Jeonghun Han

Berberine Suppresses the TPA-Induced MMP-1 and MMP-9 Expressions Through the Inhibition of PKC-α in Breast Cancer Cells

BACKGROUND Berberine (BBR) is one of the major alkaloids, and it has been reported to have a variety of pharmacologic effects, including inhibition of cell cycle progression. Here, we investigated the effect of BBR on the MMP-1 and MMP-9 expressions, which are predictors of metastasis and invasion in breast cancer cells. METHODS MMP-1 and MMP-9 mRNA expressions were analyzed by real-time PCR. The levels of MMP-1 protein and PKC-α phosphorylation were detected by Western blotting. MMP-9 protein expression was detected by gelatin zymography. Cell cycle was analyzed by FACS analysis. PKC-α knock-down was examined by PKC-α siRNA transfection. RESULTS The basal levels of both the MMP-1 and MMP-9 mRNA expressions were decreased by BBR treatment in a dose-dependent manner. In contrast, TPA, which is a tumor promoter, significantly increased the levels of the MMP-1 and MMP-9 mRNA and protein expressions in the MCF-7 breast cancer cells. We also observed that the TPA-induced MMP-1 and MMP-9 mRNA and protein expressions were prevented by BBR treatment. In addition, the TPA-induced MMP-1 and MMP-9 expressions were completely decreased by Go6983 and PKC-α siRNA, respectively. TPA-induced PKC-α phosphorylation was dose-dependently decreased by BBR treatment. CONCLUSION The TPA-induced PKC-α phosphorylation is suppressed and then the MMP-1 and MMP-9 expressions are also inhibited by berberine. Therefore, we suggest that berberine may be used as a candidate drug for the inhibition of metastasis of human breast cancer.

Zerumbone suppresses IL-1β-induced cell migration and invasion by inhibiting IL-8 and MMP-3 expression in human triple-negative breast cancer cells.

Inflammation is a key regulatory process in cancer development. Prolonged exposure of breast tumor cells to inflammatory cytokines leads to epithelial‐mesenchymal transition, which is the principal mechanism involved in metastasis and tumor invasion. Interleukin (IL)‐1β is a major inflammatory cytokine in a variety of tumors. To date, the regulatory mechanism of IL‐1β‐induced cell migration and invasion has not been fully elucidated. Here, we investigated the effect of zerumbone (ZER) on IL‐1β‐induced cell migration and invasion in breast cancer cells. The levels of IL‐8 and matrix metalloproteinase (MMP)‐3 mRNA were analyzed by real‐time polymerase chain reaction. The levels of secreted IL‐8 and MMP‐3 protein were analyzed by enzyme‐linked immunosorbent assay and western blot analysis, respectively. Cell invasion and migration was detected by Boyden chamber assay. The levels of IL‐8 and MMP‐3 expression were significantly increased by IL‐1β treatment in Hs578T and MDA‐MB231 cells. On the other hand, IL‐1β‐induced IL‐8 and MMP‐3 expression was decreased by ZER. Finally, IL‐1β‐induced cell migration and invasion were decreased by ZER in Hs578T and MDA‐MB231 cells. ZER suppresses IL‐1β‐induced cell migration and invasion by inhibiting IL‐8 expression and MMP‐3 expression in TNBC cells. ZER could be a promising therapeutic drug for treatment of triple‐negative breast cancer patients. Copyright

Smad7 acts as a negative regulator of the epidermal growth factor (EGF) signaling pathway in breast cancer cells

Although it has been suggested that smad7 blocks downstream signaling of TGF-β, the role of smad7 in the EGF signaling pathway has not been fully elucidated. We determined the effect of smad7 on EGF-induced MMP-9 expression in SKBR3 breast cancer cells. The expression of smad7 and MMP-9 was increased by EGF or TGF-β1, respectively, and further increased by EGF and TGF-β1 co-treatment. EGF induced the phosphorylation of EGFR, smad3, ERK, and JNK, and MMP-9 expression was decreased by the EGFR inhibitor, AG1478. In addition, EGF-induced MMP-9 expression was inhibited by UO126 (a MEK1/2 inhibitor) or SIS3 (a smad3 inhibitor), but not by SP600125 (a JNK inhibitor). Interestingly, EGF-induced smad3 phosphorylation was completely blocked by smad7 over-expression, but not the phosphorylation of ERK and JNK. EGF- or TGF-β1-induced MMP-9 expression was completely decreased by adenoviral-smad7 (Ad-smad7) over-expression. We also investigated the role of smad3 on EGF-induced MMP-9 expression and showed that EGF-induced MMP-9 expression was decreased by smad3 siRNA transfection, whereas EGF-induced MMP-9 expression was further increased by smad3 over-expression, as expected. This study showed that EGF-induced smad3 phosphorylation mediates the induction of MMP-9, whereas smad7 inhibits TGF-β1 as well as the EGF signaling pathway in SKBR3 cells.

Silibinin inhibits TPA-induced cell migration and MMP-9 expression in thyroid and breast cancer cells

Matrix metalloproteinases (MMPs) play an important role in cancer metastasis, cell migration and invasion. Herein, we investigated the effects of silibinin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced cell migration and MMP-9 expression in thyroid and breast cancer cells. Our results revealed that the levels of MMP-9 mRNA and protein expression were significantly increased by TPA but not MMP-2 in TPC-1 and MCF7 cells. To verify the regulatory mechanism of TPA-induced MMP-9 expression, we treated TPC-1 and MCF7 cells with the MEK1/2 inhibitor, UO126, and TPA-induced MMP-9 expression was significantly decreased. We also found that TPA-induced cell migration and MMP-9 expression was significantly decreased by silibinin. In addition, TPA-induced phosphorylation of MEK and ERK was also inhibited by silibinin. Taken together, we suggest that silibinin suppresses TPA-induced cell migration and MMP-9 expression through the MEK/ERK-dependent pathway in thyroid and breast cancer cells.

