et al. 2010, Dhillon et al. 2007, Lin et al. 2010). Moreover, naringenin induced membrane ERα rapid dissociation from caveolin-1 through depalmitoylation and then impeded it to binding adaptor and signaling proteins involved in the activation of the mitogenic signaling cascades (Galluzzo et al. 2008). All in all, the results obtained in our study and in that of others confirmed that naringenin induced apoptosis depends on high expression of ERα36 and GPR30 in ERα66-negative breast cancer cells, but the specific signaling pathways involved in naringenin-mediated (anti)-proliferative effects should be further explored in future. Fig. 7 Assumed schematic mechanisms of naringenin-mediated different proliferative effects involved in ROS generation, cell migration, cell cycle, and cell apoptosis in ERα66-negative breast cancer cells 5. Conclusions Collectively, our findings demonstrate that naringenin at higher doses induces cell anti-proliferation but lower-doses naringenin indeed promotes cell growth of both SKBR3 and MDA-MB-231 cells (Fig. 7) . Mechanistic investigation of the dual effects in the breast cancer cell lines reveals that higher-dose naringenin suppresses cell proliferation which is involved in cell migration inhibition, cell cycle arrest at the S phase and G2/M phase, cell apoptosis promotion, and ROS generation acceleration. However, lower-dose naringenin stimulates cell proliferation to some extent which was accompanied by 1) a decrease of E-cadherin expression but an increase of N-cadherin, MMP-9 and MMP-2, leading to cell migration, and 2) a decrease of ROS generation, p21, p53 and Bax expression, but an increase of Bcl-2 expression, resulted in cell apoptosis inhibition. Importantly, ERα36 and GPR30 are involved in the (anti)-proliferation effects of naringenin in ERα66-negative breast cancer cells. As naringenin possesses contrasting effects on ERα66-negative breast cancer cell viability depending on its dose, caution is warranted in determining its effective dose in breast cancer treatment. Naringenin, of course, could be still an alternative treatment for ERα66-negative breast cancer at high doses. Declarations Ethics approval This article does not contain any studies with human participants or animals performed by any of the authors. Consent to participate All participants of that article consent to participate this manuscript. Consent to publish All participants of that article consent to publish this manuscript. Author contributions Zhixiang Xu developed the idea, designed the experimental approach, analyzed the data, prepared the figures, and drafted the manuscript. Siyuan Hu drafted the manuscript. Nao Luo performed cell culture and flow cytometry assessment. Jun Liu performed cell viability and gene expression determination. Xiaomin Ren analyzed the results and prepared the figures. Xuejun Pan conceived and designed the study, and reviewed the manuscript. Funding This research was supported by the National Natural Science Foundation of China (grants, 22176076, 21567014), and the China Post-doctoral Science Foundation (grant, 2019M653846XB). Competing interests The authors declare no potential conflicts of interest. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3266516","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":230476642,"identity":"8defa385-9acb-4299-a94d-532ef79ac0db","order_by":0,"name":"Zhixiang Xu","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Zhixiang","middleName":"","lastName":"Xu","suffix":""},{"id":230476643,"identity":"c2db4eb6-948b-456f-8d57-a66ab042ce93","order_by":1,"name":"Siyuan Hu","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Siyuan","middleName":"","lastName":"Hu","suffix":""},{"id":230476644,"identity":"4004a7e1-5706-4cb9-8f1f-b573cbb9792b","order_by":2,"name":"Nao Luo","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Nao","middleName":"","lastName":"Luo","suffix":""},{"id":230476645,"identity":"8fbe7e82-e313-4db6-ad6a-6eb9d31599f7","order_by":3,"name":"Jun Liu","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Liu","suffix":""},{"id":230476646,"identity":"8b395ec8-1177-435a-a4cd-f0ac582fb3f3","order_by":4,"name":"Xiaomin Ren","email":"","orcid":"","institution":"Kunming University of Science and Technology","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xiaomin","middleName":"","lastName":"Ren","suffix":""},{"id":230476647,"identity":"724ecdab-0297-452b-bb28-2405b80c675e","order_by":5,"name":"Xuejun Pan","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYFCCA0BckQDlsBGt5QxICzPRWoCAsY0ULQYHzx78XDgvLdrg/PkDDB/KDjPwz27Ar0Wy4Vyy9MxtObkbbiQzMM44d5hB4s4B/Fr4Gc4YSPNuqwBqYWZg5m07zGAgkYBfCxvDGePfvHOAWs4fZmD+S4wWoC1m0rwNQIcdSGZgZiRGC9AvadY8x9JyZ95INjjYcy6dR+IGAS0GN84evs1Tk5zbd/7gwwc/yqzl+GcQ0MIgcQbBPgDEPATUAwF/D2E1o2AUjIJRMMIBAIp8RkSsVOy3AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0003-3772-6908","institution":"Kunming University of Science and Technology","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Xuejun","middleName":"","lastName":"Pan","suffix":""}],"badges":[],"createdAt":"2023-08-15 16:39:53","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3266516/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3266516/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":42721736,"identity":"66c35ead-6c11-4c99-a9f1-255a59aae850","added_by":"auto","created_at":"2023-09-06 14:34:00","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":651430,"visible":true,"origin":"","legend":"\u003cp\u003eNaringenin regulated cell proliferation and cytotoxicity in SKBR3 and MDA-MB-231 cells\u003c/p\u003e\n\u003cp\u003eSKBR3 and MDA-MB-231 cells were treated with serial doses of naringenin ranging from 1 to 1000 μM, E2 (10 nM), or its vehicle in the presence of 5% CS-FBS for specific times. Cell viability was then evaluated with \u003cstrong\u003e(A, D)\u003c/strong\u003e CCK-8 assay or \u003cstrong\u003e(B, C)\u003c/strong\u003eRTCA assay. \u003cstrong\u003e(E) \u003c/strong\u003eColony formation following treatment with low concentrations of naringenin (1, 5, 10 μM) for 14 days. \u003cstrong\u003e(F)\u003c/strong\u003e LDH was used to access cytotoxicity following treatment with higher doses of naringenin (100, 200, 500 μM) for 72 h. \u003cstrong\u003e(G)\u003c/strong\u003e A significant negative correlation between cell viability and LDH leakage following treatment with higher doses of naringenin (100, 200, 500 μM) for 72 h. \u003cstrong\u003e(H)\u003c/strong\u003e Cell morphology changes were observed using an Inversion Microscope at a magnification of 200 × following treatment with naringenin (1, 10, and 200 μM) for 48 h. Results are expressed as mean ± SD of three replicate experiments (n=6). The significant difference was set at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. Different concentrations and time courses of the same cells were compared with each other. Experiments without labeling the same letter are significant.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-3266516/v1/4900b44e3173133bebc4cf62.png"},{"id":42719808,"identity":"c7c6960c-849e-4c75-a674-7f9f00455bfb","added_by":"auto","created_at":"2023-09-06 14:26:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1949206,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of naringenin on cell migration in SKBR3 and MDA-MB-231 cells\u003c/p\u003e\n\u003cp\u003eSKBR3 and MDA-MB-231 cells were treated with serial doses of naringenin (1, 10 and 200 μM) or its vehicle for 12−48 h. Representative bright-field images for \u003cstrong\u003e(A)\u003c/strong\u003e SKBR3 and \u003cstrong\u003e(B)\u003c/strong\u003e MDA-MB-231 cells showed that naringenin regulates cell migration in a time-dependent manner. \u003cstrong\u003e(C) \u003c/strong\u003eScratch-wound closure monitored over time in SKBR3 and\u003cstrong\u003e \u003c/strong\u003eMDA-MB-231 cells was represented as a bar diagram. Results are mean ± SD of three bright-field images (n=3).\u003cstrong\u003e (D)\u003c/strong\u003e The mRNA expression levels of E-cadherin, N-cadherin, MMP-2 and MMP-9 that are involved in cell migration were measured with RT-qPCR and represented as a bar diagram. Results are mean ± SD of three replicate experiments. The significant difference was set at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. Different concentrations and time courses of the same cells were compared with each other. Experiments without labeling the same letter are significant.