Benzo-[a]-pyrene induces FAK activation and cell migration in MDA-MB-231 breast cancer cells

Benzo-[a]-pyrene (B[a]P) is a family member of polycyclic aromatic hydrocarbons and a widespread environmental pollutant. It is a mammary carcinogen in rodents and contributes to the development of human breast cancer. However, the signal transduction pathways induced by B[a]P and its role in breast cancer progression have not been studied in detail. Here, we demonstrate that B[a]P induces cell migration through a lipoxygenase- and Src-dependent pathway, as well as the activation of focal adhesion kinase, Src, and the extracellular signal-regulated kinase 2 in MDA-MB-231 breast cancer cells. However, B[a]P is not able to promote migration in the mammary nontumorigenic epithelial cells MCF12A. Moreover, B[a]P promotes an increase of αvβ3 integrin–cell surface levels and an increase of metalloproteinase (MMP)-2 and MMP-9 secretions. In summary, our findings demonstrate that B[a]P induces the activation of signal transduction pathways and biological processes involved in the invasion/metastasis process in MDA-MB-231 breast cancer cells.     

Sodium butyrate induces G2 arrest in the human breast cancer cells MDA-MB-231 and renders them competent for DNA rereplication

When exposed to sodium butyrate (NaBut), exponentially growing cells accumulate in G1 and G2 phases of the cell cycle. In the human breast cancer cell line MDA-MB-231, an arrest in G2 phase was observed when the cells were released from hydroxyurea block (G1/S interface) in the presence of NaBut. The inhibition of G2 progression was correlated with increased contents both of total p21(Waf1) and of p21(Waf1) associated with cyclin A and with an inhibition of cyclin A- and B1-associated histone H1 kinase activities measured in cell lysates, as well as with dephosphorylation of the RB protein. A decrease in the cell contents of cyclins A and B1 was also observed but this decrease was preceded by p21(Waf1) accumulation. When NaBut was removed from the culture medium of cells blocked in G2 phase, p21(Waf1) level decreased and, instead of proceeding to mitosis, these cells resumed a progression toward DNA rereplication. These results suggest that the induction of p21(Waf1) by NaBut leads to the inhibition of the sequential activation of cyclin A- and B1-dependent kinases in this cell line, resulting in the inhibition of G2 progression and rendering the cells competent for a new cell division cycle.     

Arachidonic acid promotes FAK activation and migration in MDA-MB-231 breast cancer cells

Arachidonic acid (AA) is a common dietary n-6 polyunsaturated fatty acid that is present in an esterified form in cell membrane phospholipids, and it might be present in the extracellular microenvironment. In particular, AA promotes MAPK activation and mediates the adhesion of MDA-MB-435 breast cancer cells to type IV collagen. However, the signal transduction pathways mediated by AA have not been studied in detail. Our results demonstrate that stimulation of MDA-MB-231 breast cancer cells with AA promotes an increase in the phoshorylation of Src and FAK, as revealed by site-specific antibodies that recognized the phosphorylation state of Src at Tyr-418, and of FAK at tyrosine-397 and in vitro kinase assays. In addition, AA also induces an increase in the migration of MDA-MB-231 cells. In contrast, AA does not induce phosphorylation of FAK and an increase in cell migration of non-tumorigenic epithelial cells MCF10A. Inhibition of Gi/Go proteins, LOX and Src activity prevent FAK activation and cell migration. In conclusion, our results demonstrate, for the first time, that Gi/Go proteins, LOX and Src play an important role in FAK activation and cell migration induced by AA in MDA-MB-231 breast cancer cells.     

Lysophosphatidic acid induces MDA-MB-231 breast cancer cells migration through activation of PI3K/PAK1/ERK signaling

Background

Enhanced motility of cancer cells is a critical step in promoting tumor metastasis. Lysophosphatidic acid (LPA), representing the major mitogenic activity in serum, stimulates migration in various types of cancer cells. However, the underlying signaling mechanisms for LPA-induced motility of cancer cells remain to be elucidated.

Methodology/Principal Findings

In this study, we found that LPA dose-dependently stimulated migration of MDA-MB-231 breast cancer cells, with 10 µM being the most effective. LPA also increased ERK activity and the MEK inhibitor U0126 could block LPA-induced ERK activity and cell migration. In addition, LPA induced PAK1 activation while ERK activation and cell migration were inhibited by ectopic expression of an inactive mutant form of PAK1 in MDA-MB-231 cells. Furthermore, LPA increased PI3K activity, and the PI3K inhibitor {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 inhibited both LPA-induced PAK1/ERK activation and cell migration. Moreover, in the breast cancer cell, LPA treatment resulted in remarkable production of reactive oxygen species (ROS), while LPA-induced ROS generation, PI3K/PAK1/ERK activation and cell migration could be inhibited by N-acetyl-L-Cysteine, a scavenger of ROS.