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.

TPA-induced p21 expression augments G2/M arrest through a p53-independent mechanism in human breast cancer cells

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has a differential role on the regulation of the cell cycle in a variety of tumor cells. The mechanism between TPA and the cell cycle in breast cancer is not fully understood. Therefore, we investigated the regulatory mechanism of TPA on control of the cell cycle of breast cancer cells. Our results showed that TPA increased the level of p21 expression in MCF-7 cells with wild-type p53 and MDA-MB-231 cells with mutant p53 in a dose-dependent manner. In contrast, TPA decreased the expression of p53 in MCF-7 cells, but did not affect MDA-MB-231 cells. We next examined the regulatory mechanism of TPA on p21 and p53 expression. Our results showed that the TPA-induced up-regulation of p21 and down-regulation of p53 was reversed by UO126 (a MEK1/2 inhibitor), but not by SP600125 (a JNK inhibitor) or SB203580 (a p38 inhibitor), although TPA increased the phosphorylation of ERK and JNK in MCF-7 cells. In addition, the TPA-induced arrest of the G2/M phase was also recovered by UO126 treatment. To confirm the expression of p21 through the MEK/ERK pathway, cells were transfected with constitutively active (CA)-MEK adenovirus. Our results showed that the expression of p21 was significantly increased by CA-MEK overexpression. Taken together, we suggest that TPA reciprocally regulates the level of p21 and p53 expression via a MEK/ERK-dependent pathway. The up-regulation of p21 in response to TPA is mediated through a p53-independent mechanism in breast cancer cells.

A functional comparison between the HER2(high)/HER3 and the HER2(low)/HER3 dimers on heregulin-β1-induced MMP-1 and MMP-9 expression in breast cancer cells.

Overexpression of HER2 correlates with more aggressive tumors and increased resistance to cancer chemotherapy. However, a functional comparison between the HER2high/HER3 and the HER2low/HER3 dimers on tumor metastasis has not been conducted. Herein we examined the regulation mechanism of heregulin-β1 (HRG)-induced MMP-1 and -9 expression in breast cancer cell lines. Our results showed that the basal levels of MMP-1 and -9 mRNA and protein expression were increased by HRG treatment. In addition, HRG-induced MMP-1 and -9 expression was significantly decreased by MEK1/2 inhibitor, U0126 but not by phosphatidylinositol 3-kinase (PI-3K) inhibitor, LY294002. To confirm the role of MEK/ERK pathway on HRG-induced MMP-1 and -9 expression, MCF7 cells were transfected with constitutively active adenoviral-MEK (CA-MEK). The level of MMP-1 and -9 expressions was increased by CA-MEK. MMP-1 and -9 mRNA and protein expressions in response to HRG were higher in HER2 overexpressed cells than in vector alone. The phosphorylation of HER2, HER3, ERK, Akt, and JNK were also significantly increased in HER2 overexpressed MCF7 cells compared with vector alone. HRG-induced MMP-1 and -9 expressions were significantly decreased by lapatinib, which inhibits HER1 and HER2 activity, in both vector alone and HER2 overexpressed MCF7 cells. Finally, HRG-induced MMP-1 and MMP-9 expression was decreased by HER3 siRNA overexpression. Taken together, we suggested that HRG-induced MMP-1 and MMP-9 expression is mediated through HER3 dependent pathway and highly expressed HER2 may be associated with more aggressive metastasis than the low expressed HER2 in breast cancer cells.

Effect of berberine on p53 expression by TPA in breast cancer cells

Berberine (BBR), an isoquinoline derivative alkaloid compound, has been reported to have anti-oxidant and anti-carcinogenic effects. A loss of functional p53 is involved with an increased risk of cancer proliferation and metastasis. Here, we investigated the effect of BBR on the transcriptional activity and the protein expression of p53 in p53-positive (wild- type, MCF7 cells) and p53-negative (mutant, MDA-MB231 cells) human breast cancer cells. Our results showed that the basal level of p53 mRNA and protein expression was increased by BBR treatment. However, tumor promoter, TPA, decreased the level of p53 mRNA and protein expression in MCF7 cells with wild-type p53. In addition, TPA-induced down-regulation of p53 mRNA and protein expression was increased by UO126, but not by SP600125 and SB203580. To verify the regulatory mechanism of p53 protein expression, we investigated the effects of proteasomal inhibitors (ALLN and MG132) or a lysosomal inhibitor (chloroquine) on TPA-induced down-regulation of p53. We observed that TPA-induced down-regulation of p53 protein was prevented by ALLN and MG132, but not by chloroquine. Further, we investigated the effect of BBR on TPA-induced down-regulation of p53 mRNA and protein levels. Interestingly, the levels of TPA-induced down-regulation of p53 mRNA and protein were prevented by BBR, but MDA-MB231 cells with mutated p53 were not affected. In addition, TPA-induced down-regulation of p53 mRNA was also prevented by BBR. Taken together, we suggest that BBR may be used as an effective ingredient for anticancer products, which trigger the transcriptional activity and the inhibition of the degradation of p53, a tumor suppressor gene, in human breast cancer.

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.