Genistein, a Potential Phytochemical against Breast Cancer Treatment-Insight into the Molecular Mechanisms

: Breast cancer (BC) is one of the most common malignancies in women. Although widespread successful synthetic drugs are available, natural compounds can also be considered as signiﬁcant anticancer agents for treating BC. Some natural compounds have similar effects as synthetic drugs with fewer side effects on normal cells. Therefore, we aimed to unravel and analyze several molecular mechanisms of genistein (GNT) against BC. GNT is a type of dietary phytoestrogen included in the ﬂavonoid group with a similar structure to estrogen that might provide a strong alternative and complementary medicine to existing chemotherapeutic drugs. Previous research reported that GNT could target the estrogen receptor (ER) human epidermal growth factor receptor-2 (HER2) and several signaling molecules against multiple BC cell lines and sensitize cancer cell lines to this compound when used at an optimal inhibitory concentration. More speciﬁcally, GNT mediates the anticancer mechanism through apoptosis induction, arresting the cell cycle, inhibiting angiogenesis and metastasis, mammosphere formation, and targeting and suppressing tumor growth factors. Furthermore, it acts via upregulating tumor suppressor genes and downregulating oncogenes in vitro and animal model studies. In addition, this phytochemical synergistically reverses the resistance mechanism of standard chemotherapeutic drugs, increasing their efﬁcacy against BC. Overall, in this review, we discuss several molecular interactions of GNT with numerous cellular targets in the BC model and show its anticancer activities alone and synergistically. We conclude that GNT can have favorable therapeutic advantages when standard drugs are not available in the pharma markets.

GNT is a natural phytochemical belonging to phytoestrogen and it possesses a similar structure to estrogen. Interestingly, it has both mimic and antagonized estrogen effects; simultaneously, it inhibits BC cell proliferation [76]. Estrogen receptor-mediated growth regulation of BC cells by GNT may be concentration-dependent. T.T.Y. Wang et al. summarized that GNT stimulated growth at lower concentrations (10 −8~1 0 −6 M), but inhibited cancer cell growth at higher concentrations (>10 −5 M) [77]. There are two types of estrogen receptors [78]. GNT has a structural similarity to both ER-α and ER-β receptors but binds with ER-β with higher affinity compared to ER-α [20,21]. In the case of ER-α, GNT acts as an antagonist. Thus, GNT-mediated anticancer activity is involved by suppressing the expression and activity of ER-α. E.J. Choi summarized that GNT regulates cell proliferation with apoptosis via the ER-α-dependent pathway in MCF-7 BC cells through the underlying mechanism of downregulating cyclin D1 and upregulating the Bcl-2/Bax ratio (B cell lymphoma 2/BCL associated X) at the dose of 50 µM [79]. On the other hand, in ER-β, GNT increases receptor activities as a type of agonist. Therefore, ER-β-dependent anticancer activity of GNT is mediated by activating the receptor and potentiating chemotherapeutic efficacy to treat cancer [80]. H. Jiang stated that GNT mediated anticancer activities through ER-β1 receptors in MDA-MB-231, MCF-7 cells, and BALB/c mice by inhibiting cell proliferation through arresting cells in the G2/M and G0/G1 phases, which led to cell cycle blockade at the dose of 10 −6 -10 −4 mol/L [81].
It has also been found that GNT can bind with the estrogen-responsive G proteincoupled receptor-30 (GPR-30) or G protein-coupled estrogen receptor-1 (GPER-1) [82] and inhibit cell proliferation [83]. Kim GY et al. summarized that GNT suppresses GPR-30 activation in breast cancer gene 1(BRCA-1)-mutated BC cells, resulting in G2/M phase arrest mediated by suppressing Akt phosphorylation [84]. Human epidermal growth factor receptor 2 (HER-2) is an important biomarker in BC and overexpressed in around 20-30% of BC types [85]. Thus, regulating HER-2 is a significant factor in BC treatment. Sakla et al. summarized that GNT inhibited proto-oncogenes of HER-2 and subsequently followed the HER-2 protein expression, phosphorylation, and promoter activity through an ERindependent mechanism in BC cells, aiming to delay tumor onset in transgenic mice [86].

