Targeting FAK/PYK2 with SJP1602 for Anti-Tumor Activity in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) presents significant challenges due to its aggressive nature and limited treatment options. Focal adhesion kinase (FAK) has emerged as a critical factor promoting tumor growth and metastasis in TNBC. Despite encouraging results from preclinical and early clinical trials with various FAK inhibitors, none have yet achieved clinical success in TNBC treatment. This study investigates the therapeutic potential of a novel dual inhibitor of FAK and PYK2, named SJP1602, for TNBC. In vitro experiments demonstrate that SJP1602 effectively inhibits FAK and PYK2 activities, showing potent effects on both kinases. SJP1602 shows concentration-dependent inhibition of cell growth, migration, invasion, and 3D spheroid formation in TNBC cell lines, surpassing the efficacy of other FAK inhibitors. Pharmacokinetic studies in rats indicate favorable bioavailability and sustained plasma concentrations of SJP1602, supporting its potential as a therapeutic agent. Furthermore, in TNBC xenograft models, SJP1602 exhibits significant dose-dependent inhibition of tumor growth. These promising results emphasize the potential of SJP1602 as a potent dual inhibitor of FAK and PYK2, deserving further investigation in clinical trials for TNBC treatment.

Targeting RAF Isoforms and Tumor Microenvironments in RAS or BRAF Mutant Colorectal Cancers with SJ-C1044 for Anti-Tumor Activity.

Colorectal cancer (CRC) is a significant global health issue characterized by a high prevalence of KRAS gene mutations. The RAS/MAPK pathway, involving KRAS, plays a crucial role in CRC progression. Although some RAS inhibitors have been approved, their efficacy in CRC is limited. To overcome these limitations, pan-RAF inhibitors targeting A-Raf, B-Raf, and C-Raf have emerged as promising therapeutic strategies. However, resistance to RAF inhibition and the presence of an immunosuppressive tumor microenvironment (TME) pose additional obstacles to effective therapy. Here, we evaluated the potential of a novel pan-RAF inhibitor, SJ-C1044, for targeting mutant KRAS-mediated signaling and inhibiting CRC cell proliferation. Notably, SJ-C1044 also exhibited inhibitory effects on immunokinases, specifically, CSF1R, VEGFR2, and TIE2, which play crucial roles in immune suppression. SJ-C1044 demonstrated potent antitumor activity in xenograft models of CRC harboring KRAS or BRAF mutations. Importantly, treatment with SJ-C1044 resulted in increased infiltration of T cells and reduced presence of tumor-associated macrophages and regulatory T cells within the TME. Thus, SJ-C1044 shows immunomodulatory potential and the ability to enhance antitumor responses. The study underscores the therapeutic potential of SJ-C1044 as a novel pan-RAF inhibitor capable of targeting oncogenic signaling pathways and overcoming immune suppression in CRC.

Berberine Suppresses Cell Motility Through Downregulation of TGF-ß1 in Triple Negative Breast Cancer Cells.

BACKGROUND/AIMS: Transforming growth factor-beta proteins (TGF-ßs) are multifunctional growth factors and powerful modulators of the epithelial-mesenchymal transition (EMT) in a variety of cancer types including breast and lung cancer cells. Here, we demonstrated the inhibitory effect of berberine (BBR) on tumor growth and metastasis of triple negative breast cancer (TNBC) cells via suppression of TGF-ß1 expression. METHODS: The levels of mRNA expression were analyzed by real-time PCR. The levels of MMP-2, MMP-9 and TGF-ß1 protein expression were analyzed by zymography and confocal microscopy, respectively. Cell migration was analyzed by wound healing assay. Tumorigenicity of TNBC cells such as tumor growth and metastasis was analyzed using xenograft models. RESULTS: In a clinical data set, aberrant TGF-ß1 expression was associated with poor prognosis of breast cancer patients. Our in vitro results using TNBC cells showed that the expression levels of matrix metalloproteinase (MMP)-2 and MMP-9 and the capacity for cell migration were increased by TGF-ß1 treatment. In contrast, basal levels of MMP-2 and MMP-9 were suppressed by a specific TGF-ß receptor I inhibitor, SB431542. In addition, TGF-ß1-induced MMP-2 and MMP-9 expression and cell migration were decreased by SB431542. Interestingly, we showed for the first time that BBR decreased the level of TGF-ß1, but not TGF-ß2, in TNBC cells. Furthermore, BBR significantly decreased the level of MMP-2 expression as well as the capacity for cell migration in TNBC cells. Finally, we examined the effect of BBR on in vivo tumor growth and lung metastasis in MDA-MB231 and 4T1 breast cancer xenograft models and showed that both were significantly decreased following BBR treatment. CONCLUSION: BBR suppresses tumorigenicity of TNBC cells through inhibition of TGF-ß1 expression. Therefore, we demonstrate that BBR could be a promising drug for treatment of TNBC.

