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NADPH oxidases (NOXs) are a family of membrane proteins responsible for intracellular reactive oxygen species (ROS) generation by facilitating electron transfer across biological membranes. Despite the established activation of NOXs by protein kinase C (PKC), the precise mechanism through which PKC triggers NOX activation during breast cancer invasion remains unclear. The present study aimed to investigate the role of NOX1 and NOX5 in the invasion of MCF‑7 human breast cancer cells. The expression and activity of NOXs and matrix metalloprotease (MMP)‑9 were assessed by reverse transcription‑quantitative PCR and western blotting, and the activity of MMP‑9 was monitored using zymography. Cellular invasion was assessed using the Matrigel invasion assay, whereas ROS levels were quantified using a FACSCalibur flow cytometer. The findings suggested that NOX1 and NOX5 serve crucial roles in 12‑O‑tetradecanoylphorbol‑13‑acetate (TPA)‑induced MMP‑9 expression and invasion of MCF‑7 cells. Furthermore, a connection was established between PKC and the NOX1 and 5/ROS signaling pathways in mediating TPA‑induced MMP‑9 expression and cellular invasion. Notably, NOX inhibitors (diphenyleneiodonium chloride and apocynin) significantly attenuated TPA‑induced MMP‑9 expression and invasion in MCF‑7 cells. NOX1‑ and NOX5‑specific small interfering RNAs attenuated TPA‑induced MMP‑9 expression and cellular invasion. In addition, knockdown of NOX1 and NOX5 suppressed TPA‑induced ROS levels. Furthermore, a PKC inhibitor (GF109203X) suppressed TPA‑induced intracellular ROS levels, MMP‑9 expression and NOX activity in MCF‑7 cells. Therefore, NOX1 and NOX5 may serve crucial roles in TPA‑induced MMP‑9 expression and invasion of MCF‑7 breast cancer cells. Furthermore, the present study indicated that TPA‑induced MMP‑9 expression and cellular invasion were mediated through PKC, thus linking the NOX1 and 5/ROS signaling pathways. These findings offer novel insights into the potential mechanisms underlying their anti‑invasive effects in breast cancer.

Matriptases, members of the type II transmembrane serine protease family, are cell surface proteolytic enzymes that mediate tumor invasion and metastasis. Matriptase is highly expressed in breast cancer and is associated with poor patient outcome. However, the cellular mechanism by which matriptase mediates breast cancer invasion remains unknown. The present study aimed to determine the role of matriptase in the protein kinase C (PKC)‑mediated metastasis of MCF‑7 human breast cancer cells. Matriptase small interfering RNA‑mediated knockdown significantly attenuated the 12‑ O ‑tetradecanoylphorbol‑13‑acetate (TPA)‑induced invasiveness and migration of MCF‑7 cells, and inhibited the activation of phospholipase C γ2 (PLCγ2)/PKC/MAPK signaling pathways. Matriptase‑knockdown also suppressed the expression of MMP‑9 and inhibited the activation of NF‑κB/activator protein‑1 in MCF‑7 cells. Additionally, GB83 [an inhibitor of protease‑activated receptor‑2 (PAR‑2)] inhibited PKC‑mediated MMP‑9 expression and metastatic ability in MCF‑7 cells. Furthermore, downregulation of matriptase suppressed TPA‑induced MMP‑9 expression and invasiveness via PAR‑2/PLCγ2/PKC/MAPK activation. These findings shed light on the mechanism underlying the role of matriptase in MCF‑7 cell invasion and migration ability, and suggest that matriptase modulation could be a promising therapeutic strategy for preventing breast cancer metastasis.