The Effects of Genistein on MDA-MB-231 BC Cells
Recently, an experiment conducted by Liu et al. GNT (5-20 µM) induced apoptosis through the mitochondrial-dependent pathway by reducing the Bcl-2/Bax ratio and inhibiting cell growth and increasing the expression of p73, leading to the activation of G2/M phase arrest and the ATM/Cdc25C/Chk2/Cdc2 checkpoint pathway [90]. GNT prompted the apoptotic pathway and directly inhibited the growth of cells through the prevention of NF-κB signaling by the Notch-1 pathway and by downregulating cyclin B1 and Bcl-2 expression, resulting in the arrest of the cell cycle at the G2/M phase at 5-20 µM [109], while at 5-50 µM, this phytochemical induced apoptosis by targeting the endogenous copper ion, reducing Cu(II) to Cu(I) through the production of reactive oxygen species (ROS) [110]. Before that, an in vitro study by Dampier et al. reported that GNT (10 µM) induced apoptosis and inhibited cell proliferation and cell cycle arrest at the G2 phase, degrading proto-oncogene c-Fos and prohibiting protein-1 (AP-1), and also ERK activity [111]. Another study by Yang et al. demonstrated that GNT (50 µM) exerted apoptosis by upregulating poly-(ADP-ribose)-polymerase, activating p53, and downregulating Bcl-2/Bax protein [92].
Another study conducted by Kousidou et al. reported that GNT (35-100 µM) progresses slowdown invasiveness by decreasing MMP gene expression, PTK activity, and glucose uptake rate, leading to phagocytosis of cancerous cells [113]. Apart from this, it reduces cell viability by decreasing the DNA methyltransferase activity and DNMT1 expression and affecting the expression of TSGs, i.e., APC, ATM, PTEN, and SERPINB5 at 60-100 µM of GNT [108]. Another recent study by Pons et al. summarized that GNT (1 µM) causes a considerable decrease in cell viability through the mitogen-dependent protein kinase pathway and by promoting apoptosis mechanisms [106].
In MDA-MB-231 BC cells, cell growth control is a significant target for GNT. Gong et al. stated that GNT (5-50 µM) inhibited cell growth by partly inducing apoptosis via downregulation of the Akt and NF-κB cascade pathways [114]. In another in vitro analysis, the cell growth inhibitory activity was evidenced by GNT (2.5-400 µM) through the upregulation of two crucial TSGs, p21WAF1 (p21) and p16INK4a (p16), and the downregulation of two tumor-promoting genes, c-MYC and BMI1, ultimately inhibiting cancer progression [115]. Y. Fang et al. concluded that GNT (40 µM) inhibited cellular growth by following the activation of DNA-dependent damage response and the ATR signaling pathway and activating the BRCA-1 complex, inhibiting the cohesion complex, and increasing phosphatide, which is distributed among CDK1, CDK2, and CDK3 [116]. Recently, it was established that GNT (1000 ppm) suppressed tumor growth by cell cycle regulation via maintaining the expression level of the cyclin D1 protein, leading to G0/G1 phase arrest, which causes cell cycle blockage [81]. Subsequently, Rajah et al. summarized that GNT (10-100 µM) inhibited tumor growth by downregulating MEK5, pERK5, and NF-κB proteins [117]. In the case of cell proliferation, a low dose of GNT (10 µM) slightly inhibited cell proliferation by reducing the P-STAT3/STAT-5 ratio [98]. In comparison, at a double dose, i.e., 20-40 µM, it significantly prevented cell proliferation by inducing apoptosis and suppressing Skp2 expression by upregulating the tumor suppressor genes, i.e., p21 and p27, resulting in G2/M phase arrest [118]. Li et al. investigated that GNT (5-20 µM) inhibited cell differentiation with cell cycle arrest at the G2/M phase by decreasing CDK1, cyclin B1, Cdc25C, c-Jun, and c-Fos levels [22]. GNT can also play a role in MDA-MB-231 by inhibiting mammosphere formation. A lower dose of GNT (2 µM) prevents mammosphere formation through PI3K/Akt signaling by increasing the PTEN expression [28], while at a higher dose, GNT (40 nm-2 µM) prevents the formation of mammosphere cells and promotes differentiation through the PI3K/Akt and MEK/ERK signaling pathway by reducing the CD44+/CD24-/ESA ratio and increasing E-cadherin mRNA expression [105]. Finally, GNT (50 µM) impedes primary tumor formation by downregulating chelator neocuproine and Bcl-2/Bax and by upregulating the caspase-3 pathway [110].

