Anti-invasive effect of gambogic acid in MDA-MB-231 human breast carcinoma cells

Gambogic acid (GA) has been known to have antitumor activity in vitro and in vivo. In the present study, we investigated the anti-invasive effects of GA in MDA-MB-231 human breast carcinoma cells. The results indicated that GA significantly inhibited the adhesion, migration, and invasion of the cells in vitro tested by the heterotypic adhesion assay, wound migration assay, and chamber invasion assay. Results of Western blotting and immunocytochemistry analysis showed that GA could suppress the expressions of matrix metalloproteinase 2 (MMP-2) and 9 (MMP-9) in MDA-MB-231 cells. Furthermore, gelatin zymography revealed that GA decreased the activities of MMP-2 and MMP-9. Additionally, GA exerted an inhibitory effect on the phosphorylation of ERK1/2 and JNK, while it had no effect on p38. Taken together, our results demonstrated the anti-invasive property of GA for the first time and indicated it could serve as a promising drug for the treatment of cancer metastasis.     

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.     

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.     

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.     

Oleic acid promotes migration on MDA-MB-231 breast cancer cells through an arachidonic acid-dependent pathway

An association between dietary fatty, obesity and an increased risk of developing breast cancer has been suggested. In breast cancer cells, free fatty acids (FFAs) mediate biological effects including cell proliferation and ERK1/2 activation. However, the contribution of FFAs to tumor progression and metastasis through the regulation of cell migration has not been studied. We demonstrated here that stimulation on MDA-MB-231 breast cancer cells with oleic acid (OA) promotes an increase in focal adhesion kinase (FAK) phosphorylation, as revealed by site-specific antibodies that recognize the phosphorylation state of FAK at tyrosine-397 (Tyr-397), Tyr-577 and in vitro kinase assays. OA also promotes the migration of MDA-MB-231 cells. Treatment with Gi/Go proteins, phospholipase C (PLC), lipoxygenases (LOXs) and Src inhibitor prevents FAK phosphorylation and cell migration. In summary, our findings delineate a new signal transduction pathway, where OA mediates the production of arachidonic acid (AA), and then AA metabolites mediate FAK phosphorylation and cell migration in MDA-MB-231 breast cancer cells.     

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.     

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.     

Role of arachidonic acid metabolism in Stat5 activation induced by oleic acid in MDA-MB-231 breast cancer cells

Epidemiological studies and animal models suggest an association between high levels of dietary fat intake and an increased risk of breast cancer. In breast cancer cells, the free fatty acid oleic acid (OLA) induces proliferation, migration, invasion and an increase of MMP-9 secretion. However, the role of OLA on Stat5 activation and the participation of COX-2 and LOXs activity in Stat5 activation induced by OLA remain to be investigated. We demonstrate here that stimulation of MDA-MB-231 breast cancer cells with 100 μM OLA induces Stat5 phosphorylation at Tyr-694 and an increase of Stat5–DNA complex formation. The Stat5 DNA-binding activity requires COX-2, LOXs, metalloproteinases and Src activities. In addition, OLA induces cell migration through a Stat5-dependent pathway. In summary, our findings establish that OLA induces cell migration through a Stat5-dependent pathway and that Stat5 activation requires AA metabolites in MDA-MB-231 breast cancer cells.     

Lysophosphatidylethanolamine utilizes LPA1 and CD97 in MDA-MB-231 breast cancer cells