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-3266516/v1/109d2b6d8786709fc073d4a1.png"},{"id":42721737,"identity":"e94d39b1-5eb8-4435-bd9d-b9d34408e62e","added_by":"auto","created_at":"2023-09-06 14:34:00","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":433210,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of naringenin on cell cycle distribution in SKBR3 and MDA-MB-231 cells\u003c/p\u003e\n\u003cp\u003eSKBR3 and MDA-MB-231 cells were treated with serial doses of naringenin (1, 10 and 200 μM) or its vehicle for 24 and 48 h. Percentages of cell population in different phases of the cell cycle stage for \u003cstrong\u003e(A)\u003c/strong\u003eSKBR3 and \u003cstrong\u003e(B)\u003c/strong\u003e MDA-MB-231 cells are represented as bar diagrams. Results are mean ± SD of three flow cytometry images (n=3). \u003cstrong\u003e(C)\u003c/strong\u003e The mRNA expression levels of cyclin A, cyclin B, cyclin D and cyclin E that are involved in cell cycle arrest were measured with RT-qPCR and represented as a bar diagram. Results are mean ± SD of three replicate experiments. The significant difference was set at \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05. Different concentrations and time courses of the same cells were compared with each other. Experiments without labeling the same letter are significant.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-3266516/v1/10ede94a59419070059a2aec.png"},{"id":42719806,"identity":"9628eafc-f4fd-4e50-8942-1dc26b1d69de","added_by":"auto","created_at":"2023-09-06 14:26:00","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":388197,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of naringenin on cell apoptosis in SKBR3 and MDA-MB-231 cells\u003c/p\u003e\n\u003cp\u003eSKBR3 and MDA-MB-231 cells were treated with serial doses of naringenin (1, 10 and 200 μM) or its vehicle for 24 and 48 h. Percentages of cell population in different phases of the cell apoptosis stage for (A) SKBR3 and (B) MDA-MB-231 cells are represented as bar diagrams. Results are mean ± SD of three flow cytometry images (n=3). (C) The mRNA expression levels of p21, p53, Bcl-2 and Bax that are involved in cell apoptosis were measured with RT-qPCR and represented as a bar diagram. Results are mean ± SD of three replicate experiments. The significant difference was set at p \u0026lt; 0.05. Different concentrations and time courses of the same cells were compared with each other. Experiments without labeling the same letter are significant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-3266516/v1/e5106d40ad6fb2e8c569c89f.png"},{"id":42722894,"identity":"25b44914-2b66-414f-aab6-9104af8abd96","added_by":"auto","created_at":"2023-09-06 14:42:00","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":563824,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of naringenin on ROS generation, SOD and GSH in SKBR3 and MDA-MB-231 cells\u003c/p\u003e\n\u003cp\u003eSKBR3 and MDA-MB-231 cells were treated with serial doses of naringenin (1, 10,100 and 200 μM) or its vehicle for 48 h. Percentage changes of intracellular ROS \u003cstrong\u003e(A)\u003c/strong\u003e, SOD activity \u003cstrong\u003e(B)\u003c/strong\u003e and GR activity \u003cstrong\u003e(C)\u003c/strong\u003ein SKBR3 and MDA-MB-231 cells are represented as bar diagrams. Results are mean ± SD of three replicate experiments (n=3). The significant difference was set at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. Different concentrations and time courses of the same cells were compared with each other. Experiments without labeling the same letter are significant.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-3266516/v1/00fb211965d79dcbb2a90d24.png"},{"id":42719813,"identity":"7dc72635-b639-462f-89bb-4c6e7f480c18","added_by":"auto","created_at":"2023-09-06 14:26:00","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":1972133,"visible":true,"origin":"","legend":"\u003cp\u003eNaringenin regulated mERs expression in SKBR3 and MDA-MB-231 cells\u003c/p\u003e\n\u003cp\u003eSKBR3 and MDA-MB-231 cells were treated with serial doses of naringenin (1, 10 and 200 μM) or its vehicle for 48 h. The gene expression of ERα36 and GPR30 in mRNA transcription and protein expression levels were measured with RT-qPCR \u003cstrong\u003e(A–B)\u003c/strong\u003e and western blotting \u003cstrong\u003e(C–D)\u003c/strong\u003e, respectively. Up means SKBR3 cells, and Down means MDA-MB-231 cells. Antagonists of ERα36 and GPR30 attenuate naringenin-induced antiproliferative effects in \u003cstrong\u003e(E) \u003c/strong\u003eSKBR3 and \u003cstrong\u003e(F) \u003c/strong\u003eMDA-MB-231 cells. Results are mean ± SD of three replicate experiments. The significant difference was set at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05. Different concentrations and time courses of the same cells were compared with each other. Experiments without labeling the same letter are significant.\u003c/p\u003e","description":"","filename":"6.png","url":"https://assets-eu.researchsquare.com/files/rs-3266516/v1/71f1640d337514e3439ddc34.png"},{"id":42719812,"identity":"3006d970-df16-4382-aebd-60738b55c17a","added_by":"auto","created_at":"2023-09-06 14:26:00","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":501986,"visible":true,"origin":"","legend":"\u003cp\u003eAssumed schematic mechanisms of naringenin-mediated different proliferative effects involved in ROS generation, cell migration, cell cycle, and cell apoptosis in ERα66-negative breast cancer cells\u003c/p\u003e","description":"","filename":"7.png","url":"https://assets-eu.researchsquare.com/files/rs-3266516/v1/6b1e26d325f059e808192a07.png"},{"id":43182078,"identity":"23efbe99-6b63-4bb6-9767-7ad7424d86db","added_by":"auto","created_at":"2023-09-15 09:52:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4406122,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3266516/v1/8975da16-eb38-4b6a-82af-6883e03cf710.pdf"},{"id":42719814,"identity":"5691274c-3561-4ac3-a27f-f39ad67a492c","added_by":"auto","created_at":"2023-09-06 14:26:00","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":3556864,"visible":true,"origin":"","legend":"\u003cp\u003eAppendix A. Supplementary Data\u003c/p\u003e\n\u003cp\u003eTable S1-S2 and Figure S1-S3 were presented in the Supporting Information.\u003c/p\u003e","description":"","filename":"SupplementaryMaterial.doc","url":"https://assets-eu.researchsquare.com/files/rs-3266516/v1/a42a972ceaf8ad2bc9f6cbf9.doc"},{"id":42719807,"identity":"c0cee651-7953-4e28-b9f4-01ce361c337c","added_by":"auto","created_at":"2023-09-06 14:26:00","extension":"png","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":174252,"visible":true,"origin":"","legend":"\u003cp\u003eGraphical abstract\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eNotes: \u003c/strong\u003eNaringenin-induced different proliferative effects in ERα66-negative breast cancer cells are involved in the regulation of cell migration, cell cycle and apoptosis. Such bidirectional effects are dependent on ROS-regulated p53 signaling cascade mediated by GPR30 and ERα36.\u003c/p\u003e","description":"","filename":"Graphicalabstract.png","url":"https://assets-eu.researchsquare.com/files/rs-3266516/v1/328bac4fdb413b7b9b9dd4d8.png"}],"financialInterests":"","formattedTitle":"Naringenin induces different proliferative effects in estrogen receptor-alpha-66 negative breast cancer cells","fulltext":[{"header":"Highlights","content":"\u003cul\u003e\n \u003cli\u003eNaringenin exerts different effects on the growth of SKBR3 and MDA-MB-231 cells.\u003c/li\u003e\n \u003cli\u003eCell growth is accompanied by the modulation of cell migration, cycle and apoptosis.\u003c/li\u003e\n \u003cli\u003eCell (anti)-proliferation depends on the ROS-regulated p53 signaling cascade.\u003c/li\u003e\n \u003cli\u003eER\u0026alpha;36 and GPR30 participate in the naringenin-regulated growth of ER\u0026alpha;66-negative breast cancer.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eFlavonoids (\u003cem\u003ee.g.\u003c/em\u003e, flavones, isoflavones, flavanones, and flavonols) represent a group of polyphenolic phytochemical naturally-occurring compounds that are commonly abundant in consumed fruits, vegetables, and herbs (Liu et al. 2019, Wang et al. 2012). Numerous studies have reported that flavonoids are associated with reduced risks of many chronic diseases including cancer (Liu et al. 2019, Pan et al. 2014, Wang et al. 2012). Due to the benefits of their medicinal and pharmacological properties in the prevention and treatment of several diseases, phytochemical flavonoids have been extensively investigated over the past decades (Kanno et al. 2006, Zhang and Zuo 2004). Structurally, various flavonoids resemble estrogen and therefore are commonly considered phytoestrogen (Ye and Shaw 2019). Naringenin, a natural antioxidant that is primarily present in grapefruit, oranges and the skin of tomatoes showed low antioxidant properties among other flavonoids due to its structural characteristics (Islas et al. 2015). Previous studies have shown that naringenin possessed pleiotropic health benefits, such as anti-inflammatory, anti-oxidant, and anti-tumor (Pereira et al. 2007, Totta et al. 2004). Indeed, MCF-7 proliferation assay and recombinant yeast screen have shown that naringenin exerts weak estrogenic activity relative to 17\u0026beta;-estradiol (7.7~7.8 \u0026times; 10\u003csup\u003e-3\u003c/sup\u003e).