Dimerization of EGFR and HER2 induces breast cancer cell motility through STAT1-dependent ACTA2 induction.

The dimerization of EGFR and HER2 is associated with poor prognosis such as induction of tumor growth and cell invasion compared to when EGFR remains as a homodimer. However, the mechanism for events after dimerization in breast cancer models is not clear. We found that expressions of alpha-smooth muscle actin (ACTA2) and signal transducer and activator of transcription 1 (STAT1) significantly increased with transient or stable overexpression of HER2 in EGFR-positive breast cancer cells. ACTA2 and STAT1 expression was also increased in HER2-positive breast cancer patients. In contrast, ACTA2 expression was decreased by HER2 siRNA. Next, we investigated the co-relation between STAT1 and ACTA2 expression. Basal ACTA2 expression was significantly decreased by treatment with the STAT1 inhibitor fludarabine or the JAK2 inhibitor AG490. In contrast, ACTA2 expression was increased by STAT1 overexpression. Levels of ACTA2, STAT1, and HER2 were increased and relapse free survival was decreased in high-risk breast cancer patients. We also investigated the effect of ACTA2 on cell motility, which was suppressed by ACTA2 shRNA overexpression in MDA-MB231 HER2 and 4T1 mammary carcinoma cells. The number of lung metastatic nodules was significantly decreased in ACTA2 knockdown mice. Taken together, these results demonstrated that induction of ACTA2 by EGFR and HER2 dimerization was regulated through a JAK2/STAT1 signaling pathway, and aberrant ACTA2 expression accelerated the invasiveness and metastasis of breast cancer cells.

Elevated IL-1ß expression induces invasiveness of triple negative breast cancer cells and is suppressed by zerumbone.

Aberrant interleukin-1 beta (IL-1ß) expression is associated with cancer development, metastasis, and poor prognosis. Here, we have investigated the regulatory mechanism of IL-1ß expression, and the inhibitory effect of zerumbone (ZER) on IL-1ß expression and IL-1ß-induced signatures, including cell invasion and signaling activation in triple negative breast cancer (TNBC) cells. The basal IL-1ß and cell invasiveness levels were significantly higher in TNBC cells, compared with non-TNBC cells. The invasiveness of TNBC cells was also increased following IL-1ß treatment. In contrast, the invasiveness of TNBC cells was decreased following IL-1 receptor antagonist (IL-1RA) treatment. Additionally, the basal IL-1ß level and the invasiveness of TNBC cells were decreased by Bay11-7085. In contrast, overexpression of NF-κB (p65) caused an increase in IL-1ß expression in TNBC cells. Our results showed that treatment with ZER decreased the basal IL-1ß expression level, and the phosphorylation level of NF-κB, in TNBC cells. Furthermore, we found that ZER completely suppressed IL-1ß-induced NF-κB phosphorylation, but did not suppress IL-1ß-induced Akt phosphorylation, in TNBC cells. Our results also demonstrate that IL-1ß-induced cell invasion is suppressed by ZER in TNBC cells. Taken together, we demonstrated that IL-1ß expression is regulated by the NF-κB-dependent pathway, and that elevated IL-1ß is directly influencing the invasiveness of TNBC cells. ZER down-regulates IL-1ß expression through the inhibition of NF-κB activity, and then suppresses cell invasiveness of TNBC.

STC-1 expression is upregulated through an Akt/NF-κB-dependent pathway in triple-negative breast cancer cells.