Bruton's agammaglobulinemia tyrosine kinase (BTK) is an important cytoplasmic tyrosine kinase involved in B‑lymphocyte development, differentiation, and signaling. Activated protein kinase C (PKC), in turn, induces the activation of mitogen‑activated protein kinase (MAPK) signaling, which promotes cell proliferation, viability, apoptosis, and metastasis. This effect is associated with nuclear factor‑κB (NF‑κB) activation, suggesting an anti‑metastatic effect of BTK inhibitors on MCF‑7 cells that leads to the downregulation of matrix metalloproteinase (MMP)‑9 expression. However, the effect of BTK on breast cancer metastasis is unknown. In this study, the anti‑metastatic activity of BTK inhibitors was examined in MCF‑7 cells focusing on MMP‑9 expression in 12‑ O ‑tetradecanoylphorbol‑13‑acetate (TPA)‑stimulated MCF‑7 cells. The expression and activity of MMP‑9 in MCF‑7 cells were investigated using quantitative polymerase chain reaction analysis, western blotting, and zymography. Cell invasion and migration were investigated using the Matrigel invasion and cell migration assays. BTK inhibitors [ibrutinib (10 µM), CNX‑774 (10 µM)] significantly attenuated TPA‑induced cell invasion and migration in MCF‑7 cells and inhibited the activation of the phospholipase Cγ2/PKCβ signaling pathways. In addition, small interfering RNA specific for BTK suppressed MMP‑9 expression and cell metastasis. Collectively, results of the present study indicated that BTK suppressed TPA‑induced MMP‑9 expression and cell invasion/migration by activating the MAPK or IκB kinase/NF‑κB/activator protein‑1 pathway. The results clarify the mechanism of action of BTK in cancer cell metastasis by regulating MMP‑9 expression in MCF‑7 cells.

Activation of peroxisome proliferator-activated receptor γ (PPARγ) serves as a key factor in the proliferation and invasion of breast cancer cells and is a potential therapeutic target for breast cancer. However, the mechanisms underlying this effect remain largely unknown. Heme oxygenase-1 (HO-1) is induced and overexpressed in various cancers and is associated with features of tumor aggressiveness. Recent studies have shown that HO-1 is a major downstream target of PPARγ. In this study, we investigated the effects of induction of HO-1 by PPARγ on TPAinduced MMP-9 expression and cell invasion using MCF-7 breast cancer cells. TPA treatment increased NF-κB /AP-1 DNA binding as well as MMP-9 expression. These effects were significantly blocked by 15d-PGJ2, a natural PPARγ ligand. 15d-PGJ2 induced HO-1 expression in a dose-dependent manner. Interestingly, HO-1 siRNA significantly attenuated the inhibition of TPA-induced MMP-9 protein expression and cell invasion by 15d-PGJ2. These results suggest that 15d-PGJ2 inhibits TPA-induced MMP- 9 expression and invasion of MCF-7 cells by means of a heme oxygenase-1-dependent mechanism. Therefore, PPARγ/HO-1 signaling- pathway inhibition may be beneficial for prevention and treatment of breast cancer. [BMB Reports 2020; 53(4): 212-217].

Casein kinase 2 (CK2) is a serine/threonine protein kinase that has been considered to represent an important factor in mammary tumorigenesis. Increased expression of matrix metalloproteinase‑9 (MMP‑9) via nuclear factor‑κB (NF‑κB) activation has been demonstrated to promote breast cancer cell invasion. In the present study, the involvement of CK2 in protein kinase C (PKC) induced cell invasion in MCF‑7 breast cancer cells was investigated as well as the underlying molecular mechanisms. The mRNA and protein levels of MMP‑9 in MCF‑7 cells were investigated using reverse transcription‑quantitative polymerase chain reaction, western blot analyses and a zymography assay. Cell invasiveness was investigated using a Matrigel invasion assay, and it was revealed that small interfering RNA specific for CK2 suppressed PKC induced cell invasion by regulating MMP‑9 expression via activation of the p38 kinase/c‑Jun N‑terminal kinase/NF‑κB pathway. In addition, it was demonstrated that CK2 inhibitors [apigenin (20 µM), emodin (20 µM) or 2‑dimethylamino‑4,5,6,7‑tetrabromo‑1H‑benzimidazole (2 µM)] suppressed PKC induced cell invasion and MMP‑9 expression. The results of the present study suggested that CK2 is an important factor involved in the induction of MCF‑7 breast cancer cell invasion by PKC. Therefore, CK2 may represent novel candidates for therapy intended to inhibit invasion in breast cancer.