(Breinholt and Larsen 1998) Recently, the \u003cem\u003ein silico\u003c/em\u003e molecular modeling further demonstrated that naringenin can bind to the ligand-binding cleft of estrogen receptor alpha (ER\u0026alpha;), and then mediate the estrogenic effects (Ye and Shaw 2019). However, not all flavonoids and their actions are necessarily beneficial. Due to its large-scale consumption, naringenin should be consequently ubiquitous in the aquatic environment as well as discharged from sewage treatment plants although few studies have been conducted to investigate the environmental levels, thus potentially increasing the health risks of wildlife (Sakalli et al. 2018). As an example, naringenin at 10 mg/L was reportedly teratogenic on amphibians (P\u0026eacute;rez-Coll and Herkovits 2004). Naringenin at high doses (\u0026ge; 50 \u0026mu;M) has been shown to exhibit antiproliferative activities and was able to cause death in various cancer cell lines (Ahamad et al. 2014, Arul and Subramanian 2013, Shi et al. 2015). Moreover, naringenin in combination with anticancer agents like tamoxifen has been found to enhance cancer chemotherapy for ER\u0026alpha;-positive breast cancer in our previous study (Xu et al. 2018). Above pieces of research indicate that the dual role of naringenin by producing either beneficial or toxic effects seems to depend on doses and the experimental model.\u003c/p\u003e\n\u003cp\u003eAlthough no evidence has been raised that humans can be exposed to such higher concentrations of naringenin from environmental media like water, there are still several potential risks originating from environmental exposure directly or indirectly. Moreover, humans could also expose to these phytoestrogens more or less through diet or medicines (Weng and Yen 2012, Zamora-Ros et al. 2016). Unfortunately, little is known about their impact on environmental safety as well as human health risks. Some phytoestrogens are known sometimes to have estrogenic potency at very low concentrations even though no toxic activity is evident at environmental concentrations (Breinholt and Larsen 1998). In contrast, higher concentrations of phytoestrogens exposure would also induce oxidative or toxic effects (P\u0026eacute;rez-Coll and Herkovits 2004, Sakalli et al. 2018). There are several assessment methods, covering \u003cem\u003ein vitro\u003c/em\u003e, \u003cem\u003ein vivo\u003c/em\u003e and epidemiological studies, that were used to assess the potential health risks of those phytoestrogens (Xu et al. 2017b). Among these methods, the \u003cem\u003ein vitro\u003c/em\u003e model systems using cultured cells, especially the immortalized tumor cells, treated with various compounds may be more suitable for evaluating any adverse effects on humans (Xu et al. 2017b). Moreover, the biological significance of gene expression patterns including estrogen receptors (ERs) can be systematically assessed by the \u003cem\u003ein vitro\u003c/em\u003e models (Xu et al. 2017a, Xu et al. 2017b). The \u003cem\u003ein vitro\u003c/em\u003e methods, therefore, are promising in assessing the potentially toxic effects along with exploring the underlying mechanism induced by phytoestrogens.\u003c/p\u003e\n\u003cp\u003eGenerally, anti-estrogenic drugs and phytoestrogens bind to the ERs and then participate in the regulation of cell growth via modulating the 17\u0026beta;-estradiol (E2)-induced gene transcription (Liu et al. 2008, M\u0026ouml;ller et al. 2010). Estrogen receptor alpha 66 (ER\u0026alpha;66) and estrogen receptor beta (ER\u0026beta;), the two types of nuclear estrogen receptors (nERs), are firstly recognized for their ability to influence the development of female characteristics including breast cancer mediated by genomic estrogen pathways (Xu et al. 2017a). And naringenin has been shown to exert its antiproliferative and pro-apoptotic effects only in the presence of either ER\u0026alpha;66 or ER\u0026beta; (Totta et al. 2004). Interestingly, our recent studies have demonstrated that naringenin inhibited cell growth at higher doses (\u0026ge; 200 \u0026micro;M) also involved in membrane estrogen receptors (mERs)-mediated pathways in ER\u0026alpha;66-positive breast cancer MCF-7 cells (Xu et al. 2018). However, whether naringenin showed similar effects in ER\u0026alpha;66-negative like ER\u0026alpha;66-positive breast cancer cells, and whether mERs are associated with such growth inhibition remains to be investigated. As the name implies, mERs differ from nERs in that they are primarily localized to the cytoplasm and plasma membrane and mediate membrane-initiated non-genomic signaling pathways (Xu et al. 2017a). Estrogen receptor alpha 36 (ER\u0026alpha;36) and G protein-coupled estrogen receptor 30 (GPR30) are two typical mERs. These two mERs could mediate the activation of phosphatidylinositol 3-kinase (PI3K)/serine/threonine kinase (Akt) and mitogen-activated protein kinase (MAPK)/extracellular regulated protein kinases 1/2 (ERK1/2) signaling pathways, resulting in cell proliferation.(Chimento et al. 2014, Lin et al. 2010)\u003c/p\u003e\n\u003cp\u003eIn the present study, naringenin was screened at different doses and incubation times in two kinds of ER\u0026alpha;66-negative breast cancer cells, SKBR3 and MDA-MB-231. Our results suggest naringenin could induce cell proliferation at lower doses but inhibit cell growth at higher doses in both SKBR3 and MDA-MB-231 cells. These bidirectional dose effects are associated with the regulation of cell migration, cell cycle arrest, cell apoptosis, and ROS generation that is mediated by GPR30 and ER\u0026alpha;36.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003ch2\u003e2.1. Chemicals and reagents\u003c/h2\u003e\n\u003cp\u003e17\u0026beta;-estradiol (E2) and naringenin standards were purchased from Sigma-Aldrich (St Louis, MO, USA). Stock solutions at 100 \u0026mu;M and 200 mM, respectively, were prepared in dimethyl sulfoxide (DMSO) and stored at \u0026minus;20 \u0026deg;C. The working solutions were freshly diluted in the basal medium. Dulbecco\u0026apos;s modified Eagle\u0026apos;s medium (DMEM), phosphate-buffered saline (PBS), heat-inactivated fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Thermo Fisher Scientific (USA). Charcoal-dextran stripped fetal bovine serum (CS-FBS) was ordered from Gemini Bio-Products (West Sacramento, CA, USA).\u003c/p\u003e\n\u003ch2\u003e2.2. Cell lines and cell culture\u003c/h2\u003e\n\u003cp\u003eBreast adenocarcinoma cell lines SKBR3 and MDA-MB-231 were obtained from ATCC (American Type Culture Collection, Manassas, VA, USA) and preserved by our laboratory. These cell lines were selected based on different phenotypes for signaling pathways to elucidate if certain pathways were required. Cells were routinely maintained in phenol red DMEM, supplemented with 10% FBS and 1% penicillin-streptomycin at 37 \u0026deg;C in a water-saturated atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e incubator.\u003c/p\u003e\n\u003cp\u003eBefore starting the exposure experiments, cell media were changed to phenol red-free DMEM containing 10% CS-FBS 48 h to deplete the influence of phenol and endogenous steroids (Berthois et al. 1986). Cells in the logarithmic growth phase were used to determine the proliferative or antiproliferative effects of naringenin. For all \u003cem\u003ein vitro\u003c/em\u003e assays, cells were treated with increasing doses of naringenin dissolved in phenol red-free DMEM containing 5% CS-FBS for specific times.\u003c/p\u003e\n\u003ch2\u003e2.3. Cell proliferation and cytotoxicity assessment\u003c/h2\u003e\n\u003cp\u003eCell proliferation and cytotoxicity were evaluated using both a CCK-8 assay and a real-time cell impedance analyzer (RTCA) according to our previous studies (Xu et al. 2018). Briefly, SKBR3 and MDA-MB-231 cells were seeded into 96-well microplates and 16-well E-plates at a cell density of 1\u0026times;10\u003csup\u003e4\u003c/sup\u003e and 6\u0026times;10\u003csup\u003e3\u003c/sup\u003e cells/well, respectively. Following incubation to adhere for 24 h, cells were treated with various doses of naringenin ranging from 1 to 1000 \u0026mu;M or its vehicle in phenol red-free DMEM containing 5% CS-FBS for 12\u0026minus;72 h (up to 96 h). 10 nM E2 was used as the positive control, and DMSO (0.5%) was used as the untreated control.