Stanniocalcin-1 (STC-1) is a secreted glycoprotein and its expression is strongly correlated with cancer development. However, regulatory mechanism of STC-1 expression in breast cancer cells is not clear. In the present study, we investigated whether STC-1 acts as a prognostic factor in TNBC patients and the regulation of STC-1 expression in breast cancer cells. Basal levels of STC-1 were significantly higher in TNBC cells than in non-TNBC cells. Induction of STC-1 expression was also associated with poor relapse-free survival of TNBC patients. In addition, we verified the correlation between the invasiveness of TNBC cells and the STC-1 expression. We found that recombinant human STC-1 treatment increased the invasiveness of TNBC cells. In contrast, STC-1-induced cell invasiveness was completely inhibited by anti-STC-1 monoclonal antibody treatment. We found that the basal levels of STC-1 expression in TNBC cells were decreased by treatment with LY294002 or Bay11-7085, but not SB203580. In contrast, transcript levels of STC-1 and protein secretion were increased by constitutively active Akt (CA-Akt) or NF-κB overexpression in TNBC cells. Finally, we observed that phosphorylation of NF-κB was significantly increased by CA-Akt overexpression in TNBC cells. Taken together, elevated STC-1 expression is associated with poor clinical outcome in TNBC patients, and STC-1 is directly involved in the invasiveness of TNBC cells. STC-1 expression is upregulated through a PI-3K/Akt/NF-κB­dependent signaling pathway in TNBC cells.

Elevated STC­1 augments the invasiveness of triple­negative breast cancer cells through activation of the JNK/c­Jun signaling pathway.

Stanniocalcin­1 (STC­1), a secreted glycoprotein, is highly expressed in a variety of human malignancies. However, the role of STC­1 has not been fully elucidated in breast cancer cells. Here, we investigated whether STC­1 acts as a prognostic factor in triple­negative breast cancer (TNBC) patients, and we explored the cellular mechanism in breast cancer cells. The level of STC­1 expression was directly associated with the relapse­free and overall survival of basal­type breast cancer patients. Breast cancer patients with a high level of STC­1 had poor prognosis. In addition, our results showed that the level of STC­1 expression was markedly higher in TNBC than in non­TNBC cells. Invasiveness of the TNBC cells was also significantly increased in response to recombinant human STC­1 treatment. In contrast, the invaded cell numbers were completely decreased by STC­1 siRNA overexpression in the Hs578T and MDA­MB­231 TNBC cells. Our results showed that the phosphorylation of c­Jun N­terminal protein kinase (JNK) and c­Jun was increased after STC­1 treatment but not the phosphorylation of ERK and p38 MAPKs in the Hs578T and MDA­MB­231 TNBC cells. Furthermore, expression of one invasion­related gene MMP­9, was increased by STC­1 treatment. STC­1­induced MMP­9 expression was suppressed by SP600125 (a JNK inhibitor) in the Hs578T cells. STC­1­induced cell invasiveness was also inhibited by SP600125. Taken together, we demonstrated that aberrant STC­1 expression is associated with poor prognosis and stimulates the invasiveness of TNBC cells through the JNK/c­Jun­dependent signaling pathway.

Silibinin inhibits triple negative breast cancer cell motility by suppressing TGF-ß2 expression.

Transforming growth factor-beta (TGF-ß) is a multifunctional cytokine that regulates many biological events including cell motility and angiogenesis. Here, we investigated the role of elevated TGF-ß2 level in triple negative breast cancer (TNBC) cells and the inhibitory effect of silibinin on TGF-ß2 action in TNBC cells. Breast cancer patients with high TGF-ß2 expression have a poor prognosis. The levels of TGF-ß2 expression increased significantly in TNBC cells compared with those in non-TNBC cells. In addition, cell motility-related genes such as fibronectin (FN) and matrix metalloproteinase-2 (MMP-2) expression also increased in TNBC cells. Basal FN, MMP-2, and MMP-9 expression levels decreased in response to LY2109761, a dual TGF-ß receptor I/II inhibitor, in TNBC cells. TNBC cell migration also decreased in response to LY2109761. Furthermore, we observed that TGF-ß2 augmented the FN, MMP-2, and MMP-9 expression levels in a time- and dose-dependent manner. In contrast, TGF-ß2-induced FN, MMP-2, and MMP-9 expression levels decreased significantly in response to LY2109761. Interestingly, we found that silibinin decreased TGF-ß2 mRNA expression level but not that of TGF-ß1 in TNBC cells. Cell migration as well as basal FN and MMP-2 expression levels decreased in response to silibinin. Furthermore, silibinin significantly decreased TGF-ß2-induced FN, MMP-2, and MMP-9 expression levels and suppressed the lung metastasis of TNBC cells. Taken together, these results suggest that silibinin suppresses metastatic potential of TNBC cells by inhibiting TGF-ß2 expression in TNBC cells. Thus, silibinin may be a promising therapeutic drug to treat TNBC.