Invasion and metastasis are among the main causes of death in patients with malignant tumors. Fisetin (3,3',4',7-tetrahydroxyflavone), a natural flavonoid found in the smoke tree (Cotinus coggygria), is known to have antimetastatic effects on prostate and lung cancers; however, the effect of fisetin on breast cancer metastasis is unknown. The aim of this study was to determine the anti-invasive activity of fisetin in human breast cancer cells. Matrix metalloproteinase (MMP)-9 is a major component facilitating the invasion of many cancer tumor cell types, and thus the inhibitory effect of fisetin on MMP-9 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated human breast cancer cells was investigated in this study. Fisetin significantly attenuated TPA-induced cell invasion in MCF-7 human breast cancer cells, and was found to inhibit the activation of the PKCα/ROS/ERK1/2 and p38 MAPK signaling pathways. This effect was furthermore associated with reduced NF-κB activation, suggesting that the anti-invasive effect of fisetin on MCF-7 cells may result from inhibited TPA activation of NF-κB and reduced TPA activation of PKCα/ROS/ERK1/2 and p38 MAPK signals, ultimately leading to the downregulation of MMP-9 expression. Our findings indicate the role of fisetin in MCF-7 cell invasion, and clarify the underlying molecular mechanisms of this role, suggesting fisetin as a potential chemopreventive agent for breast cancer metastasis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The Aurora kinase family of serine/threonine kinases are known to be crucial for cell cycle control. Aurora kinases are considered a target of anticancer drugs. However, few studies have assessed the effect of Aurora kinases in breast cancer. In the present study, to determine whether Aurora kinases play a role in oncogenic actions of protein kinase C (PKC), we investigated the effect of Aurora kinases on PKC-induced invasion and MMP-9 expression using breast cancer cells. Treatment of MCF-7 cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) induced the upregulation and phosphorylation of Aurora kinases via the MAPK signaling pathway. Moreover, the inhibition of Aurora kinases by their siRNAs and inhibitors suppressed TPA-induced cell invasion and expression of MMP-9 by inhibiting the activation of NF-κB/AP-1, major transcription factors for MMP-9 expression in MCF-7 cells. These results suggested that Aurora kinases mediate PKC-MAPK signal to NF-κB/AP-1 with increasing MMP-9 expression and invasion of MCF-7 cells. To the best of our knowledge, this is the first study to show that Aurora kinases are key molecules in PKC-induced invasion in breast cancer cells.

Decursin, a coumarin compound, was first isolated from the roots of Angelica gigas almost four decades ago. It was found to exhibit cytotoxicity against various human cancer cells and to possess anti-amnesic activity in vivo through the inhibition of AChE activity. However, the effect of decursin on breast cancer invasion is unknown. Matrix metalloproteinase-9 (MMP-9) is known to be an important factor for cancer cell invasion. Therefore, in this study, we investigated the inhibitory effect of decursin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion, as well as the molecular mechanisms involved in MCF-7 cells. Our results showed that decursin inhibits TPA-induced MMP-9 expression and cell invasion through the suppression of NF-κB. Furthermore, decursin repressed the TPA-induced phosphorylation of p38 MAPK and inhibited TPA-induced translocation of PKCα from the cytosol to the membrane, but did not affect the translocation of PKCδ. These results indicate that decursin-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of the PKCα, MAPK and NF-κB pathways in MCF-7 cells. Thus, decursin may have potential value in restricting breast cancer metastasis.