The Effects of Genistein on T-47D Breast Cancer Cells
Mukund et al. summarize that GNT (50 µM) lowered angiogenesis by preventing the transactivation of downstream HIF-1α effectors such as VEGF, reducing the expression of hypoxia-inducible factor-1α in the T-47D BC cell line [26]. Cell proliferation efficacy was evident by GNT (10 nm) with apoptosis induction through the mitochondrialdependent pathway via upregulating the cyt-C and oxidase activity, and downregulating the ATP synthase/cytochrome c oxidase ratio [98]. GNT at 1 nm-100 µM inhibits cell proliferation through ERK1/2-mediated signaling by the downregulation of phosphorylated p90RSK [119], while 10 µM of GNT induces apoptosis and inhibits cell proliferation through degrading proto-oncogene c-Fos levels and prohibiting protein 1 (AP-1) and ERK expression [111]. Another in vitro study by Rajah revealed that GNT (10-100 µM) inhibits cell proliferation and tumor growth by downregulating MEK5, pERK5, and NF-κB proteins [117]. Additionally, a high GNT (20 M) concentration inhibits cell proliferation by reducing ER-messenger RNA transcription and arresting the cell cycle at the G2/M phase [76]. According to Sotoca et al., GNT (500 nm) inhibited cell growth and induced apoptosis by activating cytoskeleton restructuring that results in interaction among integrins, focalized adhesion kinase, and CDC42 that leads to cell cycle arrest in the T-47D BC cell line [120], while according to Pons et al., GNT (1 µM) caused a significant decrease in cell viability by increasing Sirt1, TGFβ, and PRARγ and decreasing IL-1β expression in T-47D BC cells [106].

The Effects of Genistein on HCC1395 Breast Cancer Cells
Lee et al. demonstrated that GNT (1-200 µM) inhibited HCC1395 cell invasion and metastasis through the upregulation of TFPI-2, ATF3, DNMT1, and MTCBP-1 gene expression and the downregulation of MMP-2, MMP-7, CXCL12 genes, leading to cell cycle arrest at the G2/M phase, therefore reducing cell viability [25].

The Effects of Genistein on HCC38 Breast Cancer Cells
Donovan stated that GNT (4-10 ppm) inhibited cell growth by increasing the BRCA1 protein level and reducing CpG methylation, consequently decreasing the aryl hydrocarbon receptor (AhR) binding at BRCA1 in the HCC38 cell line [121].

The Effects of Genistein on BT-474 Breast Cancer Cells
GNT at a low concentration (1 µM) could promote cancer but at a high concentration (50 µM), it inhibits cell division by downregulating tyrosine kinase, HER2 activation, and the MAPK pathway [86]. GNT (3.125-25 M) inhibits cell replication and arrests the cell cycle in the G2/M phase, and inhibits the expression of EGFR, HER2, and ER-alpha [124].

The Effects of Genistein on BT20 Breast Cancer Cells
Cappelletti et al. revealed that GNT (15-30 µM) inhibits metastasis by lowering levels of CDKs, tyrosine kinase, DNA topoisomerase II, and paracrine stimulation in the BT20 cell line [112].  An early study showed that GNT (1-10 µg/mL) obstructed angiogenesis and cell mutation by decreasing the expression of ribosomal S6 kinases and tyrosine kinase [97]. An overview of GNT's anticancer activities is given in Table 1.

Clinical Trials
Human clinical trials have confirmed the in vitro research findings. In some cases, when consumed at a consistent dose, pure GNT had no estrogenic effect on breast tissue [31,142], although in other cases, dietary soy supplementation had pro-estrogenic effects on breast tissue [143][144][145]. Several secondary endpoints were evaluated in a recently published clinical study to determine whether purified GNT affects endometrial thickness, vaginal cytology, and breast density ( Table 2 [31,95,96]. Following the implementation of safety measures, it was possible to identify the potential estrogenic effects of 54 mg/day of purified GNT as indicators of BC risk in the research participants. Indeed, while the placebo group maintained a constant endometrial thickness, the GNT group demonstrated a timedependent reduction that reached statistical significance during the 36-month follow-up (approximately 12% reduction, p < 0.01). Moreover, levels of gene expression of BRCA-1 and 2 breast tumor suppressor genes [146,147] have been preserved for three years in the GNT-administered group, while levels of both BRCA-1 and 2 have decreased in the placebo group (Table 2) [31,142]. GNT also significantly reduced sister chromatid exchanges, implying that it may prevent genotoxicity and subsequent mutagenesis (Table 2) [142]. In this regard, based on the use of GNT in BC, two clinical trials-a phase II study entitled "Gemcitabine Hydrochloride and GNT in Treating Women with Stage IV BC" (NCT00244933) and a phase I study entitled "GNT in Preventing Breast or Endometrial Cancer in Healthy Postmenopausal Women" (NCT00099008)-have been completed, but the results are not yet published. The effects of GNT on human clinical studies against cancer are summarized in Table 2.