MEK activity controls IL-8 expression in tamoxifen-resistant MCF-7 breast cancer cells.

Although tamoxifen reduces disease progression, tamoxifen resistance occurs during the course of estrogen receptor-positive [ER+] breast cancer treatment. In the present study, we investigated the possibility that interleukin-8 (IL-8) is a prognostic marker for tamoxifen resistance and aimed to clarify the regulation of IL-8 expression in tamoxifen-resistant cells. Clinically, IL-8 expression is positively correlated with survival in luminal A type breast cancer patients, but not in luminal B type breast cancer patients. In addition, the levels of IL-8 mRNA and protein expression were significantly increased in tamoxifen-resistant (TamR) cells compared to tamoxifen-sensitive (TamS) cells. To determine the regulatory mechanism of IL-8 expression in TamR cells, we analyzed the activities of signaling molecules. Our results showed that the phosphorylation levels of MEK and Akt were markedly increased in TamR cells, but there was no change in the phosphorylation level of p38 MAPK. On the contrary, we observed that elevated IL-8 mRNA expression was suppressed by a specific MEK1/2 inhibitor, UO126, but not by the specific PI-3K inhibitor LY294002, in TamR cells, whereas, we found that overexpression of constitutively active-MEK (CA-MEK) significantly increased the levels of IL-8 mRNA expression in TamS cells. Finally, we investigated the effect of the specific CXCR1/2 inhibitor SB225002 on anchorage-independent growth of TamR cells, and found that the growth was completely suppressed by SB225002. Taken together, our results demonstrate that IL-8 expression is regulated through a MEK/ERK-dependent pathway in TamR cells, suggesting that IL-8 and its receptors may be promising therapeutic targets for overcoming tamoxifen resistance.

Zerumbone suppresses the motility and tumorigenecity of triple negative breast cancer cells via the inhibition of TGF-ß1 signaling pathway.

Aberrant transforming growth factor-ß (TGF-ß) plays an important role in the development of cancer such as tumor metastasis and invasion. TGF-ß-responsive gene signature is highly activated in chemotherapy-treated triple negative breast cancer (TNBC). Here, we investigated the effect of zerumbone (ZER) on TGF-ß1 signaling pathway and tumorigenecity of TNBC cells. Our results showed that the level of TGF-ß1 mRNA expression and cell invasiveness were higher in TNBC cells than in non-TNBC cells. On the other hand, the cell motility of TNBC cells was completely suppressed by LY2109761, a novel selective TGF-ß receptor type I/II (TßRI/II) dual inhibitor. In addition, FN and MMP-2 expression, which play an important role on cell motility in various cancer cells, were dose-dependently decreased by LY2109761. TGF-ß1 increased FN, MMP-2 and MMP-9 expression in HCC1806 TNBC cells. TGF-ß1-induced MMP-9 expression was decreased by both a MEK inhibitor, UO126, and a smad3 inhibitor, SIS3. Induction of FN and MMP-2 by TGF-ß1 was just decreased by SIS3. Overexpression of smad3 significantly increased FN, MMP-2, and MMP-9 expression. Interestingly, ZER significantly suppressed TGF-ß1-induced FN, MMP-2, and MMP-9 expression in HCC1806 cells. In addition, ZER completely decreased TGF-ß1-induced the phosphorylation of smad3. Finally, we observed that ZER suppressed the tumorigenecity such as tumor volume, weight, Ki67 expression, and metastasis in TNBC cells xenograft models. Taken together, we demonstrated that ZER suppresses TGF-ß1-induced FN, MMP-2, and MMP-9 expression through the inactivation of smad3 and inhibits the tumorigenecity of TNBC cells. Therefore, we suggest that ZER may act as a promising drug for treatment of TNBC.

MEK-dependent IL-8 induction regulates the invasiveness of triple-negative breast cancer cells.