\u003c/p\u003e\n\u003cp\u003eFor the CCK-8 assay, after being incubated with naringenin for a specific time, cells were then incubated with 20 \u0026mu;L CCK-8 solution per well for another 3 h at 37 \u0026deg;C. In the end, the absorption values were measured at 450 nm using a microplate reader (SpectraMax M5, Molecular Devices, CA, USA). To verify that ER\u0026alpha;36 and GPR30 signaling pathways are involved in naringenin-regulated antiproliferative effects, cells were treated with Broussoflavonol B and G36, respectively, for 1 h before naringenin addition. The results are reported as a percentage compared with the controls calculated from their relative absorbance, so 100% indicates no cytotoxicity. For RTCA analysis, the impedance was recorded every 5 minutes for up to 96 h, and these data were normalized as the last time point (approximately at 24 h) before objectives addition through the integrated software previously reported (Xu et al. 2018). \u003c/p\u003e\n\u003cp\u003eThe cytotoxicity of naringenin at higher doses to SKBR3 and MDA-MB-231 cells was also assessed using a Lactate dehydrogenase cytotoxicity kit (LDH, Beyotime, China). Following treatment with naringenin for 48 h, the supernatant was collected to assess the LDH activity following the manufacturer\u0026rsquo;s instructions. Morphological changes at 48 h were also observed using an inverted fluorescence microscope (IX73, Olympus) at a magnification of 200 \u0026times;.\u003c/p\u003e\n\u003ch2\u003e2.4. Colony formation assay\u003c/h2\u003e\n\u003cp\u003eThe SKBR3 (50 cells/well) and MDA-MB-231 (25 cells/well) were seeded in 6-well plates in triplicate. Following incubation to adhere for 24 h, cells were treated with various low concentrations of naringenin (1, 5 and 10 \u0026mu;M). Then, cells were allowed to grow at 37 \u0026deg;C in 5% CO\u003csub\u003e2\u003c/sub\u003e for 14 days, and the culture medium was changed every 2 days. In the end, cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet, and the colonies were photographed.\u003c/p\u003e\n\u003ch2\u003e2.5. Cell migration assessment\u003c/h2\u003e\n\u003cp\u003eCell migration was evaluated using the wound healing assay as described previously with some specific modifications (Xu et al. 2018). Briefly, SKBR3 and MDA-MB-231 cells were seeded in a 12-well plate at a density of 4\u0026times;10\u003csup\u003e5\u003c/sup\u003e and 2\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well, respectively. After forming a confluent monolayer, it was scratched with a sterile pipette tip (10 \u0026mu;L) and washed with a serum-free medium to remove the floating and detached cells. Afterward, cells were treated with lower doses (1 and 10 \u0026mu;M) and a higher dose (200 \u0026mu;M) of naringenin according to the results from the cell viability assay. The digitized images at 0, 12, 24, 36, and 48 h were monitored using an inverted fluorescence microscope (IX73, Olympus Corporation, Tokyo, Japan) at a magnification of 100 \u0026times;, and then analyzed with Image-Pro Plus software (Media Cybernetics, L.P., Silver Spring, MA, USA).\u003c/p\u003e\n\u003ch2\u003e2.6. Cell cycle and apoptosis analysis\u003c/h2\u003e\n\u003cp\u003eCell cycle and apoptosis were analyzed using the Cell cycle and apoptosis analysis kit (Beyotime, China) and Annexin V-FITC apoptosis detection kit (BD Biosciences, San Jose, CA, USA), respectively, according to the manufacturer\u0026apos;s instructions. Briefly, SKBR3 and MDA-MB-231 cells were seeded in 6-well plates at a density of 6\u0026times;10\u003csup\u003e5\u003c/sup\u003e and 3\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well, respectively. Following incubation to adhere for 24 h, cells were treated with naringenin the same as cell migration for 48 h in the condition of 5% CS-FBS. At the end of the time point, both adherent and floating cells were collected into a flow cytometry tube and centrifuged at 1000 g for 5 min to obtain cell pellets. Thereafter, cells were washed with precooling PBS.\u003c/p\u003e\n\u003cp\u003eFor cell cycle analysis, cells were firstly fixed in 70% ethanol overnight at 4 \u0026deg;C. And then, fixed cells were washed twice with precooling PBS and incubated with propidium iodide (PI) staining solution for 30 min at 37 \u0026deg;C. For cell apoptosis, cells were incubated with Annexin V-FITC/PI double staining solution. Both cell cycle and cell apoptosis were measured with NovoCyte\u0026trade; Flow Cytometer (ACEA Biosciences, California, USA) within 30 min. In each analysis, over 10, 000 events were recorded. The percentages of cell distribution for cell cycle (Sub-G0, G0/G1, S-phase, and G2/M DNA content) and cell apoptosis (early apoptosis and late apoptosis) were calculated using the built-in analysis software.\u003c/p\u003e\n\u003ch2\u003e2.7. Measurement of ROS generation, SOD activity and GSH content\u003c/h2\u003e\n\u003cp\u003eIntracellular reactive oxygen species (ROS) were examined with 2,7-dichloride-hydro fluorescein diacetate (DCFH-DA)-fluorescence probe (10 \u0026mu;M, Beyotime) as previously reported (Xu et al. 2018). Briefly, cells were treated with different doses of naringenin for 48 h. And then, cells were harvested, washed, and incubated with a DCFH-DA probe that was freshly prepared with the serum-free medium at 37 ℃ for 30 min in the dark. Subsequently, cells were washed with serum-free medium thrice to remove the unloaded probes, and the fluorescence was analyzed with the NovoCyte\u0026trade; Flow Cytometer (ACEA Biosciences).\u003c/p\u003e\n\u003cp\u003eSuperoxide dismutase (SOD) activity and glutathione reductase (GR) activity were determined with a total superoxide dismutase assay kit with NBT (Beyotime, China) and glutathione reductase assay kit (Nanjing JianCheng Bio Institute, Nanjing, China), respectively, according to previous studies.(Kapoor and Kakkar 2014) Briefly, following treatment with various doses of naringenin for 48 h, cells were collected and resuspended in PBS, and the lysates were prepared under ultrasonic and centrifuged. The supernatants were quantified using a BCA protein assay (Beyotime, China). Then, the lysates were used to determine the SOD and GR activities, and all results were normalized to protein content.\u003c/p\u003e\n\u003ch2\u003e2.8. RNA purification and RT-qPCR\u003c/h2\u003e\n\u003cp\u003eTo determine gene expression changes involved in cell proliferation and anti-proliferation, a real-time quantitative polymerase chain reaction (RT-qPCR) was performed to examine the gene expression in mRNA transcription levels. Briefly, SKBR3 and MDA-MB-231 cells were seeded in 6-well plates at a density of 6\u0026times;10\u003csup\u003e5\u003c/sup\u003e and 3\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/well, respectively. Following tread with naringenin, cells were washed with PBS and the total RNA was isolated from cell samples using the RNAiso Plus (Takara, Dalian, China) according to our previous studies (Xu et al. 2018). And then the first strand of cDNA was reverse-transcribed using the PrimeScript-RT reagent kit (Takara, Dalian, China). The mRNA transcription levels were analyzed using validated primers and SYBR \u003cem\u003ePremix Ex Taq\u003c/em\u003e\u003csup\u003eTM\u003c/sup\u003e Ⅱ (Tli RNaseH Plus) (TakaRa, Dalian, China) in the StepOnePlus Real-time PCR System (Applied Biosystems, CA, USA). Specific primers are shown in \u003cstrong\u003eTable S1\u003c/strong\u003e. The cycler was programmed with the following conditions: (i) initial denaturation at 95 \u0026deg;C for 30 sec, followed by 40 cycles, (ii) 95 \u0026deg;C for 5 sec, (iii) annealing of the primer-template at 60 \u0026deg;C for 30 sec. \u0026beta;-actin in the same incubations was used for internal normalization, and the mRNA levels were determined using the 2\u003csup\u003e\u0026minus;\u0026Delta;\u0026Delta;Ct\u003c/sup\u003e method as described in our previous studies (Xu et al. 2018).\u003c/p\u003e\n\u003ch2\u003e2.9. Western blotting\u003c/h2\u003e\n\u003cp\u003eWestern blotting was performed to analyze the protein expression levels of mERs (ER\u0026alpha;36 and GPR30) following treatment with different concentrations of naringenin (1, 10, and 200 \u0026mu;M) for 48 h. The details have been described previously (Xu et al. 2018). The information on antibodies used is shown in \u003cstrong\u003eTable S2\u003c/strong\u003e. Protein bands were quantified with the Image-Pro Plus software (Media Cybernetics, L.P., Silver Spring, MA, USA).\u003c/p\u003e\n\u003ch2\u003e2.10. Statistical analysis\u003c/h2\u003e\n\u003cp\u003eAll experiments were conducted thrice independently. Data were analyzed with SPSS 17.0 (Chicago, IL), and results were presented as the mean \u0026plusmn; standard deviation (SD). Statistical significance was assessed by one-way analysis of variance (ANOVA), followed by the \u003cem\u003epost-hoc\u003c/em\u003e test analysis. Differences at \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 were considered statistically significant.