Synergistic Properties of Genistein in the Treatment of Breast Cancer
In addition to its solid anticancer activity alone, GNT possesses synergistic properties with many other anticancer drugs, helping it overcome chemopreventive resistance mechanisms in BC treatment. The synergistic activity of GNT can be carried by many anticancer drugs such as doxorubicin, trastuzumab, tamoxifen, trichostatin A, cisplatin, capsaicin, paclitaxel, and vincristine.

Synergistic Properties of Genistein with Doxorubicin in MCF-7/Adr Cells
Doxorubicin is an antibiotic that exhibits no inhibitory effects on Adriamycin-resistant BC cell lines. However, the combination of GNT at 30 µmol/L and doxorubicin has synergistic effects on MCF-7/Adr cells. GNT enhances the cytotoxic effects of doxorubicin and decreases the chemoresistance of MCF-7/Adr BC cells. In addition, GNT and doxorubicin synergistically induced apoptosis by decreasing expression of Her2/new mRNA and c-erbB2, resulting in cell cycle arrest in MCF-7/Adr BC cells in the G2/M phase [29]. Another study by Yang et al. reported that GNT (50 µM) with a combination of doxorubicin slightly induces apoptosis by destroying the plasma membrane of cells and increasing poly (ADP-ribose) polymerase cleavage in MDA-MB-231 and MCF-7 BC cells [92].

Synergistic Effect of Genistein with Trastuzumab
GNT and trastuzumab synergistically develop antitumor activity in BT-474 BC cells. C. Lattrich et al. stated that the combination of GNT (10 µmol/L) and trastuzumab (1/10 µg/mL) enhanced the growth-inhibitory effect and reduced viable cell numbers by increasing the ER-β2 expression, which causes an antiestrogenic effect, leading to reduced cell proliferation in ER-α/β-positive and HER2-overexpressing BT-474 BC cells. Furthermore, both GNT and trastuzumab reduce cyclin A2 mRNA expression, c-Fos, HER2, and cyclin D1 expression, which suppresses the proliferation of BT-474 BC cells [152].

Synergistic Properties of Genistein with Tamoxifen
Tamoxifen is a well-established medicine for treating BC, although the development of gemcitabine resistance has hampered its efficacy. In this regard, GNT can improve the efficacy of tamoxifen. Y. Li et al. described that GNT enhanced the anticancer capacity of tamoxifen at the dose of 25 µM through the reactivation of ER-α and epigenetic pathway regulation, e.g., histone modification, resulting in the reduction in HDAC1 and DNMT1 expression both in vitro (ER-α-negative MDA-MB-231 BC cells) and in vivo, leading inhibited cell growth and cell viability [153]. On the other hand, Pons et al. concluded that 1 µM GNT and 10 µM tamoxifen decrease the ROS production in T47D and MCF-7 BC cells and upregulate the autophagic vacuole formation and PARP protein level and also reduce cell viability, resulting in autophagic cell death only in T47D BC cells [154]. Another early study reported that tamoxifen with GNT (1-10 µg/mL) impeded angiogenesis and cell mutation by downregulating ribosomal S6 kinases, tyrosine kinase, and cell cycle regulators [97]. GNT also shows a prohibitory effect with tamoxifen, which induces apoptosis by destroying the nuclear membrane [93] and arrests the cell cycle by decreasing the expression of HER2 in a dose-dependent manner [124].