Interleukin-8 (IL-8) serves as a prognostic marker for breast cancer, and its expression level correlates with metastatic breast cancer and poor prognosis. Here, we investigated the levels of IL-8 expression in a variety of breast cancer cells and the regulatory mechanism of IL-8 in triple-negative breast cancer (TNBC) cells. Our results showed that IL-8 expression correlated positively with overall survival in basal-type breast cancer patients. The levels of IL-8 mRNA expression and protein secretion were significantly increased in TNBC cells compared with non-TNBC cells. In addition, the invasiveness of the TNBC cells was dramatically increased by IL-8 treatment and then augmented invasion-related proteins such as matrix metalloproteinase (MMP)-2 or MMP-9. We observed that elevated IL-8 mRNA expression and protein secretion were suppressed by a specific MEK1/2 inhibitor, UO126. In contrast, the overexpression of constitutively active MEK significantly increased the level of IL-8 mRNA expression in BT474 non-TNBC cells. Finally, we investigated the effect of UO126 on the tumorigenecity of TNBC cells. Our results showed that anchorage-independent growth, cell invasion, and cell migration were also decreased by UO126 in TNBC cells. As such, we demonstrated that IL-8 expression is regulated through MEK/ERK-dependent pathways in TNBC cells. A diversity of MEK blockers, including UO126, may be promising for treating TNBC patients.

Elevated TGF-ß1 and -ß2 expression accelerates the epithelial to mesenchymal transition in triple-negative breast cancer cells.

The epithelial-mesenchymal transition (EMT) is a key process in tumor invasion and migration. Transforming growth factor-ßs (TGF-ßs) are multifunctional growth factors and powerful modulators of the EMT. Here, we investigated the relationship between TGF-ß expression and invasion by treated triple-negative breast cancer (TNBC) cells. Our results show that invasion capacity of TNBC cells was markedly higher than that of non-TNBC cells. In addition, EMT-related gene signatures, including vimentin (vim), fibronectin (FN), snail, and slug were highly expressed in TNBC cells. Interestingly, our results show that TGF-ß1 and ß2 mRNA expression levels were higher in TNBC cells than those in non-TNBC cells. Thus, we examined the effect of the TGF-ß receptor I/II inhibitor LY2109761 on EMT-related gene expression and cell motility. Our data show that vim, FN, and slug mRNA expression levels dose-dependently decreased in response to LY2109761. TNBC cell motility also decreased in response to LY2109761. Finally, we investigated the effect of LY2109761 on TGF-ß1 or TGF-ß2-induced E-cadherin (E-cad), vim, and FN mRNA and protein expression. The reduction in E-cad and induction of vim and FN expression by TGF-ß1 or TGF-ß2 were completely reversed by LY2109761 treatment in HCC1806 TNBC cells. Taken together, we demonstrated that elevated TGF-ß expression triggers invasion and migration by TNBCs through the EMT process. Inhibiting the TGF-ß signaling pathway is considered a promising therapeutic strategy for treating TNBC.

Induction of fibronectin by HER2 overexpression triggers adhesion and invasion of breast cancer cells.

Fibronectin (FN), an extracellular matrix ligand, plays a pivotal role in cell adhesion, migration, and oncogenic transformation. Aberrant FN expression is associated with poor prognoses in various types of cancer, including breast cancer. In the current study, we investigated the relationship between FN induction and HER2 expression in breast cancer cells. Our results showed that the level of FN expression increased in response to HER family ligands, EGF and TGF-α in a time- and dose-dependent manner. On the other hand, EGF-induced FN expression decreased in response to trastuzumab, which is a HER2-targeted monoclonal antibody. However, EGF-induced FN expression was not affected by trastuzumab in JIMT-1 breast cancer cells, which are trastuzumab insensitive cells. Next, we introduced the HER2 gene into MDA-MB231 cells to verify the relationship between FN and HER2. The level of FN expression significantly increased in HER2-overexpressed MDA-MB231 cells. In contrast, the induction of FN by HER2 was significantly decreased in response to trastuzumab treatment. In addition, the induction of FN by HER2 was down-regulated by the MEK 1/2 specific inhibitor, U0126. Using conditioned culture media of vec- and HER2-overexpressed MDA-MB231 cells, we observed the cell morphology, adhesion, and invasion of MDA-MB231 cells. Interestingly, in conditioned culture media of HER2-overexpressed MDA-MB231 cells, the cell morphology was altered, and adhesion and invasion of MDA-MB231 cells significantly increased. In addition, our results showed that recombinant human FN augmented cell adhesion and invasion of MDA-MB231 cells while these inductions decreased in response to an FN inhibitor. Therefore, we demonstrated that the induction of FN by HER2 triggers cell adhesion and invasion capacities.