\u003c/p\u003e"},{"header":"3. Results","content":"\u003ch2\u003e3.1. Lower-dose naringenin induces cell proliferation but higher doses inhibit cell growth\u003c/h2\u003e\n\u003cp\u003eTo investigate the influence of naringenin on the growth of ER\u0026alpha;66-negative breast cancer cells, SKBR3 and MDA-MB-231 cells were treated with increasing doses of naringenin ranging from 0.1 to 1000 \u0026mu;M for 48 h, and the cell viability was first analyzed with CCK-8 assay, as shown in \u003cstrong\u003eFig. 1A\u003c/strong\u003e. Interestingly, different effects were observed on cell viability. Naringenin at low doses (0.1\u0026minus;20 \u0026mu;M) promoted cell proliferation in comparison with the control group. And the maximal proliferative effects of naringenin were achieved at 10 \u0026mu;M for SKBR3 cells (112.59\u0026plusmn;1.71%) and 1 \u0026mu;M for MDA-MB-231 cells (111.69\u0026plusmn;1.67%), respectively, which were almost equivalent to the effect of 10 nM E2 \u003cstrong\u003e(Fig. 1A)\u003c/strong\u003e. However, naringenin significantly inhibited cell growth in both cell lines when the exposure doses were high than 50 \u0026mu;M. Similar results were also observed with the RTCA assessment \u003cstrong\u003e(Fig. 1B\u003c/strong\u003e\u003cstrong\u003e\u0026ndash;C)\u003c/strong\u003e. Particularly, naringenin at low concentrations significantly stimulated cell growth in both SKBR3 and MDA-MB-231 cells. The colony-forming assay was also conducted to testify that low concentrations of naringenin can increase the proliferation of ER\u0026alpha;66-negative breast cancer cells, results are shown in \u003cstrong\u003eFig. 1E\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eSince higher doses of naringenin could significantly inhibit cell proliferation, cells were then treated with higher doses of naringenin at 100, 200 and 500 \u0026mu;M for 12\u0026minus;72 h to investigate their timely responses, and results showed naringenin decreased cell viability in both time- and dose-dependent manners of both SKBR3 and MDA-MB-231 cell lines \u003cstrong\u003e(Fig. 1D)\u003c/strong\u003e. Similar to other results (Islas et al. 2015), the half-maximal inhibitory concentration (IC50) of naringenin for SKBR3 and MDA-MB-231 cells was more than 200 \u0026mu;M, accounting for 362\u0026plusmn;15 \u0026mu;M and 410\u0026plusmn;26 \u0026mu;M, respectively. LDH is located predominantly in the cytoplasm and its presence in the extracellular medium serves to detect disruption of cell membrane integrity (Dong et al. 2017). To further confirm the cytotoxic effects of higher doses of naringenin on cells, the LDH assay was also performed as another indicator of cytotoxicity. Results demonstrated that higher-doses naringenin induced a marked dose-dependent increase in LDH leakage of both SKBR3 and MDA-MB-21 cells for 72 h \u003cstrong\u003e(Fig. 1F)\u003c/strong\u003e. Thus, higher-doses naringenin enhanced against cytotoxicity by increasing LDH release and this activation was higher in the SKBR3 cell line. From \u003cstrong\u003eFig. 1G\u003c/strong\u003e, an inverse linear correlation was observed between LDH release and cell viability in SKBR3 (R\u003csup\u003e2\u003c/sup\u003e = 0.9956) and in MDA-MB-231 (R\u003csup\u003e2\u003c/sup\u003e = 0.9764) cells following treatment with higher doses of naringenin. Furthermore, the microscopic evaluation also revealed that cells exposed to 200 \u0026mu;M naringenin reduced significantly of the cell attachment number, normal morphology and adhesion capacity compared to those of the experimental control cells \u003cstrong\u003e(Fig. 1H)\u003c/strong\u003e. The above results demonstrated that cells treatment with lower doses of naringenin increased cell viability in both SKBR3 and MDA-MB-231 cells, and that exposure to higher doses of naringenin inhibit cell growth.\u003c/p\u003e\n\u003cp\u003eFig. 1 Naringenin regulated cell proliferation and cytotoxicity in SKBR3 and MDA-MB-231 cells\u003c/p\u003e\n\u003ch2\u003e3.2. Lower-dose naringenin promotes cell migration but higher doses inhibit it in SKBR3 and MDA-MB-231 cells\u003c/h2\u003e\n\u003cp\u003eTo investigate whether naringenin regulates cell migration, both SKBR3 and MDA-MB-231 cells were treated with naringenin at 1, 10 and 200 \u0026mu;M based on the results of cell viability for 12\u0026minus;48 h, and then the relative migration rates were evaluated with a wound-healing assay. In general, naringenin regulated cell migration in a cell type-, dose-, and time-dependent manner, as shown in \u003cstrong\u003eFig. 2A\u003c/strong\u003e\u003cstrong\u003e\u0026ndash;C\u003c/strong\u003e. Herein, the wound healing percentage of untreated SKBR3 cells increased to (17.63 \u0026plusmn; 1.21) % at 48 h, but higher healing ratios were observed for MDA-MB-231 cells in which the healing percentages increased to (59.90 \u0026plusmn; 1.25) %, (70.07 \u0026plusmn; 1.37) % and (99.79 \u0026plusmn; 1.24) % for 12, 24 and 36 h, respectively. Lower doses of naringenin (1 and 10 \u0026mu;M) marginally stimulated the cell migration of SKBR3 cells at 24 and 48 h, whereas no significant changes in cell migration were observed in MDA-MB-231 cells in all periods except for 24 h. Different from the potential stimulatory effects of naringenin at low-dose exposure, higher-dose naringenin significantly inhibited cell migration in both SKBR3 and MDA-MB-231 cells. However, naringenin exerted more relative inhibitory effects on cell migration in MDA-MB-231 cells than in SKBR3 cells. In detail, the percentage of migrated cells at the ending point increased from (1.30 \u0026plusmn; 0.44) % to (11.10 \u0026plusmn; 1.04) % for SKBR3 cells but only increased from (9.72 \u0026plusmn; 0.99) % to (29.40 \u0026plusmn; 1.19) % for MDA-MB-231 cells, respectively.\u003c/p\u003e\n\u003cp\u003eThen the migration-linked protein E-cadherin, N-cadherin, MMP-2 and MMP-9 were further assessed, as illustrated in \u003cstrong\u003eFig. 2D\u003c/strong\u003e. In line with the migration inhibition, the mRNA transcription levels of E-cadherin were significantly increased when cells were treated with higher-dose naringenin (200 \u0026mu;M), whereas the N-cadherin, MMP-9 and MMP-2 were decreased in both SKBR3 and MDA-MB-231 cells. As for low-dose exposure, SKBR3 cells treated with 1 \u0026mu;M naringenin significantly down-regulated the mRNA transcription of E-cadherin, but it markedly up-regulated the transcription levels of N-cadherin, MMP-9 and MMP-2 at 10 \u0026mu;M. Different regulatory manners have been found in MDA-MB-231 cells. The mRNA transcription levels of E-cadherin, N-cadherin and MMP-2 were markedly decreased, but no significance was found in the expression of MMP-9. As a whole, these results indicate that cell migration regulation is one of the ways through which naringenin regulated cell viability on ER\u0026alpha;66-negative breast cancer cells.\u003c/p\u003e\n\u003cp\u003eFig. 2 Effects of naringenin on cell migration in SKBR3 and MDA-MB-231 cells\u003c/p\u003e\n\u003ch2\u003e3.3. Naringenin shows dual effects on cell cycle in SKBR3 and MDA-MB-231 cells\u003c/h2\u003e\n\u003cp\u003eTo evaluate the impacts of naringenin on the cell cycle in SKBR3 and MDA-MB-231 cells, flow cytometry was applied after cells were treated with increasing doses of naringenin ranging from 1 to 200 \u0026mu;M for 24 and 48 h. As shown in \u003cstrong\u003eFig. 3A \u0026amp; Fig. S1\u003c/strong\u003e, despite no significant differences were observed in cell cycle distribution for SKBR3 cells after being treated with lower doses of naringenin (1 and 10 \u0026mu;M) for 24 h and 48 h compared with the control group, higher doses of naringenin (100 and 200 \u0026mu;M) caused a significant increase of cell population in the S and G2/M phases. Similar results have been observed in MDA-MB-231 cells, namely naringenin (1\u0026minus;200 \u0026mu;M) increased the percentage of cells in the S and G2/M phases in comparison to that of the control group, concomitant with this increase was a decrease in the percentage of cells in the G0/G1 phase \u003cstrong\u003e(Fig. 3B \u0026amp; Fig. S1)\u003c/strong\u003e. However, it seemed that MDA-MB-231 cells were more sensitive to naringenin than SKBR3 cells since 10 \u0026mu;M naringenin began to arrest cells in the S phase. These results are following the data obtained from the cell proliferation analysis. These results demonstrated that naringenin possibly exerted its effect on cell anti-proliferation at S phase entry.