Journal of Cancer Prevention 2019; 24(4): 197-207
Published online December 30, 2019
© Korean Society of Cancer Prevention
1Department of Molecular Medicine and Biopharmaceutical Sciences, Graduate School of Convergence Science and Technology, Seoul National University, 2Tumor Microenvironment Global Core Research Center, College of Pharmacy, Seoul National University, Seoul, 3Department of Food and Nutrition, Hannam University, Daejeon, 4Department of Preventive Medicine, Seoul National University College of Medicine, 5Department of Biomedical Science, Seoul National University Graduate School, 6Cancer Research Institute, Seoul National University, Seoul, Korea
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Expression of BRCA1, GPR30 and Nrf2 was measured by Western blot analysis. Reactive oxygen species (ROS) accumulation was monitored by using the fluorescence-generating probe, 2’,7’-dichlorofluorescein diacetate. The effects of genistein on breast cancer cell viability and proliferation were assessed by the MTT, migration and clonogenic assays. The expression of GPR30 was dramatically elevated at both transcriptional and translational levels in Lack of functional BRCA1 activates GPR30 signaling, thereby stimulating Akt phosphorylation and cell proliferation. Genistein induces G2/M phase arrest by down-regulating cyclin B1 expression, which is attributable to its suppression of GPR30 activation and Akt phosphorylation in BRCA1 impaired breast cancer cells.
Expression of BRCA1, GPR30 and Nrf2 was measured by Western blot analysis. Reactive oxygen species (ROS) accumulation was monitored by using the fluorescence-generating probe, 2’,7’-dichlorofluorescein diacetate. The effects of genistein on breast cancer cell viability and proliferation were assessed by the MTT, migration and clonogenic assays.
The expression of GPR30 was dramatically elevated at both transcriptional and translational levels in
Lack of functional BRCA1 activates GPR30 signaling, thereby stimulating Akt phosphorylation and cell proliferation. Genistein induces G2/M phase arrest by down-regulating cyclin B1 expression, which is attributable to its suppression of GPR30 activation and Akt phosphorylation in BRCA1 impaired breast cancer cells.
Keywords: BRCA1, GPR30, Genistein, Akt, Breast cancer
Breast cancer is one of the most frequently diagnosed malignancies in women, and especially the triple negative breast cancer (TNBC) type has a significantly worse prognosis as well as a higher recurrence rate . The main reasons for the poor prognosis include a higher rate of metastasis and resistance to chemotherapy and/or radiotherapy. TNBC is characterized by lack of all three genes encoding estrogen receptor (ER), progesterone receptor and Her2/neu, and often associated with
A seven-transmembrane receptor G protein-coupled receptor 30 (GPR30) has been reported to mediate rapid non-genomic signals of estrogens [9,10]. The stimulation of GPR30 in ER-negative breast cancer cells can result in the reduced cell proliferation and increased apoptosis. GPR30 has been suggested to activate Akt signals involved in cancer cell proliferation and cell cycle progression [9,11,12]. Further, a positive correlation between expression of GPR30 and a reactive oxygen species (ROS) level has been reported [11,13]. The increased ROS production is often found in cancer cells, which is strongly associated with decreased activation of Nrf2, a key transcription factor involved in regulation of antioxidant gene expression .
Epidemiological studies have demonstrated that consumption of soy foods lowers the risk of breast cancer [15,16]. Genistein, one of the most abundant isoflavones present in soybeans, has a chemopreventive effect on mammary carcinogenesis [17–19]. The chemopreventive and anticarcinogenic activities of genistein have been attributed, at least in part, to its capability to antagonize the ER. However, its effect on survival and proliferation of estrogen negative cells, especially differing by the presence or absence of BRCA1 has not been fully clarified. Genistein has been reported to block G2/M progression of the cancer cell cycle [20,21]. There have been investigations that explore the relationship between inhibition of Akt signaling and G2/M arrest . Overexpression of cyclin B1, a key player of G2/M phase cell cycle machinery, is associated with the hyperactivation of Akt signaling .
This study aimed to examine the effects of genistein on growth of TNBC cells with impaired BRCA function. We have found that genistein suppresses TNBC proliferation most likely through inactivation of the GPR30-Akt signaling.
Genistein was purchased from Sigma-Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s Medium (DMEM) and Rosewell Park Memorial Institute (RPMI) 1640 medium were obtained from Gibco BRL (Grand Island, NY, USA). FBS was supplied from GenDEPOT (Barker, TX, USA). TRIzol®, SYBR® safe DNA gel stain and Lipofectamine® RNAiMAX were purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies against BRCA1, P-Akt and Akt were obtained from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against cyclin B1 and actin were products of Santa Cruz Biotechnology (Santa Cruz, CA, USA). GPR30 and Nrf2 primary antibodies were supplied from Abcam (Cambridge, MA, USA). Anti-rabbit and anti-mouse secondary antibodies were purchased from Invitrogen (Carlsbad, CA, USA).
MDA-MB-231 and HCC1937 cells were cultured in DMEM and RPMI, respectively. Each medium was supplemented with 10% FBS and 1% antibiotic-antimycotic. The cells were maintained at 37oC with humidified atmosphere of 5% CO2 and 95% air.
MDA-MB-231 and HCC1937 cells were counted and seeded at a density of 1.6 × 104 per well in 48-well plates. After 24 hours of incubation, the cells were treated with various concentrations of genistein (10, 25, 50, or 100 μM). Cell viability was measured at 72 hours. Thiazolyl blue tetrazolium bromide (Sigma-Aldrich, St. Louis, MO, USA) was added at a concentration of 0.5 mg/mL. After 3 hours of incubation, dimethyl sulfoxide was added to solubilize the formazan crystals formed. The absorbance was measured at 570 nm using a micro-plate reader (Bio-Rad Laboratories, Hercules, California, USA).