Lysophosphatidylethanolamine (LPE) is a lyso-type metabolite of phosphatidylethanolamine (a plasma membrane component), and its intracellular Ca^2 + ([Ca^2 +]_i) increasing actions may be mediated through G-protein-coupled receptor (GPCR). However, GPCRs for lysophosphatidic acid (LPA), a structurally similar representative lipid mediator, have not been implicated in LPE-mediated activities in SK-OV3 or OVCAR-3 ovarian cancer cells or in receptor over-expression systems. In the present study, LPE-induced [Ca^2 +]_i increase was observed in MDA-MB-231 cells but not in other breast cancer cell lines. In addition, LPE- and LPA-induced responses showed homologous and heterologous desensitization. Furthermore, VPC32183 and Ki16425 (antagonists of LPA₁ and LPA₃) inhibited LPE-induced [Ca^2 +]_i increases, and knockdown of LPA₁ by transfection with LPA₁ siRNA completely inhibited LPE-induced [Ca^2 +]_i increases. Furthermore, the involvement of CD97 (an adhesion GPCR) in the action of LPA₁ in MDA-MB-231 cells was demonstrated by siRNA transfection. Pertussis toxin (a specific inhibitor of G_i/o proteins), edelfosine (an inhibitor of phospholipase C), or 2-APB (an inhibitor of IP₃ receptor) completely inhibited LPE-induced [Ca^2 +]_i increases, whereas HA130, an inhibitor of autotaxin/lysophospholipase D, did not. Therefore, LPE is supposed to act on LPA₁-CD97/G_i/o proteins/phospholipase C/IP₃/Ca^2 + rise in MDA-MB-231 breast cancer cells.     

Unravelling the antimetastatic potential of pentoxifylline, a methylxanthine derivative in human MDA-MB-231 breast cancer cells

Pentoxifylline (PTX), a methylxanthine derivative is a non-steroidal immunomodulating agent with unique hemorheologic properties. It is used in the treatment of intermittent claudication as it increases the amount of oxygen reaching tissues by increasing the flexibility of red blood cells. Recently, it has also shown to exhibit anti-metastatic and anti-angiogenic activities in B16F10 melanoma cells both in vitro as well as in vivo. As per the reports, the choice of drug in the treatment of breast cancer is paclitaxel, but the major limitation is its toxicity. However, the effects of PTX on metastatic processes in breast cancer are not currently known. Therefore, in this study, we have examined the effect of PTX in MDA-MB-231 human breast cancer cells. The MTT assay showed dose- and time-dependent decreases in cellular proliferation. The non-toxic concentration of PTX selected were 1, 2.5 and 5 mM for 24 h. PTX induced a G0-G1 cell-cycle arrest leading to apoptosis. Further, it affected adhesion to both the matrigel and collagen type-IV in a time- and dose-dependent manner. The PTX impeded the migration of MDA-MB-231 cells and also decreased the activities of both MMP-2 and MMP-9. Thus, PTX at non-toxic doses affected cellular proliferation, adhesion, migration and invasion. These results demonstrate its anti-metastatic effect on MDA-MB-231 cells, and further studies need to be carried out to understand the mechanism of action.     

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.     

Cytotoxic effect of sanguiin H-6 on MCF-7 and MDA-MB-231 human breast carcinoma cells

Sanguiin H-6 is a dimer of casuarictin linked by a bond between the gallic acid residue and one of the hexahydroxydiphenic acid units. It is an effective compound extracted from Rubus coreanus. It has an anticancer effect against several human cancer cells; however, its effect on breast cancer cells has not been clearly demonstrated. Thus, we aimed to investigate the anticancer effect and mechanism of action of sanguiin H-6 against two human breast carcinoma cell lines (MCF-7 and MDA-MB-231). We found that sanguiin H-6 significantly reduced cell viability in a concentration-dependent manner. It also increased the rates at which MCF-7 and MDA-MB-231 cells underwent apoptosis. Furthermore, sanguiin H-6 induced the cleavage of caspase-8, caspase-3, and poly(ADP-ribose) polymerase, which resulted in apoptosis. However, cleavage of caspase-9 was only detectable in MCF-7 cells. In addition, sanguiin H-6 increased the ratio of Bax to Bcl-2 in both MCF-7 and MDA-MB-231 cells. These findings suggest that sanguiin H-6 is a potent therapeutic agent against breast cancer cells. In addition, it exerts its anticancer effect in an estrogen-receptor-independent manner.     