\u003c/p\u003e\n\u003cp\u003eBased on the results of cell cycle distribution, several cell cycle-regulatory genes, including cyclin A, cyclin B, cyclin D and cyclin D, were analyzed in mRNA transcription levels. As shown in \u003cstrong\u003eFig. 3C\u003c/strong\u003e, a higher-dose naringenin (200 \u0026mu;M) exposure significantly increased the mRNA transcription levels of cyclin A and cyclin E, but it markedly decreased the mRNA transcription levels of cyclin B and cyclin D, which was consistent with the accumulation of cells in the S-phase. However, no significant difference has been found in cell cycle-regulated genes when cells were treated with lower doses of naringenin (1 and 10 \u0026mu;M).\u003c/p\u003e\n\u003cp\u003eFig. 3 Effects of naringenin on cell cycle distribution in SKBR3 and MDA-MB-231 cells\u003c/p\u003e\n\u003ch2\u003e3.4. Dose-dependent effects of naringenin on cell apoptosis in SKBR3 and MDA-MB-231 cells\u003c/h2\u003e\n\u003cp\u003eWe also investigated the influence of naringenin on cell apoptosis in SKBR3 and MDA-MB-231 cells. From \u003cstrong\u003eFig. 4A\u003c/strong\u003e\u003cstrong\u003e\u0026ndash;B\u003c/strong\u003e, lower doses of naringenin (1 and 10 \u0026mu;M) inhibited cell apoptosis both in SKBR3 (48 h) and MDA-MB-231 cells (24 and 48 h), whereas naringenin at higher doses (\u0026ge; 100 \u0026mu;M) promoted cell apoptosis in a dose-dependent manner. Moreover, the promotion of those inhibitive effects showed a time-dependent manner. For instance, 1\u0026minus;10 \u0026mu;M naringenin inhibited cell apoptosis in both SKBR3 and MDA-MB-231 cells compared to that of the control group. On the contrary, following treatment with 200 \u0026mu;M naringenin for 48 h, the percentage of viable cells was significantly decreased, whereas the apoptotic cells were increased from (16.16 \u0026plusmn; 1.22) % to (65.56 \u0026plusmn; 1.37) % for SKBR3 cells and from (16.92 \u0026plusmn; 0.16) % to (37.05 \u0026plusmn; 0.94) % for MDA-MB-231 cells, respectively.\u003c/p\u003e\n\u003cp\u003eWe then examined the expression changes of four important apoptosis-related genes (\u003cem\u003ei.e.\u003c/em\u003e, p21, p53, Bcl-2 and Bax) to explore whether this apoptosis regulation was regulated by mitochondrial pathways. As shown in \u003cstrong\u003eFig. 4C\u003c/strong\u003e, when cells were treated with lower-dose naringenin (1 and 10 \u0026mu;M), the mRNA transcription levels of proapoptotic genes (\u003cem\u003ei.e\u003c/em\u003e., p21, p53 and Bax) were significantly decreased, whereas the Bcl-2 was increased. Opposite results were observed when cells were treated with higher-dose naringenin (200 \u0026mu;M). Moreover, Bcl-2/Bax ratios were significantly increased following treatment with 1 and 10 \u0026mu;M naringenin but decreased following treatment with 200 \u0026mu;M naringenin. These results are consistent with the apoptosis status determined by flow cytometry. Therefore, apoptosis regulation is one of the ways through which naringenin regulated cell survival or death.\u003c/p\u003e\n\u003cp\u003eFig. 4 Effects of naringenin on cell apoptosis in SKBR3 and MDA-MB-231 cells\u003c/p\u003e\n\u003ch2\u003e3.5. Naringenin regulates ROS generation and oxidative stress in SKBR3 and MDA-MB-231 cells\u003c/h2\u003e\n\u003cp\u003eTo investigate whether naringenin induces oxidative stress, the effects of naringenin on ROS production were studied by employing DCFH-DA fluorescent dye. As shown in \u003cstrong\u003eFig. 5A\u003c/strong\u003e, the ROS was significantly increased after being treated with higher doses of naringenin for 48 h compared to that of the control group, whereas lower doses of naringenin decreased the ROS levels. In line with the results of ROS levels, although no significant changes were found when cells were treated with lower doses of naringenin, higher doses of naringenin treatment resulted in a significant depletion of SOD \u003cstrong\u003e(Fig. 5B) \u003c/strong\u003eand intracellular GR activity \u003cstrong\u003e(Fig. 5C) \u003c/strong\u003ein both SKBR3 and MDA-MB-231 cells. These results indicate that naringenin especially at higher doses could affect the cellular redox status.\u003c/p\u003e\n\u003cp\u003eFig. 5 Effects of naringenin on ROS generation, SOD and GSH in SKBR3 and MDA-MB-231 cells\u003c/p\u003e\n\u003ch2\u003e3.6. Naringenin modulates mERs expression in SKBR3 and MDA-MB-231 cells\u003c/h2\u003e\n\u003cp\u003eIt has been reported that naringenin can bind to nERs (ER\u0026alpha;66 and ER\u0026beta;) and acts as a mimetic of E2 to activate the pro-apoptotic cascades in the presence of either ER\u0026alpha;66 or ER\u0026beta; (Totta et al. 2004). Unlike the ER\u0026alpha;66-positive breast cancer cells, SKBR3 and MDA-MB-231 belong to ER\u0026alpha;66-negative breast cancer cells but they still expressed two membrane estrogen receptors (mERs), ER\u0026alpha;36 and GPR30. To further characterize the molecular mechanisms, we examined the influence of naringenin on mERs expression at both mRNA and protein levels in SKBR3 and MDA-MB-231 cells. As shown in \u003cstrong\u003eFig. 6A\u0026ndash;B\u003c/strong\u003e, naringenin regulated the mRNA transcription of ER\u0026alpha;36 and GPR30 in a dose-dependent manner. In detail, naringenin at 1 and 10 \u0026mu;M significantly down-regulated the mRNA transcription of ER\u0026alpha;36 and GPR30 in both SKBR3 and MDA-MB-231 cells compared with the control group. In contrast, naringenin at 200 \u0026mu;M markedly up-regulated the mRNA transcription of ER\u0026alpha;36 by up to 15.25 \u0026plusmn; 2.49% in SKBR3 cells but significantly decreased it in MDA-MB-231 cells. Opposite regulatory effects were observed on GPR30 transcription in SKBR3 and MDA-MB-231 cells, namely 200 \u0026mu;M naringenin treatments significantly up-regulated the mRNA transcription of GPR30 in MDA-MB-231 cells but slightly increased in SKBR3 cells (\u003cem\u003ep\u003c/em\u003e＞0.05). Therefore, naringenin mediates ER\u0026alpha;66-negative breast cancer cell growth via mERs signaling pathways.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 6C\u0026ndash;D\u003c/strong\u003e showed that naringenin regulated the protein expression of ER\u0026alpha;36 and GPR30 similar to mRNA transcription levels. It is noteworthy that the participation levels of ER\u0026alpha;36 and GPR30 in SKBR3 and MDA-MB-231cells have more or fewer differences. Generally, the downregulation of ER\u0026alpha;36 and GPR30 contributed to cell proliferation in both SKBR3 and MDA-MB-231 cells when they were exposed to low concentrations of naringenin. However, an upregulation of either ER\u0026alpha;36 or GPR30 would result in cell antiproliferative effects induced by higher concentrations of naringenin in SKBR3 and MDA-MB-231 cells, respectively. To verify such antiproliferative effects of naringenin are mediated by ER\u0026alpha;36 and GPR30, we treated cells with Broussoflavonol B (ER\u0026alpha;36 antagonist) and G36 (GRP30 antagonist), respectively, for 1 h before the addition of 200 \u0026mu;M naringenin. And then the cell viability was analyzed with CCK-8 assays \u003cstrong\u003e(Fig. 6E\u0026ndash;F)\u003c/strong\u003e. As expected, SKBR3 cells pretreated with Broussoflavonol B inhibited cell antiproliferation induced by high-concentration naringenin to some extent. Similar results were found in MDA-MB-231 cells when pretreated with G36. These results indicate that naringenin-induced cell apoptosis depends on high expression levels of both ER\u0026alpha;36 and GPR30 in SKBR3 cells, and high GPR30 expression in MDA-MB-231 cells, respectively.\u003c/p\u003e\n\u003cp\u003eFig. 6 Naringenin regulated mERs expression in SKBR3 and MDA-MB-231 cells\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eAs a heterogeneous disease with distinct clinical behavior and molecular properties, breast cancer can be divided into two categories based on the presence or absence of ER\u0026alpha;, namely ER\u0026alpha;66-positive and ER\u0026alpha;66-negative cancers (Rouzier et al. 2005). While the majority of breast cancers belong to ER\u0026alpha;66-positive, approximately 25\u0026ndash;30% of cases are ER\u0026alpha;66-negative (Chimento et al. 2014). Compared with the ER\u0026alpha;66-positive breast cancer patients, the ER\u0026alpha;66-negative classes have shorter disease-free intervals and worse overall survival (Pan et al. 2014). Several previous studies have shown that naringenin exerted antiproliferative effects or even caused cell death in various cancer cell lines including the ER\u0026alpha;66-positive MCF-7 cells (Ahamad et al. 2014, Arul and Subramanian 2013, Shi et al. 2015). Herein, SKBR3 and MDA-MB-231 cells were selected as the \u003cem\u003ein vitro\u003c/em\u003e models to investigate the potential regulatory effects of naringenin on ER\u0026alpha;66-negative breast cancer progression in our present study. CCK-8 and RTCA results showed naringenin induced different effects on the cell growth of the above two kinds of ER\u0026alpha;66-negative cells. In detail, naringenin at lower doses (\u0026le; 10 \u0026mu;M) induced cell proliferation, but high-doses naringenin (\u0026ge; 50 \u0026mu;M) significantly inhibited it in a dose- and time-dependent manner. These results suggest that naringenin at higher doses possesses chemotherapeutic potential against human ER\u0026alpha;66-negative breast cancer but lower doses would promote cancer development. Several specific cellular biological behaviors and molecular signaling pathways involved in cell growth promotion or inhibition were further studied.