Synergistic Effect of Genistein with Trichostatin A
GNT and trichostatin A act synergistically to inhibit PGR (progesterone receptors) expression, resulting in a significant change in cell growth in ER-positive and ER-negative BC cells. According to Li et al., the combination of GNT (25 µM) and trichostatin A (100 ng/mL) synergistically decreased ROS production through the underlying mechanism of increasing antioxidant enzymes, i.e., Mn-SOD (manganese-superoxide dismutase) and catalase, in MCF-7 and T47D BC cells. Furthermore, GNT and cisplatin also synergistically arrest the cell cycle at the S phase and cause a drop in the sub-G0/G1 phase, resulting in MCF-7 cells at 25-50 mol/L concentration [154].

Synergistic Effect of Genistein with Capsaicin
The combination of GNT and capsaicin exerted anti-inflammatory and anticarcinogenic effects in MCF-7 cells as well as in vivo in 48-week-old female Sprague-Dawley rats by modulating the mitogen-activated protein kinase (AMPK) and COX-2, as well as possibly other mitogen-activated protein kinases [30].

Synergistic Effect of Genistein with Paclitaxel and Vincristine
Paclitaxel and vincristine both are chemotherapy drugs. Together with GNT (100 µM), they can suppress cell growth and cell viability by inhibiting CDC2 and cyclin B1 kinase and inhibiting microtubule polymerization in human MDA-MB-231 and MCF-7 BC cell lines. Furthermore, cell death by inducing apoptosis via decreasing Bcl-2 phosphorylation without changing p21, p53, and Bax protein expression was also observed in combined treatment [103]. Table 3 summarizes the effects of phytoestrogens in combination with anticancer therapies that have been previously described. Table 3. Summary of the described effects of phytoestrogens in combination with anticancer therapies.

Possible Strategies to Overcome Anticancer Drug Resistance by Genistein
Numerous mechanisms are responsible for BC drug resistance, such as membrane glycoproteins acting as efflux pumps, including P-glycoprotein (P-gp), multidrug resistance (MDR) protein, and BCRP, as well as enzymatic inactivation of the anticancer drug [156]. Phytochemical-based therapy can provide a reliable safety mechanism to prevent anticancer resistance. Inhibition of P-glycoprotein (P-gp) activities or conjunction of P-gp substrate with the anticancer drug leads to the increased accumulation of the anticancer drug within the cell, producing cell cytotoxicity. However, GNT does not directly affect P-gp function in a BC cell line but indirectly increases intracellular drug concentration, including doxorubicin. For instance, Castro and Altenberg stated that GNT decreased photo-affinity labeling of P-gp with [ 3 H] azidopine, a P-gp substrate, suggesting that GNT could block P-gp-mediated drug efflux by direct interaction with P-gp and inhibited rhodamine123 efflux in human MCF-7 BC cell lines [157]. Furthermore, intracellular doxorubicin accumulation was boosted by GNT therapy, leading to cell cycle arrest and apoptosis via inhibiting HER2/neu rather than influencing P-gp function and MDR-1 expression in MCF-7/Adr cells [29]. GNT pretreatment with MDA-MB-231 and CB-17 scid/scid mice inactivated NF-κB and may contribute to increased growth inhibition and apoptosis induced by cisplatin docetaxel and doxorubicin in BC cells [158]. Targeting cyclooxygenase-2 (COX-2) can be a possible mechanism of overcoming drug resistance. There is a positive relationship between COX-2 and MDR1/P-gp. GNT significantly inhibited cyclooxygenase-2 activity [96], suggesting that GNT can inhibit MDR1/P-gp in BC, leading to improved anticancer drug efficacy [159]. In the case of BCRP, GNT and its glucuronide and sulfate conjugation are the substrates of BCRP established in in vitro cell culture models and in vivo pharmacokinetic studies [39,40]. This binding of GNT with BCRP indicates that GNT treatment increases anticancer drug concentration by decreasing efflux. However, there has been controversy with GNT and C member 1 (ABCC1) and ABCG2. Rigalli et al. reported that treatment of MCF-7 with GNT increases resistance to mitoxantrone and doxorubicin by increasing drug efflux [160].