Zerumbone suppresses EGF-induced CD44 expression through the inhibition of STAT3 in breast cancer cells.

Expression of the CD44 gene is upregulated in breast cancer cells and is correlated with patient survival. Aberrant CD44 expression promotes tumor progression and metastasis. In the present study, we investigated the role of zerumbone (ZER) on regulatory mechanisms of CD44 expression in breast cancer cells. Our results showed that CD44 expression was significantly increased by epidermal growth factor receptor (EGFR) ligands in SKBR3 breast cancer cells. In contrast, EGF-induced CD44 expression was decreased by a MEK1/2 inhibitor, UO126, or STAT3 inhibitor, STAT3 VI, respectively. Notably, ZER downregulated the basal level of CD44 expression in CD44+ breast cancer cells. In addition, the induction of CD44 expression by EGFR ligands, EGF or TGF-α, was markedly decreased by ZER treatment. Finally, we investigated the inhibitory mechanism of ZER on EGF-induced CD44 expression. Our results showed that EGF-induced phosphorylation of STAT3 was completely suppressed by ZER. Collectively, ZER suppressed EGF-induced CD44 expression through inhibition of the STAT3 pathway. Therefore, we suggested that ZER may act as a promising therapeutic drug for the treatment of breast cancer.

Induction of fibronectin in response to epidermal growth factor is suppressed by silibinin through the inhibition of STAT3 in triple negative breast cancer cells.

Fibronectin (FN) plays a major role in cell adhesion, migration and oncogenic transformation. Aberrant FN expression is associated with poor prognosis in various types of cancer, including breast cancer. In this study, we investigated the effect of silibinin on the epidermal growth factor (EGF)-induced FN expression in triple negative breast cancer (TNBC) cells. Our data showed that the levels of FN mRNA and protein expression were dose-dependently increased by EGF in MDA-MB468 and BT20 breast cancer cells. Consequently, EGF-induced FN expression was decreased by the epidermal growth factor receptor (EGFR) inhibitors AG1478 and gefitinib. EGF-induced FN expression was also decreased by MEK1/2, PI3K and STAT3 specific inhibitors. In the present study, we observed for the first time that EGF-induced FN expression was significantly decreased by silibinin treatment in TNBC cells. Furthermore, we found that silibinin suppressed the EGF-induced phosphorylation of STAT3 but not Erk and Akt. Taken together, silibinin downregulated EGF-induced FN expression through the inhibition of STAT3 phosphorylation in TNBC cells. Silibinin may be a promising anticancer drug for the treatment of TNBC.

Berberine suppresses TPA-induced fibronectin expression through the inhibition of VEGF secretion in breast cancer cells.

BACKGROUND/AIMS: Berberine (BBR) is an isoquinoline alkaloid and is beneficial for the anticancer effect on a variety of human tumor cells. However, BBR's anti-angiogenesis property and its clinical potential as an inhibitor of tumor angiogenesis in breast cancer cells have not been fully elucidated. Here, we investigated the effect of BBR on TPA-induced VEGF and fibronectin (FN) as well as VEGF-induced FN in breast cancer cells. METHODS: The secretion of VEGF protein was detected by ELISA. Fibronectin mRNA and protein expression was analyzed by Real-Time PCR and western blotting, respectively. The overexpressions of CA-MEK, and CA-Akt were examined by adenovirus system. RESULTS: Our results showed that TPA, a tumor promoter, significantly increased the level of VEGF and FN expression in both MCF7 and T47D breast cancer cells. On the other hand, TPA-induced VEGF and FN expression was suppressed by LY294002, a PI-3K inhibitor. In contrast, the level of FN expression also significantly increased by constitutively active (CA)-AKT overexpression. We also found that TPA-induced VEGF and FN expression was decreased by BBR treatment. Finally, our results showed that VEGF augmented the expression of FN whereas VEGF-induced FN expression was decreased by BBR treatment. CONCLUSION: Taken together, we suggest that BBR may suppress TPA-induced VEGF and FN as well as VEGF-induced FN through the inhibition of the PI-3K/AKT pathway in breast cancer cells. Therefore, we suggest that BBR may be used as a candidate drug for the inhibition of angiogenesis of human breast cancer.