Conclusions/Significance

Taken together, this study identifies a PI3K/PAK1/ERK signaling pathway for LPA-stimulated breast cancer cell migration. These data also suggest that ROS generation plays an essential role in the activation of LPA-stimulated PI3K/PAK1/ERK signaling and breast cancer cell migration. These findings may provide a basis for designing future therapeutic strategy for blocking breast cancer metastasis.     

Phospholipase D prevents apoptosis in v-Src-transformed rat fibroblasts and MDA-MB-231 breast cancer cells

Phospholipase D (PLD) activity is elevated in response to mitogenic and oncogenic signals. PLD also cooperates with overexpressed tyrosine kinases to transform rat fibroblasts. 3Y1 rat fibroblasts overexpressing the tyrosine kinase c-Src undergo apoptosis in response to serum withdrawal. We report here that elevated expression of either PLD1 or PLD2 in these cells prevents apoptosis induced by serum withdrawal. 3Y1 cells transformed by the activated tyrosine kinase v-Src have elevated PLD activity and are resistant to apoptosis induced by serum withdrawal. However, if PLD activity is blocked, the v-Src-transformed cells underwent apoptosis. MDA-MB-231 cells are a human breast cancer cell line with substantially elevated levels of PLD activity. Inhibiting PLD activity in these cells similarly rendered them sensitive to the apoptotic insult of serum withdrawal. These data indicate that elevated PLD activity generates a survival signal(s) allowing cells to overcome default apoptosis programs.     

Progesterone induces focal adhesion in breast cancer cells MDA-MB-231 transfected with progesterone receptor complementary DNA

Since the effects of progesterone are mediated mainly via estrogen-dependent progesterone receptor (PR), the expression of the effects of progesterone may be masked or overridden by the influence of estrogen under conditions in which priming with estrogens is required. We have established a PR-positive but estrogen receptor-alpha (ER-alpha) negative breast cancer cell model by transfecting PR cDNA into ER-alpha- and PR-negative MDA-MB-231 cells in order that the functions of progesterone can be studied independently of estrogens. We have demonstrated using this model that progesterone markedly inhibited cell growth. We have also discovered that progesterone induced remarkable changes in cell morphology and specific adhesion structures. Progesterone-treated cells became considerably more flattened and well spread than vehicle-treated control cells. This was associated with a striking increase of stress fibers, both in number and diameter, and increased focal contacts as shown by the staining of focal adhesion proteins paxillin and talin. There were also distinct increases in tyrosine phosphorylation of focal adhesion protein paxillin and focal adhesion kinase in association with increased focal adhesion. The staining of tyrosine-phosphorylated proteins was concentrated at focal adhesions in progesterone-treated cells. More interestingly, monoclonal antibody (Ab) to beta1 integrin was able to inhibit progesterone-induced cell spreading and formation of actin cytoskeleton. To our knowledge, this is the first report describing a direct effect of progesterone in inducing spreading and adhesion of breast cancer cells, and beta1-integrin appeared to play an essential role in the effect. It is known that the initial step of tumor metastasis is the breakaway of tumor cells from primary tumor mass when they lose the ability to attach. Hence, progesterone-induced cell spreading and adhesion may have significant implications in tumor metastasis.     

IGF2BP3-EGFR-AKT axis promotes breast cancer MDA-MB-231 cell growth

Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) is an emerging prognostic indicator, and its elevated expression correlates with malignancy in a broad spectrum of cancers. However, its regulatory networks have not yet been reported. In this study, we identified the regulatory targets of IGF2BP3 in breast cancer MDA-MB-231 cells using RNA immunoprecipitation sequencing (RIP-seq) and high-throughput RNA-sequencing (RNA-seq). We discovered that these targets were enriched in the inflammatory response, endoplasmic reticulum stress, cell cycle, and cancer-related pathways, providing a new perspective for better understanding the functional mechanisms of IGF2BP3. Moreover, we identified that the epidermal growth factor receptor (EGFR), a downstream target, is regulated by IGF2BP3. IGF2BP3 binds to and protects EGFR mRNA from degradation and facilitates cell proliferation via the EGFR/AKT pathway in MDA-MB-231 cells. In addition, IGF2BP3 expression was robust and could not be altered by stimulation with EGF and anti-EGFR siRNA or EGFR signaling pathway inhibitors (gefitinib, LY294002 and SL-327). These results demonstrate that IGF2BP3, as a stubborn oncogene, promotes triple-negative breast cancer MDA-MB-231 cell proliferation by strengthening the role of the EGFR-AKT axis.     