Two-well Culture-Inserts (Ibidi®) were attached to 12-well plates. MDA-MB-231 and HCC1937 cells were seeded at a density of 1.5 ×104 for MDA-MB-231 and 2 × 104 for HCC1937 cells per each well in the inserts. After 24 hours of incubation, the silicon inserts were removed, and 50 μM genistein was added. After incubation for another 24 hours, the cells were photographed under a microscope. The procedure was repeated using
MDA-MB-231 and HCC1937 cells were plated in 6-well plates at a density of 300 cells per well. Medium was changed every other day. After 7 days of incubation, genistein (10 or 50 μM) was added. Cells were further cultured for 7 days. The cells were then washed with PBS and fixed in cold methanol for 10 minutes. The colonies were stained using 0.5% crystal violet and imaged by the LAS-4000 image reader (Fuji Film, Tokyo, Japan).
MDA-MB-231 cells were reverse transfected with
Cells were harvested at designated time points. Cells were washed using cold PBS and collected as pellets. Cell pellets were suspended in 1 × cell lysis buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1 mM Na2 EDTA, 1 mM EGTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM glycerophosphate, 1 mM Na3VO4, 1 μg/mL leupeptin; Cell Signaling Technology) supplemented with a protease inhibitor and 0.1 mM PMSF. After incubation in ice for 1 hour, the mixture was centrifuged at 13,000 ×
Total RNA was isolated from MDA-MB-231 and HCC1937 cells by using TRIzol® (Invitrogen) according to the manufacturer’s protocol. To generate cDNA, 1 μg of total RNA was reverse transcribed with moloney murine leukemia virus reverse transcriptase (Promega, Madison, WI, USA) at 42°C for 50 min and 72°C for 15 min. One μl of cDNA was amplified in sequential reactions using Solg™ 2 × Taq PCR Smart mix 1 (SolGent, Seoul, Korea). The primer sequence and conditions used for each PCR reactions are as follows;
To measure the intracellular accumulation of ROS, a fluorescent probe 2,7-dichlorofluoresecein diacetate (DCF-DA) was used. MDA-MB-231 and HCC1937 cells were rinsed with Hank’s balanced salt solution (HBSS), and 10 μM of DCF-DA was loaded. After 30 minutes incubation at 37°C, cells were visualized under a live cell image microscope.
MDA-MB-231 (scrambled siRNA and
Animal experiments were conducted in accordance with institutional guidelines for the care and use of experimental animals. Each side of a BALB/c nude mice was injected subcutaneously with 1 × 107 control or
Results were expressed as the means of ± SD of three independent experiments. The statistical significance of the difference between groups was determined using Student’s
BRCA1 impaired cells are known to proliferate aggressively . In agreement with this notion, HCC1937 cells harboring mutated
Next, MDA-MB-231 and HCC1937 cells were compared with regard to their susceptibility to the cytotoxic effect of genistein. For both cell lines, treatment with genistein lowered the cell viability in a concentration-dependent manner (Fig. 2A). Compared to cells with wild-type
The anti-proliferative effect exerted by genistein was extended to its capability to inhibit cell migration and colony formation. When treated with genistein for 24 hours, both MDA-MB-231 and HCC1937 cells showed reduced migration, which was more pronounced in HCC1937 cells (Fig. 3A). Likewise, MDA-MB-231 cells transfected with
To determine whether genistein could regulate cell cycle machinery in its anti-proliferative activity, the cell cycle distribution was measured by flow cytometry. The proportion of cells in the G2/M phase increased 14% in
While expression of GPR30 was decreased in HCC1937 cells by genistein treatment, such change was not observed in MDA-MB-231 cells that barely express
Association between BRCA1 and tumorigenesis has been extensively investigated [26,27]. To determine whether BRCA1 deficiency has an effect on tumor forming capability in vivo, control and
The present study demonstrates that genistein inhibits the proliferation and growth of TNBC cells with mutant
Estrogen plays a key role in the development and progression of breast cancers. Estrogen binds to the receptor to exert its (patho)physiologic function. Besides classical ER, such as ERα and ERβ, GPR30 can also respond to estrogen. In contrast to ERα and ERβ, GPR30 is located at the cell surface membrane for a rapid response to estrogen . Breast cancers are classified ER positive, ER negative, and triple negative, depending on their receptor types. TNBC is the most common histological subtype associated with BRCA1 mutation . Since prevalence of
One of the salient features of our present study is demonstration of the possible involvement of GPR30 signaling in the aggressive growth and progression of breast cancer cells with BRCA1 impairment. The expression of GPR30 was dramatically elevated at both transcriptional and translational levels in
Genistein, a major phytochemical found in soy beans, has been extensively investigated for its chemoprevetive and anticarcinogenic potential [17–19]. In our present study, the effects of genistein on proliferation and progression of
It can be postulated that GPR30 might be a clue for differential effects of geinstein in breast cancer cells depending on the BRCA1 status. Highly expressed GPR30 and phosphorylated Akt in BRCA1 impaired breast cancer cells would render them more susceptible to genistein-induced growth suppression. Indeed, genistein exerts a stronger inhibitory effect on proliferation and/or migration in of
In conclusion, this study provides evidence for the inhibitory effect of BRCA1 on aberrant proliferation, migration and progression of breast cancer cells. BRCA1 mutation leads to activation of the GPR30-Akt signaling, resulting in aggressiveness of TNBC cells. Further study is required to unravel whether geinstein acts in BRCA1 lacking cells by directly targeting GPR30.
This work was supported by the Seoul National University Research Grant in 2016.
No potential conflicts of interest were disclosed.
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