Myeongjin Jeon

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, BBRs 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.

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 the motility and tumorigenecity of triple negative breast cancer cells via the inhibition of TGF-β1 signaling pathway

Aberrant transforming growth factor-β (TGF-β) plays an important role in the development of cancer such as tumor metastasis and invasion. TGF-β-responsive gene signature is highly activated in chemotherapy-treated triple negative breast cancer (TNBC). Here, we investigated the effect of zerumbone (ZER) on TGF-β1 signaling pathway and tumorigenecity of TNBC cells. Our results showed that the level of TGF-β1 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-β receptor type I/II (TβRI/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-β1 increased FN, MMP-2 and MMP-9 expression in HCC1806 TNBC cells. TGF-β1-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-β1 was just decreased by SIS3. Overexpression of smad3 significantly increased FN, MMP-2, and MMP-9 expression. Interestingly, ZER significantly suppressed TGF-β1-induced FN, MMP-2, and MMP-9 expression in HCC1806 cells. In addition, ZER completely decreased TGF-β1-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-β1-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.

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-κB‑dependent signaling pathway in TNBC cells.

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

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

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.

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.

Berberine Suppresses Cell Motility Through Downregulation of TGF-β1 in Triple Negative Breast Cancer Cells

Background/Aims: Transforming growth factor-beta proteins (TGF-βs) 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-β1 expression. Methods: The levels of mRNA expression were analyzed by real-time PCR. The levels of MMP-2, MMP-9 and TGF-β1 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-β1 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-β1 treatment. In contrast, basal levels of MMP-2 and MMP-9 were suppressed by a specific TGF-β receptor I inhibitor, SB431542. In addition, TGF-β1–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-β1, but not TGF-β2, 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-β1 expression. Therefore, we demonstrate that BBR could be a promising drug for treatment of 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.