\u003c/p\u003e\n\u003cp\u003eIn agreement with the cell viability results, morphology and cell migration assessment showed lower doses of naringenin (1 and 10 \u0026mu;M) induced cell migration to some extent as well as increased cell numbers but without any changes in cell morphology. Conversely, cell attachment number and morphology, and migration capacity of both SKBR3 and MDA-MB-231 cells significantly declined following treatment with higher doses of naringenin (200 \u0026mu;M). It is worth mentioning that the migration capacity of MDA-MB-231 cells was greater than that of SKBR3 cells due to their different growth characteristics (Alamer and Darbre 2018). Epithelial to mesenchymal transition (EMT) plays a crucial role in cell migration and metastasis of breast cancer, and the characteristic feature of EMT is the switch of the epithelial E-cadherin to mesenchymal N-cadherin (Wei et al. 2018, Weng and Yen 2012). Consistent with this theory, higher-dose naringenin treatment resulted in an increase of E-cadherin but a decrease of N-cadherin in ER\u0026alpha;66-negative cells, whereas contrary results were obtained when cells were treated with lower doses of naringenin. The activation of matrix metalloproteinases (MMPs), including MMP-2 and MMP-9, could also facilitate cells to invade the extracellular matrix, resulting in the promotion of tumor metastasis and deterioration (Kessenbrock et al. 2010, Weng and Yen 2012). As expected, lower-doses naringenin up-regulated the expression of MMP-2 and MMP-9 in SKBR3 cells but not in MDA-MB-231 cells, whereas higher-doses naringenin significantly down-regulated their expression. Similar results have been found by Magee and co-workers in MDA-MB-231 cells treated with genistein (Magee et al. 2004). In addition, the p21/p53 tumor transcription factor was also considered to suppress cell migration and invasion (Kim et al. 2017), which is consistent with the cell migration results of our present study.\u003c/p\u003e\n\u003cp\u003eDysregulation of the cell cycle is a hallmark of tumorigenesis and development (Evan and Vousden 2001). Naringenin has been found to induce cell cycle arrest in various cells (\u003cem\u003ee.g.\u003c/em\u003e, THP-1, HepG2 and A431 cells) but in different manners (Ahamad et al. 2014, Arul and Subramanian 2013, Shi et al. 2015), resulting in different cell antiproliferative effects. Herein, higher doses of naringenin (100 and 200 \u0026mu;M) caused a significant accumulation of cell population in the S and G2/M phases along with a decrease in the G0/G1 phase in both SKBR3 and MDA-MB-231 cells. Moreover, no significant difference was observed in cell cycle distribution for SKBR3 cells following treatment with lower doses of naringenin (1 and 10 \u0026mu;M) for 24 h and 48 h compared with the control group, whereas it still gently increased the percentage of cells in the S and G2/M phases in MDA-MB-231cells. The cell cycle, of course, is controlled at multiple checkpoints which are regulated by various kinds of cell cycle-regulating proteins, including cyclin A, cyclin B, cyclin D and cyclin E (Evan and Vousden 2001, Xie et al. 2016, Zhao et al. 2017). Consistent with the cell cycle distribution results, cyclin A and cyclin E were significantly increased following treatment with 200 \u0026mu;M naringenin, whereas the expression of cyclin D and cyclin B were markedly decreased. Additionally, no significant changes in mRNA transcription of cycle-regulatory genes were observed.\u003c/p\u003e\n\u003cp\u003eOther than cell cycle phase arrest, cell apoptosis is also involved in cell growth regulation (Evan and Vousden 2001). Apoptosis is a common form of cell death morphologically characterized by cellular shrinkage, chromatin condensation, DNA fragmentation, and cell disruption into apoptotic bodies (Kapoor and Kakkar 2014). The death receptor pathway and the mitochondria pathway are two well-established mechanisms involving apoptotic cell death (Dong et al. 2017). Among these, the death receptor pathway is mediated by caspases, and the mitochondrial-dependent apoptosis is regulated by the Bcl-2 family proteins, particularly Bax and Bcl-2 (Adams and Cory 1998). As important tumor suppressor genes, p21, p53 and their p53/p21 complex can induce cell apoptosis by regulating the expression of Bcl-2 and Bax, thus suppressing tumor cell proliferation and preventing tumor development (Fridman and Lowe 2003, Kim et al. 2017). In the present study, when cells were exposed to high doses-induced oxidative stress for 24\u0026ndash;48 h, cell apoptosis induction became evident owing to a decline of Bcl-2 protein, but to an elevation of Bax, p21 and p53 proteins. Moreover, changes in Bax/Bcl-2 ratio also result in significant activation of caspases and lead to cell death through the mitochondrial death pathway (Adams and Cory 1998). The Bcl-2/Bax ratio, of course, also markedly declined following treatment with 200 \u0026mu;M naringenin, whereas contrary results were observed when cells were exposed to lower doses of naringenin.\u003c/p\u003e\n\u003cp\u003eROS acted as an early signal in various biological circumstances, but an excessive generation of ROS that cannot be attenuated by intracellular redox systems would lead to a variety of biochemical and physiological lesions such as lipid peroxidation, protein oxidation, enzyme inactivation and oxidative DNA damage, resulted in cell cycle arrest and apoptosis induction as well as metastatic properties suppression (Islas et al. 2015, Simon et al. 2000). Previously, we found naringenin at a high level of 200 \u0026mu;M caused oxidative stress-induced apoptosis by p53 activation in MCF-7 cells, but pre-treatment with antioxidants blocked cell death induced by naringenin (Xu et al. 2018). An increased generation of ROS would destroy the mitochondrial membrane potential (MMPs), and then induce the release of LDH (Dong et al. 2017, Holder et al. 2012), and cytochrome c from mitochondria into the cytosol (Li et al. 2010). Moreover, the cytochrome c also triggered caspase-9 signaling pathways activation and ultimately caused cell death (Li et al. 2010). Those intracellular ROS production, SOD activity, GSH content and LDH release were then investigated. Results demonstrated that SKBR3 and MDA-MB-231 cells treated with higher doses of naringenin resulted in significant production of ROS and LDH release, but depletion of SOD and intracellular GSH compared to that of the control group. Contrary results were obtained when cells were treated with lower doses of naringenin. In addition, high levels of ROS also induced Bax expression but suppressed Bcl-2 expression through the activation of p38 and JNK signaling pathways, resulting in cell apoptosis (Chen et al. 2010). Herein, high doses of naringenin promoted Bax expression but suppressed Bcl-2 expression. The above results support the assumption that naringenin can induce cell growth inhibition in ER\u0026alpha;66-negative breast cancer cells through the increasing rates of apoptosis mediated by ROS accumulation.\u003c/p\u003e\n\u003cp\u003ePhytoestrogens usually act both as agonists and antagonists of estrogens at different concentrations (Totta et al. 2004). Naringenin can mimic natural steroid hormones (\u003cem\u003ee.g.\u003c/em\u003e, E2) and enable it to interact with ERs as an agonist or antagonist. Liu \u003cem\u003eet al.