Impaired G2/M cell cycle arrest induces apoptosis in pyruvate carboxylase knockdown MDA-MB-231 cells

BackgroundPrevious studies showed that suppression of pyruvate carboxylase (PC) expression in highly invasive breast cancer cell line, MDA-MB-231 inhibits cell growth as a consequence of the impaired cellular biosynthesis. However, the precise cellular mechanism underlying this growth restriction is unknown.MethodsWe generated the PC knockdown (PCKD) MDA-MB-231 cells and assessed their phenotypic changes by fluorescence microscopy, proliferation, apoptotic, cell cycle assays and proteomics.ResultsPC knockdown MDA-MB-231 cells had a low percentage of cell viability in association with accumulation of abnormal cells with large or multi-nuclei. Flow cytometric analysis of annexin V-7-AAD positive cells showed that depletion of PC expression triggers apoptosis with the highest rate at day 4. The increased rate of apoptosis is consistent with increased cleavage of procaspase 3 and poly (ADP-Ribose) polymerase. Cell cycle analysis showed that the apoptotic cell death was associated with G2/M arrest, in parallel with marked reduction of cyclin B levels. Proteomic analysis of PCKD cells identified 9 proteins whose expression changes were correlated with the degree of apoptosis and G2/M cell cycle arrest in the PCKD cells. STITCH analysis indicated 3 of 9 candidate proteins, CCT3, CABIN1 and HECTD3, that form interactions with apoptotic and cell cycle signaling networks linking to PC via MgATP.ConclusionsSuppression of PC in MDA-MB-231 cells induces G2/M arrest, leading to apoptosis. Proteomic analysis supports the potential involvement of PC expression in the aberrant cell cycle and apoptosis, and identifies candidate proteins responsible for the PC-mediated cell cycle arrest and apoptosis in breast cancer cells.General significanceOur results highlight the possibility of the use of PC as an anti-cancer drug target.     

Cycling hypoxia up-regulates thioredoxin levels in human MDA-MB-231 breast cancer cells

The thioredoxin system is a key cellular antioxidant system and is highly expressed in cancer cells, especially in more aggressive and therapeutic resistant tumors. We analysed the expression of the thioredoxin system in the MDA-MB-231 breast cancer cell line under conditions mimicking the tumor oxygen microenvironment. We grew breast cancer cells in either prolonged hypoxia or hypoxia followed by various lengths of reoxygenation and in each case cells were cultured with or without a hypoxic cycling preconditioning (PC) phase preceding the hypoxic growth. Flow cytometry-based assays were used to measure reactive oxygen species (ROS) levels. Cells grown in hypoxia showed a significant decrease in ROS levels compared to normoxic cells, while a significant increase in ROS levels over normoxic cells was observed after 4 h of reoxygenation. The PC pre-treatment did not have a significant effect on ROS levels. Thioredoxin levels were also highest after 4 h of reoxygenation, however cells subjected to PC pre-treatment displayed even higher thioredoxin levels. The high level of intracellular thioredoxin was also reflected on the cell surface. Reporter assays showed that activity of the thioredoxin and thioredoxin reductase gene promoters was also highest in the reoxygenation phase, although PC pre-treatment did not result in a significant increase over non-PC treated cells. The use of a dominant negative Nrf-2 negated the increased thioredoxin promoter activity during reoxygenation. This data suggests that the high levels of thioredoxin observed in tumors may arise due to cycling between hypoxia and reoxygenation.     

Docosahexaenoic acid (omega-3) blocks voltage-gated sodium channel activity and migration of MDA-MB-231 human breast cancer cells

Omega − 3 polyunsaturated fatty acids have been suggested to play an important role in cancer prevention/progression, on the one hand, and in modulation of membrane ion channels on the other. We investigated whether docosahexaenoic acid would influence the in vitro migration of MDA-MB-231 human breast cancer cells. An important follow-up question was whether any effect would involve voltage-gated Na⁺ channels, shown previously to occur in human breast cancer in vitro and in vivo and to correlate with metastatic potential. Short-term (acute) and long-term (24–72 h) application of docosahexaenoic acid suppressed the activity of the channel activity in a dose-dependent manner. At the working concentrations of docosahexaenoic acid used (0.05–0.5 μM), there was no effect on proliferation. Long-term treatment with docosahexaenoic acid down-regulated mRNA and protein (total and plasma membrane) levels of neonatal Nav1.5 voltage-gated Na⁺ channel, known to be predominant in these cells. Docosahexaenoic acid suppressed migration of the MDA-MB-231 cells to the same extent as tetrodotoxin, a highly specific blocker of voltage-gated Na⁺ channels, but the two effects were not additive. It was concluded that the docosahexaenoic acid-induced suppression of cellular migration occurred primarily via down-regulation of voltage-gated Na⁺ channel (neonatal Nav1.5) mRNA and functional protein expression.     