Sulforaphane [1-isothiocyanato-4-(methylsulfinyl)-butane] is an isothiocyanate found in some cruciferous vegetables, especially broccoli. Sulforaphane has been shown to display anti-cancer properties against various cancer cell lines. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix (ECM), plays an important role in cancer cell invasion. In this study, we investigated the effect of sulforaphane on 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion in MCF-7 cells. TPA-induced MMP-9 expression and cell invasion were decreased by sulforaphane treatment. TPA substantially increased NF-κB and AP-1 DNA binding activity. Pre-treatment with sulforaphane inhibited TPA-stimulated NF-κB binding activity, but not AP-1 binding activity. In addition, we found that sulforaphane suppressed NF-κB activation, by inhibiting phosphorylation of IκB in TPA-treated MCF-7 cells. In this study, we demonstrated that the inhibition of TPA-induced MMP-9 expression and cell invasion by sulforaphane was mediated by the suppression of the NF-κB pathway in MCF-7 cells.

Cell invasion is required for neoplastic metastasis. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix, is a major component in the process of cancer cell invasion. Sulfuretin is one of the major flavonoids isolated from Rhus verniciflua. Sulfuretin has been used to reduce oxidative stress, platelet aggregation, the inflammatory response and mutagenesis. However, the effect of sulfuretin on breast cancer metastasis is unknown. In this study, we investigated the inhibitory effect of sulfuretin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion in MCF-7 cells. Sulfuretin inhibited TPA-induced transcriptional activation of nuclear factor-κB (NF-κB). We demonstrated that sulfuretin mediated the inhibition of TPA-induced MMP-9 expression and that cell invasion in MCF-7 cells involved suppression of the NF-κB pathway. Therefore, inhibiting MMP-9 expression by sulfuretin may have therapeutic potential for controlling breast cancer invasiveness.

Νuclear factor-κB (NF-κB) and activator protein-1 (AP-1) are major transcription factors that have been associated with breast cancer metastasis by inducing matrix metalloproteinase-9 (MMP-9) expression. In this study, we investigated the inhibitory effects of guggulsterone isomers (cis or trans) on 12-O-tetradecanoylpho-bol-13-acetate (TPA)-induced MMP-9 expression. Cis-guggulsterone inhibited TPA-induced MMP expression by blocking IκB kinase (IKK)/NF-κB signaling, whereas trans-guggulsterone blocked mitogen-activated protein kinase (MAPK)/AP-1 signaling. Cis-guggulsterone was more potent than trans-guggulsterone in the inhibition of TPA-induced MMP-9 expression and invasion of MCF-7 cells. Furthermore, we found that the combination of these isomers exerted an additive effect on the inhibition of MCF-7 cell invasion. These results suggest that guggulsterone isomers downregulate MMP-9 expression and tumor cell invasion through the isomer-specific suppression of IKK/NF-κB and MAPK/AP-1 activation. In addition, the suppression of MMP-9 expression correlated well with the inhibition of cell invasion.

Curcumin (diferuloylmethane) is a polyphenol derived from the plant turmeric (Curcuma longa), which is commonly used as a spice. Although anti-carcinogenic, anti-oxidant, anti-inflammation, and anti-angiogenic properties have been reported, the effect of curcumin on breast cancer metastasis is unknown. Matrix metalloproteinase-9 (MMP-9) is a major component in cancer cell invasion. In this study, we investigated the inhibitory effect of curcumin on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced MMP-9 expression and cell invasion and the molecular mechanisms involved in MCF-7 cells. Our results showed that curcumin inhibits TPA-induced MMP-9 expression and cell invasion through suppressing NF-κB and AP-1 activation. Also, curcumin strongly repressed the TPA-induced phosphorylation of p38 and JNK and inhibited TPA-induced translocation of PKCα from the cytosol to the membrane, but did not affect the translocation of PKCδ. These results indicate that curcumin-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involves the suppression of the PKCα, MAPK and NF-κB/AP-1 pathway in MCF-7 cells. Curcumin may have potential value in restricting breast cancer metastasis., (Copyright © 2012 Elsevier GmbH. All rights reserved.)