Search

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.

Metastatic cancers spread from their site of origin (the primary site) to other parts of the body. Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix, is important in metastatic cancers as it plays a major role in cancer cell invasion. The present study examined the inhibitory effect of an ethanol extract of Peucedanum japonicum Thunb. (PJT) on MMP-9 expression and the invasion of MCF-7 breast cancer cells induced by 12-O-tetradecanoylphorbol-13-acetate (TPA). Western blot analysis, gelatin zymography, and reverse transcription-quantitative PCR revealed that PJT significantly suppressed MMP-9 expression and activation in a dose-dependent manner. Furthermore, PJT attenuated TPA-induced nuclear translocation and the transcriptional activation of nuclear factor (NF)-κB. The results indicated that the PJT-mediated inhibition of TPA-induced MMP-9 expression and cell invasion involved the suppression of the PKCα/NF-κB pathway in MCF-7 cells. Thus, the inhibition of MMP-9 expression by PJT may have potential value as a therapy for restricting the invasiveness of breast cancer.

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.

Autophagy is known as an important regulatory mediator for cell survival or death and its important role in cancer. Pemetrexed (PTX) has been used in chemotherapy for lung cancer. However, the underlying molecular mechanisms have not been fully clarified. To investigate the role of autophagy induced by PTX in A549 cells, we performed MTT assay, acridine orange staining, western blotting, Annexin V/PI by using the 3-MA autophagy inhibitor. PTX induced autophagy after 48 h treatment in A549 cells. Furthermore, PTX showed acidic vesicular organelles (AVOs) and expressed LC3-II in A549 cells. The induction of autophagy by PTX was inhibited by 3-MA which was confirmed by reduced AVOs. When the autophagy was inhibited, Annexin V was increased. In addition, PARP cleavage was increased as shown by western blotting. Taken together, PTX induced autophagy in A549 cells and these cellular events possibly cause the apoptotic and/or necrotic cell death of A549 cells.

Obesity is a risk factor associated with numerous disorders, such as type 2 diabetes, hypertension, dyslipidemia and coronary heart disease. In this study, we investigated the inhibitory effects of Pericarpium zanthoxyli extract (PZE) on the adipocytic differentiation of OP9 cells. During adipocyte differentiation, the OP9 cells were treated with 0, 10 and 20 µg/ml of PZE at various time intervals, followed by the examination of lipid droplet formation and the mRNA expression of adipogenesis-related genes. The cells treated with PZE during the early period (days 0-2) showed a significant reduction in the accumulation of lipid droplets, which were induced by a standard adipogenic cocktail, as well as a decrease in the expression of the adipogenesis-related transcription factor, peroxisome proliferator-activated receptor γ (PPARγ) and PPARγ-target genes, such as adipocyte protein 2 (aP2), fatty acid synthase (FAS) and other adipocyte markers. Adipocyte differentiation was not inhibited by treatment with PZE during the late stage of differentiation (days 3-5). Thus, the inhibitory effects of PZE on adipocyte differentiation occurred during the early stages of adipogenesis, which was confirmed by the decrease in the levels of CCAAT/enhancer-binding protein β (C/EBPβ) in a dose-dependent manner when the OP9 cells were exposed to PZE. Taken together, our results indicate that PZE inhibit the early stages of adipogenic differentiation by inhibiting C/EBPβ expression.

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.