Stable transfection of an estrogen receptor beta cDNA isoform into MDA-MB-231 breast cancer cells

We previously reported stable transfection of estrogen receptor alpha (ERalpha) into the ER-negative MDA-MB-231 cells (S30) as a tool to examine the mechanism of action of estrogen and antiestrogens [J. Natl. Cancer Inst. 84 (1992) 580]. To examine the mechanism of ERbeta action directly, we have similarly created ERbeta stable transfectants in MDA-MB-231 cells. MDA-MB-231 cells were stably transfected with ERbeta cDNA and clones were screened by estrogen response element (ERE)-luciferase assay and ERbeta mRNA expression was quantified by real-time RT-PCR. Three stable MDA-MB-231/ERbeta clones were compared with S30 cells with respect to their growth properties, ability to activate ERE- and activating protein-1 (AP-1) luciferase reporter constructs, and the ability to activate the endogenous ER-regulated transforming growth factor alpha (TGFalpha) gene. ERbeta6 and ERbeta27 clones express 300-400-fold and the ERbeta41 clone express 1600-fold higher ERbeta mRNA levels compared with untransfected MDA-MB-231 cells. Unlike S30 cells, 17beta-estradiol (E2) does not inhibit ERbeta41 cell growth. ERE-luciferase activity is induced six-fold by E2 whereas neither 4-hydroxytamoxifen (4-OHT) nor ICI 182, 780 activated an AP-1-luciferase reporter. TGFalpha mRNA is induced in response to E2, but not in response to 4-OHT. MDA-MB-231/ERbeta clones exhibit distinct characteristics from S30 cells including growth properties and the ability to induce TGFalpha gene expression. Furthermore, ERbeta, at least in the context of the MDA-MB-231 cellular milieu, does not enhance AP-1 activity in the presence of antiestrogens. In summary, the availability of both ERalpha and ERbeta stable breast cancer cell lines now allows us to compare and contrast the long-term consequences of individual signal transduction pathways.     

Saturated fatty acid-induced apoptosis in MDA-MB-231 breast cancer cells. A role for cardiolipin

Little is known about the biochemical basis of the action of free fatty acids (FFA) on breast cancer cell proliferation and apoptosis. Here we report that unsaturated FFAs stimulated the proliferation of human MDA-MB-231 breast cancer cells, whereas saturated FFAs inhibited it and caused apoptosis. Saturated FFA palmitate decreased the mitochondrial membrane potential and caused cytochrome c release. Palmitate-induced apoptosis was enhanced by the fat oxidation inhibitor etomoxir, whereas it was reduced by fatty-acyl CoA synthase inhibitor triacsin C. The non-metabolizable analog 2-bromopalmitate was not cytotoxic. This indicates that palmitate must be metabolized to exert its toxic effect but that its action does not involve fat oxidation. Pharmacological studies showed that the action of palmitate is not mediated via ceramides, reactive oxygen species, or changes in phosphatidylinositol 3-kinase activity. Palmitate caused early enhancement of cardiolipin turnover and decreased the levels of this mitochondrial phospholipid, which is necessary for cytochrome c retention. Cosupplementation of oleate, or increasing beta-oxidation with the AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide-1-beta-D-ribonucleoside, both restored cardiolipin levels and blocked palmitate-induced apoptosis. Oleate was preferentially metabolized to triglycerides, and oleate cosupplementation channeled palmitate esterification processes to triglycerides. Overexpression of Bcl-2 family members blocked palmitate-induced apoptosis. The results provide evidence that a decrease in cardiolipin levels and altered mitochondrial function are involved in palmitate-induced breast cancer cell death. They also suggest that the antiapoptotic action of oleate on palmitate-induced cell death involves both restoration of cardiolipin levels and redirection of palmitate esterification processes to triglycerides.