\u003c/em\u003e found that nERs (ER\u0026alpha;66 and ER\u0026beta;) played important roles in low-dose naringenin-induced cell growth (Liu et al. 2008). Generally, naringenin induced cell apoptotic cascade in the presence of either ER\u0026alpha;66 or ER\u0026beta; in ER-positive cells (Totta et al. 2004). Nevertheless, whether naringenin exerted the same effects in ER\u0026alpha;66-negative cancer as in ER\u0026alpha;66-positive cancer cells is unclear. ER\u0026alpha;36 as a truncated variant of the classical estrogen receptor\u0026mdash;ER\u0026alpha;66, is expressed in several breast cancer cells but not in normal mammary epithelial cells MCF10A (Zou et al. 2009). Compared with the ER\u0026alpha;66-positive breast cancer cells including MCF-7 and T47D, however, ER\u0026alpha;36 was highly expressed in ER\u0026alpha;66-negative breast cancer cells such as the SKBR3 and MDA-MB-231 cell lines (Zhang et al. 2012, Zou et al. 2009). The different expression patterns of ER\u0026alpha;66 and ER\u0026alpha;36 in these cancer cells further indicated that ER\u0026alpha;36 is transcriptionally regulated differently from ER\u0026alpha;66. Moreover, GPR30 was also highly expressed in ER\u0026alpha;66-negative breast cancer and participated in the proliferation/apoptosis processes (Zhou et al. 2016). Naringenin has been said to selectively impair the membrane-initiating signals of the ER\u0026alpha; subtype in earlier studies (Galluzzo et al. 2008). In the present study, lower-dose naringenin inhibited the expression of ER\u0026alpha;36 and GPR30 in both SKBR3 and MDA-MB-231 cells, but higher-dose naringenin promoted ER\u0026alpha;36 expression in SKBR3 cells and GPR30 in MDA-MB-231 cells, resulted in the regulation of cell growth and apoptosis, respectively. Similar results by others also showed that the activation of ER\u0026alpha;36 and GPR30 pathways are involved in the anti-proliferation and pro-apoptosis of both ER\u0026alpha;66-positive and ER\u0026alpha;66-negative breast cancer cells (Chimento et al. 2014, Pan et al. 2014, Wei\u0026szlig;enborn et al. 2014). Previous studies indicated that mERs, especially ER\u0026alpha;36 and GPR30, participated in cellular processes through rapid activation of second messenger pathways, such as an increase in intracellular free calcium or ROS. And then, they triggered functional responses or activated multiple downstream kinase cascades (\u003cem\u003ei.e.\u003c/em\u003e, PI3K/Akt and MAPKs), resulting in cell proliferation, differentiation, apoptosis, or survival (Xu et al. 2017a). The PI3K/Akt and MAPKs, such as ERK1/2, JNK and p38, played critical roles in converting extracellular stimuli into cellular responses (Bulzomi et al. 2010, Chen et al. 2010, Dhillon et al. 2007, Lin et al. 2010). Moreover, naringenin induced membrane ER\u0026alpha; rapid dissociation from caveolin-1 through depalmitoylation and then impeded it to binding adaptor and signaling proteins involved in the activation of the mitogenic signaling cascades (Galluzzo et al. 2008). All in all, the results obtained in our study and in that of others confirmed that naringenin induced apoptosis depends on high expression of ER\u0026alpha;36 and GPR30 in ER\u0026alpha;66-negative breast cancer cells, but the specific signaling pathways involved in naringenin-mediated (anti)-proliferative effects should be further explored in future.\u003c/p\u003e\n\u003cp\u003eFig. 7 Assumed schematic mechanisms of naringenin-mediated different proliferative effects involved in ROS generation, cell migration, cell cycle, and cell apoptosis in ER\u0026alpha;66-negative breast cancer cells\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eCollectively, our findings demonstrate that naringenin at higher doses induces cell anti-proliferation but lower-doses naringenin indeed promotes cell growth of both SKBR3 and MDA-MB-231 cells \u003cstrong\u003e(Fig. 7)\u003c/strong\u003e. Mechanistic investigation of the dual effects in the breast cancer cell lines reveals that higher-dose naringenin suppresses cell proliferation which is involved in cell migration inhibition, cell cycle arrest at the S phase and G2/M phase, cell apoptosis promotion, and ROS generation acceleration. However, lower-dose naringenin stimulates cell proliferation to some extent which was accompanied by 1) a decrease of E-cadherin expression but an increase of N-cadherin, MMP-9 and MMP-2, leading to cell migration, and 2) a decrease of ROS generation, p21, p53 and Bax expression, but an increase of Bcl-2 expression, resulted in cell apoptosis inhibition. Importantly, ER\u0026alpha;36 and GPR30 are involved in the (anti)-proliferation effects of naringenin in ER\u0026alpha;66-negative breast cancer cells. As naringenin possesses contrasting effects on ER\u0026alpha;66-negative breast cancer cell viability depending on its dose, caution is warranted in determining its effective dose in breast cancer treatment. Naringenin, of course, could be still an alternative treatment for ER\u0026alpha;66-negative breast cancer at high doses.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval\u003c/p\u003e\n\u003cp\u003eThis article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003eConsent to participate\u003c/p\u003e\n\u003cp\u003eAll participants of that article consent to participate this manuscript.\u003c/p\u003e\n\u003cp\u003eConsent to publish\u003c/p\u003e\n\u003cp\u003eAll participants of that article consent to publish this manuscript.\u003c/p\u003e\n\u003cp\u003eAuthor contributions\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eZhixiang Xu\u003c/strong\u003e developed the idea, designed the experimental approach, analyzed the data, prepared the figures, and drafted the manuscript. \u003cstrong\u003eSiyuan Hu\u003c/strong\u003e drafted the manuscript. \u003cstrong\u003eNao Luo\u003c/strong\u003e performed cell culture and flow cytometry assessment. \u003cstrong\u003eJun Liu\u003c/strong\u003e performed cell viability and gene expression determination. \u003cstrong\u003eXiaomin Ren\u003c/strong\u003e analyzed the results and prepared the figures. \u003cstrong\u003eXuejun Pan\u003c/strong\u003e conceived and designed the study, and reviewed the manuscript.\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research was supported by the National Natural Science Foundation of China (grants, 22176076, 21567014), and the China Post-doctoral Science Foundation (grant, 2019M653846XB).\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper\u003c/p\u003e\n\u003cp\u003eData availability statement\u003c/p\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdams JM, Cory S (1998) The Bcl-2 protein family: arbiters of cell survival. Science 281, 1322\u0026ndash;1326\u003c/li\u003e\n\u003cli\u003eAhamad MS, Siddiqui S, Jafri A, Ahmad S, Afzal M, Arshad M (2014): Induction of apoptosis and antiproliferative activity of naringenin in human epidermoid carcinoma cell through ROS generation and cell cycle arrest. 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FEBS Lett 583, 1368\u0026ndash;1374\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Naringenin, Bidirectional dose effects, In vitro, ERα66-negative, Reactive oxygen species, Membrane estrogen receptors","lastPublishedDoi":"10.21203/rs.3.rs-3266516/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3266516/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Naringenin is a flavanone able to suppress the growth of various cancer cells including estrogen receptor-alpha-66 (ERα66)-positive breast cancer cells, but the anti-tumorigenic roles of naringenin in ERα66-negative breast cancer remain unclear. This study aims to determine the potential effects of naringenin on two ERα66-negative breast cancer cells (SKBR3 and MDA-MB-231) and to define their mechanisms of action. Herein, several cellular biological behaviors, including cell viability, migration, cycle, apoptosis, reactive oxygen species (ROS) generation, and oxidative stress (e.g., LDH, SOD and GSH), were evaluated in the present study. Then the gene expression levels involved in cell migration, cycle and apoptosis, as well as two membrane estrogen receptors (ERα36 and GPR30) were analyzed with real-time quantitative polymerase chain reaction (RT-qPCR) assay and western blotting. Results showed that naringenin exerted a concentration-dependent response on the growth of both ERα66-negative cell lines. The cell growth regulation was accompanied by modulation of cell migration, cycle and apoptosis, which were dependent on the ROS-regulated p53 signaling cascade. Importantly, ERα36 and GPR30 were involved in the (anti)-proliferative effects. These findings indicate naringenin possesses contrasting effects on ERα66-negative breast cancer cell growth depending on its dose","manuscriptTitle":"Naringenin induces different proliferative effects in estrogen receptor-alpha-66 negative breast cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-09-06 14:25:55","doi":"10.21203/rs.3.rs-3266516/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"8198c64c-1a21-43f9-93bc-489833209c02","owner":[],"postedDate":"September 6th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2023-11-20T10:14:40+00:00","versionOfRecord":[],"versionCreatedAt":"2023-09-06 14:25:55","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3266516","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3266516","identity":"rs-3266516","version":["v1"]},"buildId":"cBFmMYwuxLRRLfASyISRj","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}