The majority of chemotherapy treatments for lung cancer use cisplatin; however, its use is limited as it has several side-effects. Autophagy (or type II cell death) is an important mechanism by which programmed cell death occurs. The purpose of this study was to determine whether low-dose cisplatin treatment induces autophagy in lung cancer cells. We also examined whether autophagy inhibition results in p53-mediated apoptosis. NCI-H460 (wild-type p53) and NCI-H1299 (null-type p53) cells were treated with 5 or 20 µM cisplatin for 12, 24 or 48 h. An MTT assay was performed to measure the cell viability following cisplatin treatment. To detect cisplatin-induced autophagy, cell morphology (autophagic vacuole) and LC3 localization were examined. The outcome of autophagy inhibition was determined using 3-methyladenine (3-MA) to detect Annexin V (+), propidium iodide (PI) (-) and acridine orange (+) cells by FACS analysis. To determine whether cisplatin induced autophagy, we examined the role of p53 as a cell survival regulator in autophagy. Low-doses of cisplatin (5 µM) induced cell death and this was augmented by 3-MA in both cell lines. Autophagic vacuoles and cytoplasmic LC3 formation was more evident in H460 cells than in H1299 cells. The induction of autophagy by low-dose cisplatin was increased by 2-fold in H460 compared to H1299 cells. However, the tests for apoptosis showed no difference between the 2 cell lines. Following 3-MA pretreatment, cisplatin-induced autophagy was found to be markedly reduced (a 3-fold reduction) in wild-type p53 compared to null-type p53 cells. However, cisplatin-induced apoptosis increased in wild-type p53 compared to null-type p53 cells. Autophagy induction and apoptotic shift after autophagy inhibition may be mediated by p53 activation in lung cancer cells treated with low-dose cisplatin.

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.

Decursin, a coumarin compound, was originally isolated from the roots of Angelica gigas almost four decades ago, and it was found to exhibit cytotoxicity against various types of human cancer cells and anti-amnesic activity in vivo through the inhibition of AChE activity. However, the anti-skin photoaging effects of decursin have not been reported to date. In the present study, we investigated the inhibitory effects of decursin on the expression of matrix metalloproteinase (MMP)-1 and MMP-3 in human dermal fibroblast (HDF) cells. Western blot analysis and real-time PCR revealed that decursin inhibited the ultraviolet (UV)B-induced expression of MMP-1 and MMP-3 in a dose-dependent manner. Decursin significantly blocked the UVB-induced activation of nuclear factor-κB (NF-κB). However, decursin showed no effect on MAPK or AP-1 activity. In this study, decursin prevented the UVB-induced expression of MMPs via the inhibition of NF-κB activation. In conclusion, decursin may be a potential agent for the prevention and treatment of skin photoaging.

The tumor suppressor protein, PTEN (phosphatase and tensin homologue), has been reported to play a crucial role in a variety of tumor cells. Recent studies indicate that the transcription factor, PPARgamma (peroxisome proliferator-activated receptor gamma) up-regulates PTEN expression transcriptionally through the binding of two response elements in the genomic sequence upstream of PTEN in tumor cells. Here, we determined the PTEN and PPARgamma expression in human leiomyomas, and whether estrogen can change their protein expressions in cultured leiomyoma cells. Immunohistochemical staining and Western blot analysis revealed that PTEN protein levels in leiomyomas were higher than that the adjacent normal myometrial tissues, which was paralleled with decrease in phospho-Akt (ser-473) and phosphatidylinositol 3,4,5-tris phosphate (PIP3) level. Interestingly, leiomyomas exhibit high PPARgamma expression compared to the adjacent normal myometrial tissues. In addition, we found that 17beta-estradiol (E2) significantly stimulated PTEN and PPARgamma expression in cultured leiomyoma cells, but did not change both protein levels in normal cells. This report shows the first evidence that PTEN and PPARgamma are up-regulated in leiomyoma tissues, and estrogen stimulates expression of PTEN and PPARgamma in leiomyoma cells, but not in normal cells.

Matrix metalloproteinase-9 (MMP-9), which degrades the extracellular matrix (ECM), plays an important role in breast cancer cell invasion. NF-kappaB and AP-1 are known to induce MMP-9 expression. We investigated whether cordycepin, an NF-kappaB or AP-1 inhibitor, can modulate MMP-9 expression and cell invasion in MCF-7 cells. Toxicity of cordycepin was determined by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. MMP-9 expression was determined by real-time PCR, Zymography, and Western blot analysis. AP-1 activation was assayed by electrophoretic mobility shift assay (EMSA). MAPK signaling was evaluated by Western blotting with specific p-ERK, and ERK, p-p38, p38, p-JNK, JNK antibodies. Cordycepin suppressed AP-1 activation, but not NF-kappaB activation in 12-O-tetradecanoylpho-bol-13-acetate (TPA)-treated MCF-7 cells. Cordycepin inhibits TPA-induced MMP-9 expression and cell invasion by suppressing AP-1 activation. Also, cordycepin suppressed the MAPK signaling pathway. Cordycepin is a potent inhibitor of TPA-induced MMP-9 expression and blocks strongly the ability of AP-1 activation via MAPK signaling pathway in MCF-7 cells.