MicroRNA-125b induces metastasis by targeting STARD13 in MCF-7 and MDA-MB-231 breast cancer cells

MicroRNAs (miRNAs) are a class of small noncoding RNAs that regulate gene expression by targeting mRNAs to trigger either translation repression or mRNA degradation. miR-125b is down-regulated in human breast cancer cells compared with the normal ones except highly metastatic tumor cells MDA-MB-231. However, few functional studies were designed to investigate metastatic potential of miR-125b. In this study, the effects of miR-125b on metastasis in human breast cancer cells were studied, and the targets of miR-125b were also explored. Transwell migration assay, cell wound healing assay, adhesion assay and nude mice model of metastasis were utilized to investigate the effects of miR-125b on metastasis potential in vitro and in vivo. In addition, it was implied STARD13 (DLC2) was a direct target of miR-125b by Target-Scan analysis, luciferase reporter assay and western blot. Furthermore, activation of STARD13 was identified responsible for metastasis induced by miR-125b through a siRNA targeting STARD13. qRT-PCR, immunofluorescent assay and western blot was used to observe the variation of Vimentin and α-SMA in breast cancer cells. In summary, our study provided new insights into the function of miR-125b during the metastasis of breat cancer cells and also suggested the role of miR-125b in pro-metastasis by targeting STARD13.     

Diosgenin inhibits the migration of human breast cancer MDA-MB-231 cells by suppressing Vav2 activity

Diosgenin, a naturally occurring steroidal saponin, possess tumor therapeutic potential. However, the effect of diosgenin on cancer metastasis remains poorly understood. In this study, we performed in vitro experiments to investigate the inhibitory activity of diosgenin on human breast cancer MDA-MB-231 cell migration, and reveal the possible mechanism. Diosgenin caused a marked inhibition of cell migration in MDA-MB-231 cell by transwell assay. In addition, diosgenin significantly impacted MDA-MB-231 cell migratory behavior under real-time observation. We also found diosgenin significantly inhibited actin polymerization, Vav2 phosphorylation and Cdc42 activation, which might be, at least in part, attributed to the anti-metastatic potential of diosgenin. These findings reveal a new therapeutic potential of diosgenin for human breast cancer metastasis therapy.     

6]-Gingerol inhibits metastasis of MDA-MB-231 human breast cancer cells

Gingerol (Zingiber officinale Roscoe, Zingiberaceae) is one of the most frequently and heavily consumed dietary condiments throughout the world. The oleoresin from rhizomes of ginger contains [6]-gingerol (1-[4′-hydroxy-3′-methoxyphenyl]-5-hydroxy-3-decanone) and its homologs which are pungent ingredients that have been found to possess many interesting pharmacological and physiological activities, such as anti-inflammatory, antihepatotoxic and cardiotonic effects. However, the effects of [6]-gingerol on metastatic processes in breast cancer cells are not currently well known. Therefore, in this study, we examined the effects of [6]-gingerol on adhesion, invasion, motility, activity and the amount of MMP-2 or -9 in the MDA-MB-231 human breast cancer cell line. We cultured MDA-MB-231 cells in the presence of various concentrations of [6]-gingerol (0, 2.5, 5 and 10 μM). [6]-Gingerol had no effect on cell adhesion up to 5 μM, but resulted in a 16% reduction at 10 μM. Treatment of MDA-MB-231 cells with increasing concentrations of [6]-gingerol led to a concentration-dependent decrease in cell migration and motility. The activities of MMP-2 or MMP-9 in MDA-MB-231 cells were decreased by treatment with [6]-gingerol and occurred in a dose-dependent manner. The amount of MMP-2 protein was decreased in a dose-dependent manner, although there was no change in the MMP-9 protein levels following treatment with [6]-gingerol. MMP-2 and MMP-9 mRNA expression were decreased by [6]-gingerol treatment. In conclusion, we have shown that [6]-gingerol inhibits cell adhesion, invasion, motility and activities of MMP-2 and MMP-9 in MDA-MB-231 human breast cancer cell lines.