Nano-Formulation of Genistein for Breast Cancer Treatment
GNT research for cancer treatment has been extended in recent years due to evidence of lower disease risk associated with its administration and a quest for pharmacological medicines that impact growth factor signaling pathways in cells. A significant drawback of GNT as a natural substance is its low water solubility. This may necessitate modifying its chemical structure to increase solubility and boost bioavailability [161].
However, the advancement of nanomedicines has the potential to overcome phytochemical limitations and allied health concerns, such as improved solubility, increased bioavailability, targeted treatment of tumor cells or tissues while avoiding healthy cell damage, and increased cell take-up. Nanomedicines could provide new avenues for circumventing these concerns. Additional advantages may include improved blood stability, multifunctional nanomedicine design, minimal interaction with synthetic medications, and improved anticancer activity [162]. Furthermore, multidrug resistance (MDR) is one of the most important variables contributing to the failure of phytochemical therapy in cancer. MDR can be circumvented using a new technique including nanocarriers and phytochemical delivery. Modifying the biophysical interaction between the nanomedicines and cancer cell membrane lipids can increase phytochemical delivery and overcome drug resistance. This is accomplished by improving the transport of phytochemicals to target tissues through surface modification of nanomedicines [163,164]. Currently, advancements in treatment efficiency through nanomedicines have received much attention because of the increased delivery of phytochemicals to tumors and cancer cells. Numerous highly successful nanomedicines have been employed to enhance phytochemicals' physicochemical qualities and anticancer activity [165]. BC treatment with doxorubicin and GNT is improved by using multifunctional hybrid nano-constructs that enable intracellular localization of the drugs [166]. A research study by Jimmy Pham and his colleagues demonstrated that mitochondriotropic nano-emulsified genistein-loaded vehicles showed more effective potential against hepatic and colon carcinomas than the control drugs [167].In one study, cervical cancers were treated with bioflavonoid genistein-loaded chitosan nanoparticles targeted to the folate receptor, which had a significant anticancer effect. The naturally derived chitosan nanoparticles exhibited potent biodegradability and biocompatibility when coated with the GNT [168]. Additionally, genistein-loaded biodegradable TPGS-b-PCL nanoparticles possessed enhanced therapeutic effects in cervical cancer cells [169]. Moreover, the nanoformulation of GNT promoted selective apoptosis in the cell line of oral squamous cancer by suppressing the expression of a 3PK-EZH2 signaling pathway [170].

Concluding Remarks and Future Directions
The evidence provided from available scientific literature (in vitro and in vivo) detailed in this review offers a comprehensive summary of the anticancer activities of GNT. Overall, information from our study would help in the identification of the mechanisms of GNT against BC pathogenesis that will aid in drug development in anti-BC therapy. Mechanisms of GNT are related to multiple molecular pathways, including regulating miRNA, several proteins such as apoptosis, transcription factor and tumor suppressorrelated proteins, enzymes including kinase, several growth factors, receptors, and other numerous targets (Figure 2). Successful conventional treatment of BC is limited due to rising resistance to some chemotherapeutic drugs, but GNT may bring therapeutic advantages by sensitizing multidrug-resistant BC cells and mediating some synergistic effects with conventional anticancer drugs. Therefore, using GNT as a regular food supplement may help in the near future to treat BC. However, we still need further research, including clinical trials, regarding drug interactions, accurate pharmacokinetics, accurate therapeutics doses, routes of administration, and established nanoformulation of GNT. The successful performance of all approaches will make GNT a novel candidate for drug development against BC.

Concluding Remarks and Future Directions
The evidence provided from available scientific literature (in vitro and in vivo) detailed in this review offers a comprehensive summary of the anticancer activities of GNT. Overall, information from our study would help in the identification of the mechanisms of GNT against BC pathogenesis that will aid in drug development in anti-BC therapy. Mechanisms of GNT are related to multiple molecular pathways, including regulating miRNA, several proteins such as apoptosis, transcription factor and tumor suppressorrelated proteins, enzymes including kinase, several growth factors, receptors, and other numerous targets (Figure 2). Successful conventional treatment of BC is limited due to rising resistance to some chemotherapeutic drugs, but GNT may bring therapeutic advantages by sensitizing multidrug-resistant BC cells and mediating some synergistic effects with conventional anticancer drugs. Therefore, using GNT as a regular food supplement may help in the near future to treat BC. However, we still need further research, including clinical trials, regarding drug interactions, accurate pharmacokinetics, accurate therapeutics doses, routes of administration, and established nanoformulation of GNT. The successful performance of all approaches will make GNT a novel candidate for drug development against BC.