Ultraviolet (UV)B irradiation induces the production of matrix metalloproteinases (MMPs), which are responsible for the degradation of collagenous extracellular matrix in connective tissues, causing skin photoaging. Although Radix clematidis is commonly used in Chinese medicine for the treatment of arthralgia, the anti-skin photoaging effects of Radix clematidis have not yet been reported. In the present study, we investigated the inhibitory effects of Radix clematidis extract (RCE) on MMP-1 and -3 expression of human dermal fibroblast cells via various in vitro experiments and elucidated the pathways of inhibition. Western blot analysis and real-time PCR revealed RCE inhibited UVB-induced MMP-1 and -3 expressions in a dose-dependent manner. UVB strongly activated nuclear factor-kappaB (NF-kappaB) activity, which was determined by IkappaBalpha degradation, nuclear localization of p50 and p65 subunit, and NF-kappaB binding activity. However, UVB-induced NF-kappaB activation was completely blocked by RCE pretreatment. These findings suggest that RCE prevents UVB-induced MMP expression through inhibition of NF-kappaB activation. In conclusion, RCE is a potential agent for the prevention and treatment of skin photoaging.

Scutellaria barbata has been used to treat cancer in Chinese medicine. The responsible anticancer mechanism, however, is not clear. Here we demonstrated an inhibitory mechanism due to a Scutellaria barbata extract (SBE) on a human promyelocytic leukemia cell line (HL-60) that has a mutation in the tumor suppressor gene p53. HL-60 cells were incubated with various concentrations of SBE. After a 24-h incubation, cytotoxicity and apoptosis were determined by MTT and DNA fragmentation assay, respectively. After treatment with SBE, cell cycle arrest was determined by measuring the cell number stained by 5'-bromo-2'-deoxyuridine (BrdU) and 7-amino-actinomycin D (7-AAD). Treatment of cells with SBE resulted in a concentration- and time-dependent inhibition of growth and a G1 phase arrest of the cell cycle. This effect was associated with a marked decrease in the protein expression of cyclin A, D1, D2, D3, and E and their activating partners, cyclin-dependent kinases (CDK) 2, 4, and 6 with concomitant upregulation of p21, cyclin-dependent kinase inhibitor. Downstream of the CDK inhibitory protein-CDK/cyclin cascade, SBE decreased phosphorylation level of retinoblastoma protein. SBE treatment also resulted in apoptosis evidenced by an increase of sub-G1 phase cells, DNA fragmentation and degradation of the inhibitory protein for the caspase-activated deoxyribonuclease. The molecular mechanism during SBE-mediated growth inhibition in HL-60 cells may be due to modulation of the cell-cycle machinery and the induction of apoptosis.

Adipocyte differentiation is regulated by the sequential activation of transcription factors such as the CAAT/enhancer binding protein and peroxisome proliferator- activated receptor-gamma (PPAR-gamma). Several recent studies have shown that regulators of chromatin structure are also involved in adipocyte differentiation. Here we investigated the effects of diallyl disulfide (DADS), an oil-soluble sulfur compound found in processed garlic and an inhibitor of histone deacetylase (HDAC), on adipogenesis. Treatment with DADS accelerated terminal differentiation of 3T3-L1 cells into adipocytes as evidenced by Oil red O staining and cellular triglyceride assay results. Notably, the inhibition of HDAC during the first 2 days was sufficient to stimulate adipogenesis. Western blot analysis revealed that DADS increased the level of acetylated histones H3 and H4. In addition, DADS increased the expression of adipogenesis-related genes; LPL, FAS, SREBP1c, aP2 and PPAR-gamma, and decreased the expression of pref-1, a preadipocyte marker gene. Taken together, our results suggest that DADS affects adipocyte differentiation through histone acetylation at an early phase of adipocyte differentiation.